骨髓间充质干细胞中环氧化酶2及聚蛋白多糖酶1沉默与胰岛素样生长因子1过表达

doi:10.3969/j.issn.2095-4344.2015.10.003

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (10): 1488-1494.doi: 10.3969/j.issn.2095-4344.2015.10.003

• 骨髓干细胞 bone marrow stem cells • 上一篇下一篇

骨髓间充质干细胞中环氧化酶2及聚蛋白多糖酶1沉默与胰岛素样生长因子1过表达

袁钰祺¹，张海宁¹，孔霞²，隋爱华³，王英振¹

青岛大学附属医院，¹关节外科，山东省创伤骨科研究所，³中心实验室，山东省青岛市 266061，²单县中心医院肿瘤科，山东省菏泽市 274300

出版日期:2015-03-05 发布日期:2015-03-05
通讯作者: 王英振，硕士，博士生导师，青岛大学附属医院关节外科，山东省创伤骨科研究所，山东省青岛市 266061
作者简介:袁钰祺，男，1989年生，山东省单县人，汉族，2015年青岛大学毕业，硕士，主要从事骨外科的基础和临床研究。
基金资助:
国家自然科学基金资助项目(81171774，81272056)

Lentivirus-mediated cyclooxygenase 2 and aggrecanase 1 silencing and insulin-like growth factor 1 overexpression in human bone marrow mesenchymal stem cells

Yuan Yu-qi¹, Zhang Hai-ning¹, Kong Xia², Sui Ai-hua³, Wang Ying-zhen¹

1 Department of Joint Surgery, Shandong Institute of Orthopaedics and Traumatology, Affiliated Hospital of Qingdao University Medical College, Qingdao 266061, Shandong Province, China
2 Department of Oncology, Shanxian Central Hospital, Heze 274300, Shandong Province, China

3 Central Laboratory, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China

Online:2015-03-05 Published:2015-03-05
Contact: Wang Ying-zhen, Master, Doctoral supervisor, Department of Joint Surgery, Shandong Institute of Orthopaedics and Traumatology, Affiliated Hospital of Qingdao University Medical College, Qingdao 266061, Shandong Province, China
About author:Yuan Yu-qi, Master, Department of Joint Surgery, Shandong Institute of Orthopaedics and Traumatology, Affiliated Hospital of Qingdao University Medical College, Qingdao 266061, Shandong Province, China
Supported by:
the National Natural Science Foundation of China, No. 81171774, 81272056

摘要/Abstract

摘要：

背景：环氧化酶2、聚蛋白多糖酶1和胰岛素样生长因子1参与关节软骨病理损伤过程。

目的：观察携带基因环氧化酶2、聚蛋白多糖酶1的shRNA干扰载体和携带胰岛素样生长因子1的过表达慢病毒载体在骨髓间充质干细胞中的表达情况。

方法：应用重组慢病毒技术构建携带沉默基因环氧化酶2、聚蛋白多糖酶1、过表达基因胰岛素样生长因子1和绿色荧光蛋白基因的重组慢病毒表达载体，并用其转染体外培养的第3代人骨髓间充质干细胞(实验组)，并以无目的基因的慢病毒载体转染人骨髓间充质干细胞和未做处理的人骨髓间充质干细胞分别作为阴性对照组和空白组。

结果与结论：环氧化酶2和聚蛋白多糖酶1在转染重组慢病毒的人骨髓间充质干细胞中的mRNA和蛋白水平有受到了明显抑制，胰岛素样生长因子1 mRNA和蛋白表达水平则明显上升。说明应用慢病毒可在人骨髓间充质干细胞成功沉默环氧化酶2和聚蛋白多糖酶1基因，同时将胰岛素样生长因子1高表达，为系统性治疗关节炎提供基因治疗的基础。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

关键词: 干细胞, 骨髓干细胞, 聚蛋白多糖酶1, 胰岛素样生长因子1, 骨髓间充质干细胞, 慢病毒, 关节炎, 国家自然科学基金

Abstract:

BACKGROUND: Cyclooxygenase 2, aggrecanase 1, and insulin-like growth factor 1 are involved in pathological injury of the articular cartilage.

