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06 August 2013, Volume 17 Issue 32 Previous Issue    Next Issue

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Methylcobalamin induces differentiation of rat bone mesenchymal stem cells into neuron-like cells in vitro Yang Ming-zhi, Peng Li-jun, Hu Wen-kai 2013, 17 (32):  5741-5748.  doi: 10.3969/j.issn.2095-4344.2013.32.001 Abstract ( 445 )   PDF (2314KB) ( 392 )   Save BACKGROUND: Currently, transplantation of bone marrow mesenchymal stem cells into the spinal cord is very limited to the recovery of animals following spinal cord injury. Methylcobalamin is a common drug for the treatment of neurological diseases and injuries, but its effects on bone marrow mesenchymal stem cells are unclear. OBJECTIVE: To study the feasibility of bone marrow mesenchymal stem cells differentiating into neuron-like cells induced by methylcobalamin in vitro and to observe the cell viability and proliferation of differentiated cells.  Methods: Rat bone marrow mesenchymal stem cells were isolated, cultured and purified by density gradient centrifugation and adherent culture. The fourth to fifth generation of bone marrow mesenchymal stem cells were treated for 24, 48 and 72 hours with different concentrations (25, 50 and 100 mg/L) of methylcobalamin. The morphological changes and cell growth were continuously observed under an inverted phase constract microscope. The viability of induced cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The expressions of Nestin and neuron-specific enolase were identified by reverse transcription PCR and western blot.  RESULTS AND CONCLUSION: Most of bone marrow mesenchymal stem cells could differentiate into neuron-like cells after induction with methylcobalamin. The expressions of Nestin and neuron-specific enolase were up-regulated after 48 hours of methylcobalamin treatment at different concentrations, especially after treatment with 100 mg/L methylcobalamin. Similarly, the expressions of Nestin and neuron-specific enolase could be increased significantly after 100 mg/L methylcobalamin treatment for 24, 48 and 72 hours, especially for 72 hours. It is indicated that methylcobalamin can induce bone marrow mesenchymal stem cells differentiating into neuron-like cells, and the optimal concentration of methylcobalamin is 100 mg/L. Figures and Tables | References | Related Articles | Metrics

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Bone marrow mesenchymal stem cells are involved in tissue repair of A549 lung adenocarcinoma Xu Feng, Zhang Lei, Pan Jin-kun, Xue Li-li, Zhao Xiao-yan, Li Bao-ping 2013, 17 (32):  5749-5756.  doi: 10.3969/j.issn.2095-4344.2013.32.002 Abstract ( 396 )   PDF (2336KB) ( 370 )   Save BACKGROUND: Tumor has been considered as a specific nonhealing trauma. Bone marrow mesenchymal stem cells participate in tumor mesenchymal reconstitution by tumor tissue homing and differentiation into mesenchyme, resulting in changing tumor microenvironment and affecting tumor growth and transfer. OBJECTIVE: To explore the mechanisms of participation of bone marrow mesenchymal stem cells in tumor tissue repair in an A549 lung cancer-bearing mouse model. METHODS: Bone marrow mesenchymal stem cells were isolated in vitro, cultured, and identified using flow cytometry, and then used to establish a mouse model of A549 lung cancer-bearing. In the experimental group, human bone marrow mesenchymal stem cells were injected into tissue surrounding the tumor. In the control group, an equal volume of PBS was injected. Animal survival condition and tumor size were compared. At 4 weeks, the specimens were harvested. Hematoxylin-eosin staining was used to compare tumor tissue. Masson staining was utilized to compare collagen fiber content. Reverse transcription-PCR was employed to detect the expression of α-smooth muscle actin. Immunohistochemistry was used to examine the expression of fibroblast specific protein and fibroblast activation protein to reflect the degree of interstitial fibers in tumor tissue in both groups. The expression levels of vascular endothelial growth factor, hepatocyte growth factor, interleukin-6 and tenescin-C were compared between the two groups using immunohistochemistry. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells promoted tumor growth in tumor-bearing mice. The growth rate of tumor tissue in experimental group was faster than the control group (P < 0.05). Compared with the control group, α-smooth muscle actin mRNA expression was significantly higher in the experimental group. Immunohistochemistry was used to detect the expression of tumor angiogenesis factors markers (fibroblast specific protein and fibroblast activation protein) in tumor tissue of experimental group. The expression levels of vascular endothelial growth factor, hepatocyte growth factor, interleukin-6 and tenescin-C were significantly greater in the experimental group than in the control group (P < 0.05). Results indicated that bone marrow mesenchymal stem cells differentiated into fibroblasts in tumor microenvironment, participated in the formation and construction of tumor stroma as well as promoted the growth and repair of tumor via the secretion of vascular endothelial growth factor, hepatocyte growth factor, interleukin-6 and tenescin-C. Figures and Tables | References | Related Articles | Metrics

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Induction ways of bone marrow mesenchymal stem cells differentiating into nerve cells Chen Zeng-sheng, Chu Qiang, Liu Yan-feng, Song Xuan, Li Ping 2013, 17 (32):  5757-5764.  doi: 10.3969/j.issn.2095-4344.2013.32.003 Abstract ( 343 )   PDF (453KB) ( 361 )   Save BACKGROUND: Currently, bone marrow mesenchymal stem cells can differentiate into nerve cells via many approaches. Different methods for inducing bone marrow mesenchymal stem cells differentiating into nerve cells have different ratios. OBJECTIVE: To investigate the difference between chemical method and co-culture method to induce the differentiation of rat bone marrow mesenchymal stem cells into nerve cells. METHODS: Rat bone marrow mesenchymal stem cells were isolated and purified using whole bone marrow culture method, and then randomly divided into two groups: chemical group, β-mercaptoethanol was added; co-culture group, co-cultured in a Transwell chamber. RESULTS AND CONCLUSION: Visible protrusions from induced cells showed radiation growth at 1 week of induced culture, and neuron-specific enolase staining was positive at 2 weeks of culture. Star-like structure of nerve cells was visible in the co-culture group within 4-5 days of culture, and then more protrusions formed. Meanwhile, the positive rate of neuron-specific enolase was (70.82±2.46)%. After 6-7 days of culture, neuron-like cells formed and were interconnected in the chemical group; while, the positive rate of neuron-specific enolase was (52.37±1.83)%. These findings suggest that cell microenvironment plays a leading role in the differentiation of bone marrow mesenchymal stem cells into nerve cells, and chemical induction method is inferior to the co-culture method. Figures and Tables | References | Related Articles | Metrics