OBJECTIVE: To observe the expression of shRNA vectors carrying cyclooxygenase 2, aggrecanase 1 and overexpression vectors carrying insulin-like growth factor 1 in bone marrow mesenchymal stem cells.

METHODS: Lentiviral vectors carrying the silencing gene cyclooxygenase 2, aggrecanase 1, the over-expressing gene insulin-like growth factor 1 and binding green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect passage 3 human bone marrow mesenchymal stem cells cultured in vitro (experimental group). The human bone marrow mesenchymal stem cells transfected with no target gene lentivirals were used as negative control group. The human bone marrow mesenchymal stem cells transfected with no treatment served as blank group.

RESULTS AND CONCLUSION: Cyclooxygenase 2 and aggrecanase 1 transfected in human bone marrow mesenchymal stem cells were significantly inhibited at gene and protein levels, while the expression of insulin-like growth factor 1 was increased significantly at gene and protein levels. We confirmed that cyclooxygenase 2 and aggrecanase 1 were successfully silenced while insulin-like growth factor 1 overexpressed by using lentiviral vectors in human bone marrow mesenchymal stem cells, which brings a new hope for the systemic gene treatment of arthritis.

全文链接：

Key words: Mesenchymal Stem Cells, Lentivirus Infections, Cyclooxygenase 2, Insulin-Like Growth Factor I

中图分类号:

R394.2

袁钰祺，张海宁，孔霞，隋爱华，王英振. 骨髓间充质干细胞中环氧化酶2及聚蛋白多糖酶1沉默与胰岛素样生长因子1过表达[J]. 中国组织工程研究, 2015, 19(10): 1488-1494.

Yuan Yu-qi, Zhang Hai-ning, Kong Xia, Sui Ai-hua, Wang Ying-zhen. Lentivirus-mediated cyclooxygenase 2 and aggrecanase 1 silencing and insulin-like growth factor 1 overexpression in human bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(10): 1488-1494.

图/表（结果） 4

Morphology of cultured human bone marrow mesenchymal stem cells

After the suspension of human bone marrow mesenchymal stem cells inoculated in culture bottles, the cells were adherent completely after 4-6 hours. When cultured for 24 hours after half culture medium was changed, part of the cells were adherent and oval, and many non-adherent suspended cells and blood cells were visible (Figure 2).

Identification of lentivirus-infected human bone marrow mesenchymal stem cells

Some cells expressed enhanced green fluorescent protein at 2 days after transfection. And the fluorescence intensity reached the brightest after 72 hours, and then it was kept constantly. Under the light microscope, the full, stereo sense of cells increased, virus effect was apparent, and a few apoptosis cells floated with particles increased in the cell cytoplasm. Three 100-fold vision fields were randomly selected from each hole, to count total cells and green fluorescent protein positive cells, and calculate the percentage of green fluorescent protein-positive cells. The average of three view percentages was considered as the transfection efficiency. When multiplicity of infection=10, the transfection efficiency of experimental group and negative control group reached 90%, as shown in Figure 3.

Changes in mRNA expression of COX-2, Aggrecanase-1 and IGF-1 in transfected human bone marrow mesenchymal stem cells

The RT-PCR results showed that compared with the negative control group and blank group, the COX-2 and aggrecanase-1 mRNA in human bone marrow mesenchymal stem cells of the experimental group expressed lower (P < 0.05), and the IGF-1 gene of the experimental group showed a higher expression (P < 0.05). And there was no significant difference in the expression of these three genes between the blank group and the negative control group (P > 0.05; Figure 4).