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Human umbilical cord mesenchymal stem cells: Comparison of hematopoiesis supporting capacity before and after cryopreservation Chang Xiang-ping, Ma Yan, Bi Xiao-juan, Song Li-juan, Duan Xian-lin, Jiang Ming 2013, 17 (32):  5765-5771.  doi: 10.3969/j.issn.2095-4344.2013.32.004 Abstract ( 374 )   PDF (2229KB) ( 667 )   Save BACKGROUND: Cryopreservation of human umbilical cord mesenchymal stem cells has been a hot research issue currently, but the studies concerning their effects on expansion of hematopoietic stem/progenitor cells after cryopreservation are seldom. OBJECTIVE: To investigate the effects of human umbilical cord mesenchymal stem cells before and after cryopreservation as feeder layer on expansion of human bone marrow mononuclear cells in vitro. METHODS: 2.5g/L mitomycin C processed human umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells at passage 3 were used as the feeder layer to expand adult allogeneic bone marrow mononuclear cells in culture. Up to day 35, methylcellulose assay was used to detect hematopoietic stem/progenitor cell colony proliferation. RESULTS AND CONCLUSION: There were no differences in the morphology and size of colonies in the cryopreserved human umbilical cord mesenchymal stem cell group, bone marrow mesenchymal stem cell group and non-cryopreserved human umbilical cord mesenchymal stem cell group. However, these parameter described above were significantly higher in these three groups than the blank control group (P < 0.05). There were fewer colonies in the cryopreserved human umbilical cord mesenchymal stem cell group than the non-cryopreserved human umbilical cord mesenchymal stem cell group (P < 0.05). These findings indicate that human umbilical cord mesenchymal stem cells before and after cryopreservation have the ability as feeder layer on expansion of bone marrow mononuclear cells in vitro similar to bone marrow mesenchymal stem cells. But this ability of human umbilical cord mesenchymal stem cells may decrease after cryopreservation. Figures and Tables | References | Related Articles | Metrics

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Human umbilical cord-derived mesenchymal stem cells co-cultured with hepatocytes can differentiate into hepatocyte-like cells Li Hua, Wen Feng, Qi Zhong-chun, Zhou Jin-jun, Zhu Ya-jie, Cheng Peng, Wei Dong, Su Xiao-mei, Tan Yong, Peng Jing-jing, Luo Qiao-li, Li Dong, Zhang Tao 2013, 17 (32):  5772-5777.  doi: 10.3969/j.issn.2095-4344.2013.32.005 Abstract ( 424 )   PDF (1874KB) ( 569 )   Save BACKGROUND: The studies have shown that the mesenchymal stem cells derived from bone marrow and umbilical cord can be continuously cultured in vitro, and maintain the characteristics of stem cells. The mesenchymal stem cells can differentiate into hepatocyte-like cells after “cocktail” induction by various cytokines. OBJECTIVE: To further identify whether umbilical cord-derived mesenchymal stem cells in vitro co-cultured with normal hepatocytes can differentiate into hepatocyte-like cells, and to investigate the differentiation method. METHODS: Mesenchymal stem cells were isolated from human umbilical cord with adherent method, and the surface markers of umbilical cord-derived mesenchymal stem cells were detected with flow cytometry. The umbilical cord-derived mesenchymal stem cells were co-cultured with liver LO2 cells without adding exogenous inducers. The expressions of alpha-fetoprotein, albumin and human cytokeratin 19 mRNA of hepatocyte specific markers were detected with reverse transcription PCR at 7, 14 and 21 days after culture, and periodic acid-Schiff staining was used to identify the functions. RESULTS AND CONCLUSION: Mesenchymal stem cells could isolated from human umbilical cord successfully, showing fibroblastic morphology and adherent cell characterization. Among these cells, 96.02% cells were CD29 positive cells and 96.6% cells were CD105 positive cells. The percentage of CD34 negative cells was 99.65%. The percentage of CD105+CD29+ double positive cells was 94.84%. The mRNA of alpha-fetoprotein was found on the 7th day after co-cultured with LO2 cells, and the mRNA of albumin and human cytokeratin 19 were found on the 14th day. After co-cultured for 21 days, the alpha-fetoprotein mRNA could not be observed in the co-culture group. The expressions of albumin and human cytokeratin 19 were increased at 14 days. After co-cultured for 21 days, the glycogen staining was positive. Umbilical cord-derived mesenchymal stem cells can differentiate into hepatocyte-like cells after co-cultured with normal hepatocytes. Figures and Tables | References | Related Articles | Metrics

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Adipose stem cell-derived growth factors and proliferation of oral mucosa fibroblasts Zhao Jia-jia, Hu Li, Liu Jia-rong, Gong Ni-ya, Chen Li-li 2013, 17 (32):  5778-5784.  doi: 10.3969/j.issn.2095-4344.2013.32.006 Abstract ( 450 )   PDF (387KB) ( 447 )   Save BACKGROUND: Adipose stem cells have been confirmed to promote the repair of soft tissue after damage, and the action mechanism is possibly related to the paracrine of adipose stem cells, that is adipose stem cells secrete a variety of cytokines, which may promote the restoration of damaged cells. However, little report addressed the types of adipose stem cells secreted factors, contents of each factor, and role in soft tissue repair after damage, especially oral mucosa. OBJECTIVE: To observe the influence of vascular endothelial growth factor, platelet-derived growth factor, and basic fibroblast growth factor in adipose stem cell-conditioned medium on the proliferation of oral mucosa fibroblasts. METHODS: Protein microarray analysis was used to analyze the contents of vascular endothelial growth factor, platelet-derived growth factor, and basic fibroblast growth factor in adipose stem cell-conditioned medium. CCK8 analysis was used to analyze the effects of adipose stem cell-conditioned medium with different concentrations of vascular endothelial growth factor, platelet-derived growth factor, and basic fibroblast growth factor (0, 1, 10, 20, 50 and 100 μg/L) or the neutralizing antibody of the three kinds of cytokines on the proliferation of oral mucosa fibroblast on 1, 2, 3, 4 and 5 days. RESULTS AND CONCLUSION: High contents of vascular endothelial growth factor, platelet-derived growth factor, and basic fibroblast growth factor were contained in adipose stem cell-conditioned medium (signal value > 300). Adipose stem cell-conditioned medium could promote the proliferation of oral mucosa fibroblasts, wherein, the promotion effects of platelet-derived growth factor and basic fibroblast growth factor were very significant, and the peak changes could be observed with variation of cytokines concentrations. The optimal concentrations of platelet-derived growth factor and basic fibroblast growth factor were 50 μg/L and 1 μg/L, respectively (P ≤ 0.05). The neutralizing antibody of platelet-derived growth factor and basic fibroblast growth factor inhibited the promotion effect of adipose stem cell-conditioned medium. On the other hand, vascular endothelial growth factor had no significant effect on the proliferation of oral mucosa fibroblasts. Adipose stem cell-conditioned medium can promote the proliferation of oral mucosa fibroblasts, platelet-derived growth factor and basic fibroblast growth factor in adipose stem cell-conditioned medium have obvious promotion effects, which are dependent on the cytokine concentrations. Therefore, we should pay attention to choose the optimal concentrations of cytokines, thereby effectively promoting the proliferation of fibroblasts. Figures and Tables | References | Related Articles | Metrics

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Oxidative stress effect on viability of umbilical cord-derived mesenchymal stem cells in storage solution of transplantation Niu Yu-hu, Chen Yan, Zhang Jian-lin, Lei Xin, Dong Yan-ting, Cui Lei, Niu Bo 2013, 17 (32):  5785-5792.  doi: 10.3969/j.issn.2095-4344.2013.32.007 Abstract ( 488 )   PDF (2236KB) ( 413 )   Save BACKGROUND: The viability of human umbilical cord-derived mesenchymal stem cells is often declined with the commonly used transplantation storage solution in clinics, which may influence the therapeutic effects of cellular transplantation. However, reasons for this are still unknown. OBJECTIVE: To investigate the role of oxidative stress in the reduction of human umbilical cord-derived mesenchymal stem cells viability in the storage process during clinical transplantation and to observe the effects of radical scavenger on the results. METHODS: Human umbilical cord-derived mesenchymal stem cells were harvested and cultured in normal saline for 0, 2, 4 and 6 hours at room temperature. Intracellular reactive oxygen levels were detected at those time points. Antioxidant enzyme activities and levels of malondialdehyde were measured to determine the intracellular oxidative stress levels after storage. Cell adhesion rate changes were retested after adding N-acetyl cysteine to the storage solution. RESULTS AND CONCLUSION: The reactive oxygen levels in human umbilical cord-derived mesenchymal stem cells were increased significantly after normal saline storage and levels of malondialdehyde were increased in a time-dependent manner. Activities of superoxide dismutase, catalase and glutathione peroxidase were all reduced. Addition of N-acetyl cysteine into the storage medium decreased the reactive oxygen levels and improved the human umbilical cord-derived mesenchymal stem cells viabilities. Experimental findings indicate that, increased reactive oxygen species in human umbilical cord-derived mesenchymal stem cells is one of the reasons for reduced cell viability. Adding the radical scavenger N-acetyl cysteine can improve the storage effects of human umbilical cord-derived mesenchymal stem cells. Figures and Tables | References | Related Articles | Metrics

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Chondrogenic potential of adipose-derived stem cells versus bone marrow mesenchymal stem cells An Rong-ze, Zhao Jun-yan, Wang Zhao-jie 2013, 17 (32):  5793-5798.  doi: 10.3969/j.issn.2095-4344.2013.32.008 Abstract ( 545 )   PDF (2087KB) ( 531 )   Save BACKGROUND: Adipose-derived stem cells and bone marrow mesenchymal stem cells are used widely in cartilage tissue engineering, and there are many similarities in biological characteristics between two kinds of cells. OBJECTIVE: To compare the chondrogenic potential of bone marrow mesenchymal stem cells and adipose-derived stem cells in vitro. METHODS: Adipose-derived stem cells were isolated from the 3-month-old New Zealand white rabbits’ abdomen. Bilateral femurs of rabbits were obtained, and then the bone marrow mesenchymal stem cells were separated with the adherence screening method. The growth curve of the passage 3 adipose-derived stem cells and bone marrow mesenchymal stem cells were drawn, and the doubling time of two kinds of cells was compared. Then the passage 3 adipose-derived stem cells and bone marrow mesenchymal stem cells were treated with chondrogenic induction. After induced for 14 days, the adipose-derived stem cells and bone marrow mesenchymal stem cells were treated with toluidine blue staining and type Ⅱ immunohistochemistry staining respectively.  RESULTS AND CONCLUSION: Primary bone marrow mesenchymal stem cells showed aggregative growth, while the primary adipose-derived stem cells were in single and scattered growth. The proliferation speed of adipose-derived stem cells was faster than that of bone marrow mesenchymal stem cells, while the doubling time of adipose-derived stem cells was shorter than that of the bone marrow mesenchymal stem cells. After chondrogenic induction for 14 days, both adipose-derived stem cells and bone marrow mesenchymal stem cells could express glycosaminoglycans and type Ⅱ collagen, and the expression level of type Ⅱ collagen in bone marrow mesenchymal stem cells after chondrogenic induction was higher than that in the adipose-derived stem cells. The in vitro proliferation of adipose-derived stem cells and bone marrow mesenchymal stem cells was rapid and stable, but the proliferative ability of adipose-derived stem cells was faster than that of bone marrow mesenchymal stem cells. When cultured in single layer, both adipose-derived stem cells and bone marrow mesenchymal stem cells could transform into chondrocytes under certain conditions, but bone marrow mesenchymal stem cells seemed to be more potential than adipose-derived stem cells. Figures and Tables | References | Related Articles | Metrics