Changes in the protein expression of COX-2 and Aggrecanase-1 in transfected human bone marrow mesenchymal stem cells

The protein expression of COX-2 and Aggrecanase-1 in the experimental group was significantly lower than that in the blank group and the negative control group (P < 0.05). There was no statistically significance in the protein expression between the blank group and the negative control group (P > 0.05). These results were similar to the results of RT-PCR (Figure 5).

hIGF-1 expression in supernatants after transfection

ELISA was used to detect the expression of the secreted hIGF-1 in the supernatants of bone marrow mesenchymal stem cells at 7 days after transfection. The expression of hIGF-1 in the supernatants was significantly higher in the experimental group [(39.41±1.18) ng/mL] than the blank group

[(1.27±0.16) ng/mL] and negative control group [(1.13±0.10) ng/mL] (P < 0.05). But there was no difference between the blank group and the negative control group (P > 0.05). These findings were roughly similar to those detected by RT-PCR in the gene expression.

参考文献

[1]Jung HS, Kwon BS, Lee SW. Tumor-specific gene delivery using RNA-targeting Tetrahymena group I intron. Biotechnol Lett. 2005;27(8):567-574.
[2]He T, Wang Y, Xiang J, et al. In vivo tracking of novel SPIO-Molday ION rhodamine-B™-labeled human bone marrow-derived mesenchymal stem cells after lentivirus- mediated COX-2 silencing: a preliminary study. Curr Gene Ther. 2014;14(2):136-145.
[3]Sarzi-Puttini P, Cimmino MA, Scarpa R, et al. Osteoarthritis: an overview of the disease and its treatment strategies. Semin Arthritis Rheum. 2005;35(1 Suppl 1):1-10.
[4]Jain A, Stein BE, Skolasky RL, et al. Total joint arthroplasty in patients with rheumatoid arthritis: a United States experience from 1992 through 2005. J Arthroplasty. 2012; 27(6):881-888.
[5]de Boer TN, Huisman AM, Polak AA, et al. The chondroprotective effect of selective COX-2 inhibition in osteoarthritis: ex vivo evaluation of human cartilage tissue after in vivo treatment. Osteoarthritis Cartilage. 2009;17(4): 482-488.
[6]Tseng SS, Lee MA, Reddi AH. Nonunions and the potential of stem cells in fracture-healing. J Bone Joint Surg Am. 2008;90 Suppl 1:92-98.
[7]Thirunavukkarasu K, Pei Y, Wei T. Characterization of the human ADAMTS-5 (aggrecanase-2) gene promoter. Mol Biol Rep. 2007;34(4):225-231.
[8]Wang X, Grogan SP, Rieser F, et al. Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study. Biomaterials. 2004;25(17): 3681-3678.
[9]Tortorella MD, Liu RQ, Burn T, et al. Characterization of human aggrecanase 2 (ADAM-TS5): substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4). Matrix Biol. 2002;21(6):499-511.
[10]Miller JA, Liu RQ, Davis GL, et al. A microplate assay specific for the enzyme aggrecanase. Anal Biochem. 2003; 314(2):260-265.
[11]Debnath T, Kim da H, Lim BO. Natural products as a source of anti-inflammatory agents associated with inflammatory bowel disease. Molecules. 2013;18(6):7253-7270.
[12]Depboylu C, Weihe E, Eiden LE. COX1 and COX2 expression in non-neuronal cellular compartments of the rhesus macaque brain during lentiviral infection. Neurobiol Dis. 2011;42(1):108-115.
[13]Attur M, Al-Mussawir HE, Patel J, et al. Prostaglandin E2 exerts catabolic effects in osteoarthritis cartilage: evidence for signaling via the EP4 receptor. J Immunol. 2008;181(7): 5082-5088.
[14]Hagmann S, Moradi B, Frank S, et al. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells. BMC Musculoskelet Disord. 2013;14:223.
[15]Ulivi V, Lenti M, Gentili C, et al. Anti-inflammatory activity of monogalactosyldiacylglycerol in human articular cartilage in vitro: activation of an anti-inflammatory cyclooxygenase-2 (COX-2) pathway. Arthritis Res Ther. 2011;13(3):R92.