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Ginsenoside Rb1 affects the proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro Luo Zhi-jun, Li Hong-mian, Wang He-geng, Liu Da-lie, Nan Hua 2013, 17 (32):  5799-5805.  doi: 10.3969/j.issn.2095-4344.2013.32.009 Abstract ( 301 )   PDF (468KB) ( 413 )   Save BACKGROUND: Various factors can affect the osteogenic differentiation of human adipose-derived stem cells, and the osteoinductive factor of traditional Chinese medicine is very important for the research of human adipose-derived stem cells. OBJECTIVE: To investagate the effects of ginsenoside Rb1 on the proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro. METHODS: The human adipose-derived stem cells were isolated and cultured in vitro. After passaqed to the third generation, human adipose-derived stem cells at 2×103/well were incubated in a 96-well plate, and treated with 200 μL of 0.5, 1.0, 2.0, 4.0，6.0 μmol/L ginsenoside Rb1 medium. The human adipose-derived stem cells in the control group were treated with an equal volume of Dulbecco’s modified Eagle medium. Growth curves were examined by 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide T colorimetric assay. Alkaline phosphatase activity and osteocalcin content were detected by alkaline phosphatase kit and radio-immunity method, respectively. Calcified nodules were observed using alizarin red O staining. RESULTS AND CONCLUSION: The proliferation viability of human adipose-derived stem cells was significantly increased after cultured with 0.5 μmol/L ginsenoside Rb1. With the increasing of the concentration of ginsenoside Rb1, the mitogenic activity of the cells was decreased. The 6.0 μmol/L ginsenoside Rb1 showed a depressant effect on proliferation. Ginsenoside Rb1 could promote alkaline phosphatase activity and osteocalcin expression in human adipose-derived stem cells and showed a dose-dependent manner. Calcified nodule formation induced by 4.6 and 6.0 μmol/L ginsenoside Rb1 were better when compared with 0.5, 1.0 and 2.0 μmol/L ginsenoside Rb1. Ginsenoside Rb1 can promote the proliferation of human adipose-derived stem cells cultured in vitro in a certain concentration, and in the high concentration, the ginsenoside Rb1 can promote the osteogenic differentiation of human adipose-derived stem cells. So ginsenoside Rb1 can be used as an osteoinductive factor. Figures and Tables | References | Related Articles | Metrics

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Culture and identification of human embryo-derived myoblasts Liu Gui-ying, Yang Li-ye, Li Wen-yu, Zheng Jia-kun, Chen Qiang 2013, 17 (32):  5806-5812.  doi: 10.3969/j.issn.2095-4344.2013.32.010 Abstract ( 368 )   PDF (1929KB) ( 563 )   Save BACKGROUND: There are myoblasts in human embryonic skeletal muscle. It remains poorly understand whether myoblasts in vitro can form myotube and what are the corresponding markers for identifying myoblasts and myotubes. OBJECTIVE: To investigate whether in vitro cultured myoblasts from human embryonic skeletal muscle can form myotube and whether they can express neural markers. METHODS: Human embryonic muscle-derived myoblasts were cultured in serum-containing medium. When the primary culture was established, cultured cells were identified with immunocytochemistry for neural markers, such as β-tubulin Ⅲ, nestin, neuron specific enolase, neurofilament 200, and glial fibrillary acidic protein, and muscle markers (desmin, myogenin, smooth muscle actin and myosin). RESULTS AND CONCLUSION: A population of myoblasts could migrate from human embryonic muscle tissues. They could express the markers for skeletal muscle such as desmin and myogenin, and they could express neuron specific enolase, nestin and neurofilament 200. They could form myotubes in vitro, and myotubes expressed βⅢ-tubulin, neurofilament 200 and glial fibrillary acidic protein. The data support the hypothesis that myoblasts from human embryonic muscle express neural markers and muscle markers, and cultured myoblasts and myotubes expressed neuron specific enolase, β-tubulin Ⅲ, nestin, neurofilament 200 and glial fibrillary acidic protein. This indicates that these markers could not be used for cell identification of trans-differentiation study from muscle origin to nervous system. Figures and Tables | References | Related Articles | Metrics

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Mouse embryonic hepatic stem cells differentiate into cardiomyocyte-like cells Wu Bi-gang, Chang Jing, Zhang Xiao-gang2 2013, 17 (32):  5813-5818.  doi: 10.3969/j.issn.2095-4344.2013.32.011 Abstract ( 312 )   PDF (2291KB) ( 383 )   Save BACKGROUND: In recent years, embryonic hepatic stem cells have attracted more attention, but there are few reports on the potential of embryonic hepatic stem cells to differentiate into cardiomyocyte-like cells as well as the related differentiation conditions.  OBJECTIVE: To investigate the moderate condition to induce mice embryonic hepatic stem cells to differentiate into cardiomyocyte-like cells in vitro with chemical reagents. METHODS: Dimethylsulfoxide in combination with 5-azacytidine with different concentrations and time were used to induce the embryonic hepatic stem cells of 13.5 days mice and to observe the differentiation effect. RESULTS AND CONCLUSION: Under in vitro conditions, 0.8% dimethylsulfoxide+5 μmol/L 5-azacytidine could induce the mouse embryonic hepatic stem cells to express the specific markers of myocardial cells, while increasing the concentration of the inducer and extending the induction time could not improve the induction efficacy. Figures and Tables | References | Related Articles | Metrics

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Low-frequency electromagnetic fields enhance the recovery of spinal cord injured rats undergoing bone mesenchymal stem cell transplantation Feng Yu, Bai Wen-fang, Xu Wei-cheng, Li Xin-ping, Bai Li-ming, Liang Ling, Wang Xin, Zhang Ming-sheng 2013, 17 (32):  5819-5826.  doi: 10.3969/j.issn.2095-4344.2013.32.012 Abstract ( 340 )   PDF (2939KB) ( 514 )   Save BACKGROUND: Bone marrow mesenchymal stem cell transplantation is considered as a promising therapy for spinal cord injury. How to more effectively promote the survival of bone marrow mesenchymal stem cells in the area of spinal cord injury and to accelerate the recovery of motor function after spinal cord injury is a current study focus. Previous studies have found that low-frequency electromagnetic fields can promote bone marrow mesenchymal stem cell proliferation and differentiation, but whether the low-frequency electromagnetic fields can be applied to bone marrow mesenchymal stem cell transplantation for treatment of spinal cord injury requires further studies. OBJECTIVE: To discuss the effects of low-frequency electromagnetic fields on motor function of spinal cord injury rats after transplantation of bone mesenchymal stem cells. METHODS: Sixty-four rat models of incomplete spinal cord injury at T10 were established by compression method and then randomized into control group, transplantation group (bone mesenchymal stem cell transplantation), electromagnetic field group and combination group (electromagnetic field+bone mesenchymal stem cell transplantation). After successful modeling, bone mesenchymal stem cells labeled with 5-bromo-2'-deoxyuridine were injected into the original injured site in the transplantation group and combination group, which were isolated and purified with the fast adherence method; while alpha-minimum essential medium was injected into the electromagnetic field group and control group for instead. At 24 hours post-operation, the electromagnetic field group and combination group were explored to low-frequency electromagnetic fields (frequency 50 Hz, magnetic indaction intensity 5 mT) for 60 minutes per day. RESULTS AND CONCLUSION: After cell transplantation for 21 days, the Basso, Beattie, and Bresnahan scores in the combination group was higher than the other groups (P < 0.05). 5-Bromo-2'-deoxyuridine positive cells grew well, and integrated into the normal spine; syringomyelia was reduced, and the number of spinal neural cells was increased in the combination group. In addition, glial fibrillary acidic protein expression was decreased in the combination group, while matrix metalloproteinase 2 expression was increased. It indicates that low-frequency electromagnetic fields could promote recovery of motor function in the spinal cord injury rats transplanted with bone mesenchymal stem cells, which could be associated that low-frequency electromagnetic fields facilitate the survival of transplanted bone mesenchymal stem cells, up-regulate the expression of matrix metalloproteinase 2, and reduce glial scar formation in the spinal cord injured site. Figures and Tables | References | Related Articles | Metrics