[16]Wakefield AP, Ogborn MR, Ibrahim N, et al. A dietary conjugated linoleic acid treatment that slows renal disease progression alters renal cyclooxygenase-2-derived prostanoids in the Han: SPRD-cy rat. J Nutr Biochem. 2012; 23(8):908-914.
[17]Kurland ES, Rosen CJ, Cosman F, et al. Insulin-like growth factor-I in men with idiopathic osteoporosis. J Clin Endocrinol Metab. 1997;82(9):2799-2805.
[18]Lazowski DA, Fraher LJ, Hodsman A, et al. Regional variation of insulin-like growth factor-I gene expression in mature rat bone and cartilage. Bone. 1994;15(5):563-576.
[19]Paveli? J, Matijevi? T, Knezevi? J. Biological & physiological aspects of action of insulin-like growth factor peptide family. Indian J Med Res. 2007;125(4):511-522.
[20]Xu ZY, Wang YZ, Zhang HN, et al. Construction and Detection of Bicistronic Eukaryotic Vector pIRSES2-EGFP- hIGF-1. Xiandai Shengwu Yixue Jinzhan. 2013;13(15): 2837-2840.
[21]He T, Zhang HN, Xiang JY, et al. Cyclooxygenase 2 silencing has no effects on oriented differentiation of human bone marrow mesenchymal stem cells. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(14):2140-2146.
[22]Du QF, Zhang HN, Wang YZ, et al. Effects of aggrecanase-2 shRNA transfection on chondrocytes of rheumatoid arthritis patient leaded by lentivirus. Zhonghua Guke Zazhi. 2014;34(9):936-944.
[23]Wang DL, Xing DG, Wang L, et al. Effects of transplanting bone marrow mesenchymal stem cells transfected by insulin- like growth factor-1 gene on fracture healing of rats with diabetes. Zhonghua Shiyan Waike Zazhi. 2012;29(3):518-520.
[24]Shao M, Hollar S, Chambliss D, et al. Targeting the insulin growth factor and the vascular endothelial growth factor pathways in ovarian cancer. Mol Cancer Ther. 2012;11(7): 1576-1586.
[25]Chen C, Xu Y, Song Y. IGF-1 gene-modified muscle-derived stem cells are resistant to oxidative stress via enhanced activation of IGF-1R/PI3K/AKT signaling and secretion of VEGF. Mol Cell Biochem. 2014;386(1-2):167-175.
[26]Olmos A, Díaz L, Avila E, et al. Associations between insulin-like growth factor I, vascular endothelial growth factor and its soluble receptor 1 in umbilical serum and endothelial cells obtained from normotensive and preeclamptic pregnancies. Growth Factors. 2013;31(4):123-129.
[27]Vane JR, Botting RM. Anti-inflammatory drugs and their mechanism of action. Inflamm Res. 1998;47 Suppl 2:S78-87.
[28]Vane J. Towards a better aspirin. Nature. 1994;367(6460): 215-216.
[29]Yan X, Yin L, Wang Y, et al. The low binding affinity of ADAMTS4 for citrullinated fibronectin may contribute to the destruction of joint cartilage in rheumatoid arthritis. Clin Exp Rheumatol. 2013;31(2):201-206.
[30]Berenbaum F. Signaling transduction: target in osteoarthritis. Curr Opin Rheumatol. 2004;16(5):616-622.
[31]Shi S, Mercer S, Trippel SB. Effect of transfection strategy on growth factor overexpression by articular chondrocytes. J Orthop Res. 2010;28(1):103-109.
[32]Liu J, Jones KL, Sumer H, et al. Stable transgene expression in human embryonic stem cells after simple chemical transfection. Mol Reprod Dev. 2009;76(6): 580-586.
[33]Pfeifer A, Hofmann A. Lentiviral transgenesis. Methods Mol Biol. 2009;530:391-405.
[34]Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res. 2005;320(2):269-276.

引言

Currently, multiple gene therapy is a new field with respect to the single-gene and double-gene systemic therapy for arthritis^[1-2]. Clinical drugs to treat arthritis include narcotic analgesics, non-steroidal anti-inflammatory drugs, glucocorticoids and slow-acting medicine, and their role is mainly aimed at mitigation of the illness^[3]. Even though these drugs can control the inflammation and pain, inner cartilage and bone tissue are still undermined^[4-6].