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Effects of low-frequency electromagnetic fields on skin wound healing after the transplantation of gene modified epidermal stem cells Liang Ling, Li Xin-ping, Bai Wen-fang, Bai Li-ming, Zhu Hong-xiang, Xu Wei-cheng, Feng Yu, Wang Xin, Chen Yi, Zhang Ming-sheng 2013, 17 (32):  5827-5833.  doi: 10.3969/j.issn.2095-4344.2013.32.013 Abstract ( 321 )   PDF (2412KB) ( 423 )   Save BACKGROUND: The repair and management of full-thickness skin defects resulting from burns and chronic wounds remain a significant unmet clinical challenge. Using epidermal stem cells and keratinocyte growth factor for full-thickness wound repair is a promising approach. Low-frequency electromagnetic fields which are a non-invasive physical stimulation therapy have been recognized as a good method to enhance wound healing. OBJECTIVE: To develop a new strategy to accelerate wound healing by transplanting transfected epidermal stem cells and keratinocyte growth factor and treating with low-frequency electromagnetic fields in a mouse model. METHODS: Epidermal stem cells from Sprague-Dawley neonatal rats were isolated and cultured in vitro, then the   cells were labeled with 5-bromo-2-deoxyuridine and transfected by Ad-KGF, a recombinant adenovirus carrying the keratinocyte growth factor. Mice were given to create full thickness skin wound on the dorsum and randomly assigned to four groups: control group, transplantation of epidermal stem cells group, transplantation of keratinocyte growth factor gene modified epidermal stem cells group, and transplantation of keratinocyte growth factor gene modified epidermal stem cells plus low-frequency electromagnetic field exposure group. RESULTS AND CONCLUSION: The best healing pattern was observed in the keratinocyte growth factor gene modified epidermal stem cells plus low-frequency electromagnetic field exposure group (P < 0.05) at days 9 and 16. 5-Bromo-2-deoxyuridine labeled cells existed in the wound in the treated groups at day 9. A significantly increased expression of endogenous keratinocyte growth factor was detected in the transplantation of Keratinocyte Growth Factor gene modified epidermal stem cells group, and transplantation of keratinocyte growth factor gene modified epidermal stem cells plus low-frequency electromagnetic field exposure group at day 16. A well-advanced epithelialization was observed in transplantation of keratinocyte growth factor gene modified epidermal stem cells plus low-frequency electromagnetic field exposure group at days 16 and 30. These results suggest that low-frequency electromagnetic fields enhanced wound healing following the transplantation of keratinocyte growth factor gene modified epidermal stem cells. Figures and Tables | References | Related Articles | Metrics

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Quantitative analysis of transplanted effect of human amniotic epithelial cells in mice with acute liver injury Luo Hong-wu, Xun Quan, Huang Xiang-jun, Huang Fei-zhou 2013, 17 (32):  5834-5839.  doi: 10.3969/j.issn.2095-4344.2013.32.014 Abstract ( 262 )   PDF (1917KB) ( 317 )   Save BACKGROUND: There are many preliminary studies on the survival, metaptosis, and correlation characteristics of human amniotic epithelial cells after transplanted into the animals, but there are no reports on the quantitative analysis of the transplantation effect. OBJECTIVE: To make quantitative analysis on serum biochemical function of liver and the expression of human albumin in mice received passaged human amniotic epithelial cells transplantation in spleen. METHODS: Forty nude mice were randomly divided into four groups (n=10 in each group): hepatectomy+cell transplantation 2 weeks group, hepatectomy+cell transplantation 4 weeks group, hepatectomy+normal saline group (treated with partial hepatectomy) and hepatectomy+cell transplantation group (transplanted with 0.2 mL passaged human amniotic epithelial cells with 5×106 under spleen, and the blood were collected at 2 and 4 weeks after transplantation). The mice in the hepatectomy+normal saline group were treated with splenic injection of  0.2 mL normal saline; the cell transplantation group did not receive hepatectomy, and transplanted with 0.2 mL passaged human amniotic epithelial cells with 5×106 under spleen. The histological and morphological changes of the liver and spleen in each group as well as the expressions of serum alanine aminotransferase, aspartate aminotransferase and human serum albumin in each group were detected, and the quantitative analysis of human serum albumin expression was performed. RESULTS AND CONCLUSION: There was no obvious morphological change after human amniotic epithelial cells transplanted into the acute liver injury mice for 4 weeks, but specific cells could be detected by histological method. The serum levels of alanine aminotransferase, aspartate aminotransferase and human serum albumin were improved obviously, and the human albumin could be detected in serum, the level of human albumin at 4 weeks after transplantation was significantly increased than 2 weeks after transplantation. Human amniotic epithelial cells can survive for more than 4 weeks after transplanted into the liver injury mice, and can still express partial characteristics and functions of hepatocyte-like cells, improve the liver function, thus treating acute liver injury. Figures and Tables | References | Related Articles | Metrics