It has been confirmed that the loss of proteoglycan (aggrecan) is one of the earliest events in the process of articular cartilage damage^[7-9]. Aggrecanase 1, playing a main role in proteoglycan metabolism of newborn articular cartilage, has primary responsibility in the loss of proteoglycans and the pathological damage to the articular cartilage^[10].

Cyclooxygenase 2 (COX-2), as the main medium participating in the pathological changes, is the key enzyme in the initial synthesis of prostaglandin, especially prostaglandin E2. Prostaglandin E2 can the release of interleukin 1α, interleukin-1β, tumor necrosis factor-α from chondrocytes, increase the degradation of aggrecans, and cause the degradation of articular cartilage, which plays an important role in the onset and development of osteoarthritis^{[5, 11-13]}. Released COX-2 plays an important role in the development of synovitis and cartilage damage^[14-16]. Insulin-like growth factor 1 (IGF-1) is the first growth factor confirmed to have autocrine regulation effect on articular cartilage^[17-18]. It can significantly improve the proliferation of chondrocytes from diverse sources, without affecting the cell differentiation, but promoting the synthesis of aggrecan and the expression of matrix metalloproteinases and slowing the degradation of aggrecan^[19]. In vitro, it has been found that in the presence of interleukin-1 beta and tumor necrosis factor, IGF-1 can still promote the aggrecan synthesis of chondrocytes.

Presently, in the gene therapy of arthritis, human bone marrow stem cells with multi-directional differentiation ability are potential target cells. In this experiment, by using lentivirus as carrier, the silencing gene COX-2, aggrecanase-1 and the over-expressing gene IGF were infected into human bone marrow mesenchymal stem cells, and the expressions of these three genes were investigated, to search for a new method for gene therapy of arthritis.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
全文链接：

材料方法

Design

In vitro gene transfection experiment.

Time and setting

This experiment was started in June 2014 and completed at the end of December 2014, in the Central Laboratory of the Affiliated Hospital of Qingdao University.

Materials

Cells

Human bone marrow mesenchymal stem cells were isolated from the bone marrow of 21 donors, with a mean age of (54±7.1) years, 13 males and 8 females, with no infectious or blood diseases. Bone marrow aspiration was conducted in the femur (n=21) within the framework of total hip replacement. All patients provided informed consent according to the latest version of the Helsinki Declaration. This protocol was approved by the ethics committee of the Affiliated Hospital of Qingdao University, China.

Methods

Culture of bone marrow mesenchymal stem cells

Whole bone marrow culture method was used, and the bone marrow was provided by the Qingdao University Hospital. 5 mL bone marrow mixed with equal volume of DMEM-L in 15 mL sterile centrifuge tube was pipetted to make uniform suspensions. 5 mL suspension was absorbed and plated in 25 cm² cell culture flasks, and the cells were maintained in an incubator at 37 ℃ and 5% CO₂, called the primary cells. After 24 hours, half of medium was replaced with 2 mL fresh configuration culture (DMEM: serum: double antibody=89:10:1), while the suspension cells and the residue was taken out of the culture flasks, and hereafter the full medium was changed on alternate days. After 7-8 days, when the cells reached 80% confluence, the adherent cells were harvested and passaged (by 1:2). The passage cells were plated in culture flasks until passage 3, which were collected and transfected by virus.

Lentiviral vectors construction

From GenBank, the gene serial numbers of homo COX-2 (hCOX-2), homo Aggrecanase-1 (hAggrecanase-1) and homo IGF-1 (hIGF-1) were found. Lentivirus vectors with interference sequences sh-hCOX2, sh-hAggrecanase-1 and overexpression sequence hIGF-1, named accordingly hCOX-2-shRNA, hAggrecanase1-shRNA and hIGF-1-shRNA (Table 1), were designed and synthesized by Shanghai GenePharma Co., Ltd., using software, on the basis of early screening experiments in which some gene sequences had been designed and used for transfection successfully^[20-22]. The designed shRNA template was cloned into a self-inactivating lentivirus vector that harbored a green fluorescent protein (GFP) reporter and a H1 promoter upstream of the insertion sites. The cloned oligonucleotides could then produce in vitro shRNA, namely pLL2G (pGLV3/H1/GFP+puro vector) (Figure 1).