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Effect of bone marrow mononuclear cell transplantation on angiogenesis and expression of cytokines following myocardial infarction Tang Jie, Chen Tao, Mi Jie, Xu Ai-guo, Wang Yong-de, Zhang Jian, Qi Xiang-qian 2013, 17 (32):  5840-5846.  doi: 10.3969/j.issn.2095-4344.2013.32.015 Abstract ( 257 )   PDF (2279KB) ( 433 )   Save BACKGROUND: Cell transplantation offers a new promise of rebuilding the damaged myocardium. But the results of them are not consistent. It is not clear if the transplanted cells can permanently improve heart function and the mechanism underlying this therapeutic effect. OBJECTIVE: To study the effect of intracoronary autologous bone marrow mononuclear cell transplantation on cardiac function, and angiogenesis and cytokine production in canines with acute myocardial infarction. METHODS: Left anterior descending coronary artery ligation was used to produce acute myocardial infarction models in hybrid canines. Bone marrow mononuclear cells were harvested by using puncture of anterior crest and posterior superior iliac spine to prepare cell suspension. Sixteen hybrid canines were randomly divided into transplantation group (n=10) and control group (n=6). Bone marrow mononuclear cells (transplantation group, n=10) or normal saline (control group, n=6) were intracoronarily infused into infarction-related arteries 2 hours after acute myocardial infarction. To evaluate the heart function, we used echocardiography at 2 hours and 6 weeks after acute myocardial infarction. Capillary density was assessed 6 weeks after transplantation by using von Willebrand factor test. The mRNA levels of vascular endothelial growth factor 188, vascular endothelial growth factor 164, basic fibroblast growth factor and matrix metalloproteinase-9 in the infarct area were determined by reverse transcription-PCR at 6 weeks after transplantation. RESULTS AND CONCLUSION: In contrast to the control group, ejection fraction and stroke volume at 6 weeks after transplantation increased significantly in the transplantation group. The transplantation group had a greater amount of new vessels in the peri-infarct area than the control group. Compared with the control group, the mRNA levels of vascular endothelial growth factor 188, vascular endothelial growth factor 164, and basic fibroblast growth factor significantly increased in the transplantation group, but the mRNA level of matrix metalloproteinase-9 significantly decreased in the transplantation group. These findings suggest that intracoronary transplantation of autologous bone marrow mononuclear cells may improve the cardiac function, and increase capillary density, especially in the border zone of infarcted myocardium. Otherwise, bone marrow mononuclear cell transplantation can increase the mRNA levels of vascular endothelial growth factor 188, vascular endothelial growth factor 164, and basic fibroblast growth factor, but decrease the mRNA level of matrix metalloproteinase-9. Figures and Tables | References | Related Articles | Metrics

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Evaluation indexes for the viability of umbilical cord-derived mesenchymal stem cells before transplantation Lei Xin, Chen Yan, Zhang Jian-lin, Cui Lei, Niu Yu-hu, Niu Bo 2013, 17 (32):  5847-5854.  doi: 10.3969/j.issn.2095-4344.2013.32.016 Abstract ( 560 )   PDF (2347KB) ( 611 )   Save BACKGROUND: Umbilical cord-derived mesenchymal stem cells are gaining more attention in clinical treatments. Cell viability prior to transplantation has a direct impact on clinical prognosis. Despite trypan blue staining is a widely performed procedure to assess the viability of umbilical cord-derived mesenchymal stem cells, it cannot reflect the functional capacity of those cells accurately because of some subjective  factors. OBJECTIVE: To explore sensitive and accurate assay for the functions of umbilical cord-derived mesenchymal stem cells. METHODS: Human umbilical cord-derived mesenchymal stem cells were isolated and cultured in vitro. Cultured umbilical cord-derived mesenchymal stem cells were preserved in 0.9% saline for 0, 2, 4 and 6 hours at 4 ℃. Various methods (trypan blue staining, AnnexinV-PI, terminal deoxynucleotidyl transferase dutp nick end labeling, cell counting kit-8, live-dead assay, cell adherent assay) were used to determine the viability of post-storage umbilical cord-derived mesenchymal stem cells, and the results were compared with colony-forming efficiency, a measure of cell function. RESULTS AND CONCLUSION: Human umbilical cord-derived mesenchymal stem cells cultured in vitro showed a spindle shape and attached growth, the third-generation umbilical cord-derived mesenchymal stem cells were positive for CD29, CD44, CD105, and negative for CD 34 and CD 45. Umbilical cord-derived mesenchymal stem cells incubated in the adipogenic and osteogenic medium were both positive. Cell viability measured with trypan blue correlated moderately with colony-forming efficiency, while the percentage of viable cells measured with other methods correlated better with colony-forming efficiency, among which adherent assay was the most obvious. It is proved that cell adherent assay-measured viability is the most accurate indicator. Figures and Tables | References | Related Articles | Metrics

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Periodontal ligament stem cells expansion in vitro under different cryopreservation systems Wang Xuan, Liu Yi-shan, Ma Yan, Bi Xiao-juan, Zheng Shu-tao, Liu Jia, Li Bo-qi, Sun Da-lei, Zhao Jin 2013, 17 (32):  5855-5862.  doi: 10.3969/j.issn.2095-4344.2013.32.017 Abstract ( 371 )   PDF (2354KB) ( 357 )   Save BACKGROUND: The cryopreservation is the key step to ensure the successful stem cell transplantation treatment. The traditional cryopreservation is to place the cells directly into the cryopreservation solution, but dimethyl sulfoxide in the cryopreservation liquid likes a double-edged sword during the repeated cryopreservation and recovery process, that is, although dimethyl sulfoxide can reduce the cell membrane mechanical injury by ice crystals in the recovery process, at the same time, it can enhance the toxic effects on cells directly and affect cell survival situation and is not conducive to clinical transplantation therapy. In recent years, extracted stem cells from frozen tissue had become another research direction. OBJECTIVE: To look for the best solution of periodontal ligament stem cells expansion in vitro by the appropriate cryopreservation periodontal tissue. METHODS: Periodontal tissue was scraped from healthy human teeth and divided them into three equal parts: fresh group, harvesting periodontal ligament stem cells from fresh tissue; 5% dimethyl sulfoxide group, 5% dimethyl sulfoxide added into the cryopreservation system; 10% dimethyl sulfoxide group, 10% dimethyl sulfoxide added into the cryopreservation system. One month later, periodontal ligament stem cells were extracted from the cryopreserved periodontal ligament from the latter two groups. RESULTS AND CONCLUSION: In the time that cells swam out of tissue mass and cell harvest amount, the 5% dimethyl sulfoxide group was inferior to the fresh group but better than the 10% dimethyl sulfoxide group (P < 0.05). No differences were found among the three groups in the following aspects (P > 0.05): colony formation rate of passage 1 periodontal ligament stem cells, cell survival rate, proliferation ability of passage 3 periodontal ligament stem cells, cell growth curve and surface marker expression of periodontal ligament stem cells. The results suggest that the 5% dimethyl sulfoxide added cryopreserved system for periodontal ligament tissue cannot only shorten the time of periodontal ligament stem cells amplification in vitro, ensure cell harvest and maintain basic cellular biological characteristics, but also reduce the total amount of the dimethyl sulfoxide and the direct damage to the cells caused by repeatedly frozen cells, thereby providing a more secure guarantee for the future implementation of clinical transplantation therapy. So, the 5% dimethyl sulfoxide added cryopreserved system may be the new selection for donor tissue storage. Figures and Tables | References | Related Articles | Metrics

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Tougu Xiaotong Granule containing serum induces cartilage differentiation of bone marrow stromal stem cells Liu Bo-ling, Li Xi-hai, Xiao Li-chun, Liang Gui-qing, Wu Guang-wen, Li Zhao-hui, Chen Qi-yong 2013, 17 (32):  5863-5870.  doi: 10.3969/j.issn.2095-4344.2013.32.018 Abstract ( 311 )   PDF (2618KB) ( 345 )   Save BACKGROUND: Bone marrow stromal stem cells have multilineage differentiation capacity and can differentiate into transparent chondrocytes under certain conditions, which can provide new thoughts for treatment of osteoarthritis.  OBJECTIVE: To investigate the effects of Tougu Xiaotong Granule containing serum on the cartilage differentiation of bone marrow stromal stem cells. METHODS: Bone marrow stromal stem cells from Sprague-Dawley rat limbs were cultured in vitro, and those cells at passage 3 were used in the study. Cells were divided into six groups: saline serum group, Tougu Xiaotong Granule water extract group, Tougu Xiaotong Granule alcohol extract group, chondroinductive group, Tougu Xiaotong Granule water extract and chondroinductive group, Tougu Xiaotong Granule alcohol extract and chondroinductive group. The Sox9, collagen Ⅱ, and collagen X mRNA and protein expression levels were tested by real-time polymerase chain reaction and western blot analysis. RESULTS AND CONCLUSION: After cells were intervened with drug-containing serum for 14 days, the Sox9, collagen Ⅱ, and collagen X mRNA and protein expression in Tougu Xiaotong Granule water extract group, Tougu Xiaotong Granule alcohol extract group, chondroinductive group, Tougu Xiaotong Granule water extract and chondroinductive group, Tougu Xiaotong Granule alcohol extract and chondroinductive group were significantly higher than that in saline serum group (P < 0.05, P < 0.01). In addition, the chondroinductive group, Tougu Xiaotong Granule water extract serum and chondroinductive group, Tougu Xiaotong Granule alcohol extract and chondroinductive group showed significantly higher expression levels than Tougu Xiaotong Granule water extract serum group, Tougu Xiaotong Granule alcohol extract group (P < 0.01). Sox9 expression in Tougu Xiaotong Granule water extract serum and chondroinductive group were significantly higher than that in the chondroinductive group. Experimental findings indicate that, Tougu Xiaotong Granule containing serum can accelerate bone marrow stromal stem cells differentiate into cartilage cells by up-regulation of Sox9 expression. Figures and Tables | References | Related Articles | Metrics

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Effect of bone marrow mesenchymal stem cells combined with Danhong injection on expression of GAP-43 and Bcl-2 after cerebral infarction  Li Jin-yan, Zhang Zhe-cheng 2013, 17 (32):  5871-5876.  doi: 10.3969/j.issn.2095-4344.2013.32.019 Abstract ( 408 )   PDF (1974KB) ( 434 )   Save BACKGROUND: Danhong injection, scavenging free radicals and inhibiting lipid peroxidation, can improve microenvironment injury after cerebral infarction.     OBJECTIVE: To explore the influence of bone marrow mesenchymal stem cells combined with Danhong injection on expression of GAP-43 and Bcl-2 after cerebral infarction in rats. METHODS: Sixty Wistar rats were selected to prepare models of cerebral infarction by middle cerebral artery occlusion and then randomly divided into control group, bone marrow mesenchymal stem cell group, and combination group. Control group received tail vein injection of PBS. Bone marrow mesenchymal stem cell group received tail vein injection of 2.5×109/L bone marrow mesenchymal stem cell suspension. Combination group received injection of 2.5× 109/L bone marrow mesenchymal stem cell suspension+2 mL/kg Danhong injection, for 5 consecutive days, once a day. RESULTS AND CONCLUSION: There were no significant differences in the neurological dysfunction scores among the three groups at 24 hour and 3 days after implantation (P > 0.05). The neurological dysfunction scores in the ombination group were significantly lower than those in the bone marrow mesenchymal stem cell group and control group at 1 and 2 weeks after transplantation (P < 0.05). In the combination group, GAP-43 and Bcl-2 expression was significantly higher than the bone marrow mesenchymal stem cell group and control group (P < 0.05). Bone marrow mesenchymal stem cell transplantation combined with Danhong injection can significantly promote the local expression of GAP-43 and Bcl-2 after cerebral infarction, and has obvious inhibitory effects on cell apoptosis in rats with cerebral infarction. Figures and Tables | References | Related Articles | Metrics

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Differentiation potential of umbilical cord blood-derived mesenchymal stem cells Chen Ming-xing, Ouyang Gui-fang 2013, 17 (32):  5877-5882.  doi: 10.3969/j.issn.2095-4344.2013.32.020 Abstract ( 317 )   PDF (613KB) ( 421 )   Save BACKGROUND: Umbilical cord blood-derived mesenchymal stem cells are multipotential stem cells in the mesoderm in early development stage, and have been paid great attention due to its properties of multi-directional differentiation. OBJECTIVE: To summarize the potential of induced differentiation of umbilical cord blood-derived mesenchymal stem cells. METHODS: We retrieved PubMed Database for articles concerning the differentiation potential of umbilical cord blood-derived mesenchymal stem cells published from January 1999 to December 2012. In titles and abstracts, the key words were “umblical cord blood, mesenchymal stem cells, potential, differentiation”. Totally, 52 articles addressing the differentiation potential of umbilical cord blood-derived mesenchymal stem cells were reviewed. RESULTS AND CONCLUSION: Numerous studies have confirmed that human umbilical cord blood-derived mesenchymal stem cells can successfully differentiate into multiple kinds of cell lines, but their understanding remains minor. If we can master the characteristics of the differentiation potential of umbilical cord blood-derived mesenchymal stem cells, it would be used to repair bone and myocardium detects. Present studies remain in a starting stage. Isolation and purification, regulation of differentiation direction, in vitro amplification and immunogenicity require further investigations. Figures and Tables | References | Related Articles | Metrics

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Studies on p53 in induced pluripotent stem cells Lin Tong-xiang, Lin Yi 2013, 17 (32):  5883-5888.  doi: 10.3969/j.issn.2095-4344.2013.32.021 Abstract ( 325 )   PDF (650KB) ( 504 )   Save BACKGROUND: Induced pluripotent stem cells can bypass the ethical issues of embryonic stem cells, and become the hotspot of stem cell research. OBJECTIVE: To explore the research progress and problems of induced pluripotent stem cells. METHODS: A retrospective analysis on the findings, research progress and problems of induced pluripotent stem cells in recent years was performed. The Thomson Reuters Web of Science was searched for the articles related to the induced pluripotent stem cells and p53 gene. RESULTS AND CONCLUSION: In recent years, domestic and foreign scholars have conducted a lot of researches on induced pluripotent stem cells. For example, a Japanese group is establishing the stem cell bank to provide a basis for the treatment of retinal diseases. However, the safety issues of induced pluripotent stem cells need to be solved before routine cell treatment application, in which the functional research of related p53 gene is one of the essential concerns. The other member of p53 gene, p73 gene, also participates in the generation and differentiation of induced pluripotent stem cells, and the in-depth studies are needed. The finding of p53 gene function will promote the in-depth development of regenerative medicine and translational medicine. Figures and Tables | References | Related Articles | Metrics

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Stem cell transplantation for ischemic heart disease: Clinical feasibility and safety Hu Ya-guang 2013, 17 (32):  5889-5894.  doi: 10.3969/j.issn.2095-4344.2013.32.022 Abstract ( 276 )   PDF (771KB) ( 623 )   Save BACKGROUND: The stem cells transplanted into the damaged heart tissues can differentiate into cardiomyocytes, which bring new hope for the research of ischemic heart disease. OBJECTIVE: To explore the feasibility and safety of stem cell transplantation for the treatment of ischemic heart disease. METHODS: Various experimental methods to the feasibility and safety of stem cell transplantation for the treatment of ischemic heart disease were analyzed. REPAIR-AMI experiment was a randomized and double-blind and placebo-controlled multi-center study that used to analyze the therapeutic effect of intracoronary transplantation of bone marrow progenitor cells immediately after acute myocardial infarction. MAGIC Cell-3-DES experiment was used to evaluate the safety of granulocyte colony-stimulating factor mobilized stem cell therapy and the effect of intracoronary injection of mobilized peripheral blood stem cells on the treatment of acute myocardial infarction and old myocardial infarction. BOOST experiment was the randomized controlled study on autologous bone marrow cell transplantation through coronary vein after myocardial infarction. PROTECT-CAD experiment was the randomized controlled clinical trial about the direct injection of stem cells into the myocardial for the treatment of ischemic heart disease. RESUTLS AND CONCLUSION: Stem cell transplantation may improve the left ventricular systolic function and left ventricular diastolic function as well as the coronary flow reserve, and the related studies have been confirmed. Stem cell transplantation for the treatment of ischemic heart disease can increase the left ventricular jection fraction. As the less clinical accident, stem cell therapy cannot increase the risk of restenosis based on the treatment of drug-eluting stents. It is safe and feasible of stem cell transplantation for the treatment of ischemic heart disease. Large sample, long scale and multi-center randomized controlled studies are needed to further evaluate the effect and risk of stem cell transplantation. Figures and Tables | References | Related Articles | Metrics

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Umbilical cord blood mesenchymal stem cell transplantation for the treatment of renal anemia: Hot spots and issues Wang Kai, Jiao Hong-liang, Li Jian-bin, Wu Xian-ming 2013, 17 (32):  5895-5900.  doi: 10.3969/j.issn.2095-4344.2013.32.023 Abstract ( 300 )   PDF (892KB) ( 478 )   Save BACKGROUND: Umbilical cord blood as a source of hematopoietic stem cells has become a hot topic. Adjuvant therapy of umbilical cord blood transfusion has been used in China to correct renal anemia of uremia patients. OBJECTIVE: To evaluate the efficacy and safety of umbilical cord blood mesenchymal stem cells in the treatment of renal anemia, and to compare with the effects of different sources of mesenchymal stem cells with different transplantation methods on the treatment of kidney diseases. METHODS: A retrospective analysis was conducted on six renal anemia patients in the Department of Outpatient, Henan Red Cross Blood Center between January 2010 and December 2012. The experiment was approved by Medical Ethics Committee, six patients were informed consent for treatment programs, and parturients and their families have signed the informed consent. The newborns umbilical cord blood of 80-140 mL were collected with closed sterile plastic blood bags, and then the separated umbilical cord blood mesenchymal stem cells were transfused into the renal anemia patients through the superficial vein in the back of the hand with the number of ≥1×108/copy, two copies per time, and re-transfused after 4 days, a total of three times. The blood hematocrit, hemoglobin, red blood cells in urine, renal blood flow changes were observed before and after treatment. RESULTS AND CONCLUSION: The hemoglobin, blood hematocrit, red blood cells in urine and renal blood flow were significantly increased before and after treatment (P < 0.05). Transfusion of multiple copies of umbilical cord blood mesenchymal stem cells through the superficial vein in the back of the hand is convenient and safe, which is considered as a new method for the treatment of renal anemia. But there are certain limitation in the data and the conclusion as the clinical research was designed as self-control, so further confirm is needed. Figures and Tables | References | Related Articles | Metrics