Preliminary experiments

The third generation of human bone marrow mesenchymal stem cells were digested with 2.5 g/L trypsin, plated in 96-well culture plates (5×10⁵ cells per well), and virus solutions of hCOX-2, hAggrecanase-1and hIGF-1 were added respectively (according to multiplicities of infection=5, 10, 20, 30). The original medium was replaced by complete medium after 48 hours. The cells with GFP were counted under fluorescence microscope and the transfection efficiency in human bone marrow mesenchymal stem cells was calculated after 72 hours. When the multiplicity of infection=10, the transfection efficiency of hCOX-2, hAggrecanase-1, hIGF-1 all achieved the highest.

Cell Grouping

Cells were divided into three groups: the group of human bone marrow mesenchymal stem cells transfected by lentivirus respectively carrying hCOX-2, hAggrecanase-1, hIGF-1 (experimental group, lenti-COX2-Aggre1-IGF1 group), the group of human bone marrow mesenchymal stem cells transfected by lentivirus excluding genes (negative control group, lenti-NC group), and the group of human bone marrow mesenchymal stem cells with no treatment (blank group).

Gene transfection and observation

The third generation of human bone marrow mesenchymal stem cells were digested with 2.5 g/L trypsin, counted, and plated in 6-well culture plates (4×10⁵ cells per well) containing serum-free DMEM medium. The cells were maintained in the incubator, and the original medium was changed by fresh medium every other day. The lentivirus was removed from the refrigerator at -80 ℃ and melted. Then the lentivirus was successively joined into the cells, according to multiplicity of infection=10, and blundered. The cells were maintained in culture at 37 ℃, and taken out after 12 hours to change the medium containing virus with 10% serum fresh medium, and observed after 72 hours by inverted fluorescence microscope. Then the transfection efficiency of each group was calculated.

RT-PCR detection

The cells were harvested 5 days after transfection. Total RNA was extracted using the RNAiso kit. The concentration and purity of the RNA were evaluated using a spectrophotometer. RT-PCR was performed using the SYBR Premix Ex Taq II kit in a RT-PCR machine according to the manufacturer’s instructions. IGF-1 upstream primer: 5’-TCT TCA GTT CGT GTG TGG AGA C-3’, downstream primer: 5’-TCC GAC TGC TGG AGC CAT A-3’. Aggrecanase-1 upstream primer: 5’-GAC ACG CTG GGT ATG GCT GA-3’, downstream primer: 5’-ACT GAT GCA TGG CTT GGA GTT G-3; COX-2 upstream primer: 5’-GGT TGC TGG TGG TAG GAA TGT-3’, downstream primer: 5’-GTA TTT CAT CTG CCT GCT CTG GT-3’. Internal GAPDH upstream primer: 5’-GCA CCG TCA AGG CTG AGA AC-3’, downstream primer: 5’-TGG TGA AGA CGC CAG TGG A-3’. The PCR reaction conditions: SYBR Premix Ex Taq II (Tli RNaseH Plus) (2×)12.5 μL, Primer F (10 μmol/L) 0.5 μL, Primer R

(10 μmol/L) 0.5 μL, cDNA (negative control reaction, adding RNase free dH₂O) 2 μL, dH₂O 9.5 μL, total 25 μL. Positive reaction, to do two parallel samples: 95 ℃ for 30 seconds, 95 ℃ for 5 seconds, 60 ℃ for 30 seconds, 40 cycles. The comparative ??CT method was used to evaluate the gene expression of hCOX-2, hAggrecanase-1 and hIGF-1.

Western blot analysis

Western blot was used to test the protein expression of COX-2, aggrecanase-1 in human bone marrow mesenchymal stem cells. The cells were harvested 7 days after transfection, washed with PBS for three times, then suspended with PBS for centrifugal separation. Precipitation cells were cracked in the mixture of cracking fluid RIPA and protease inhibitor PMSF (100:1) for 1 hour on the ice, then to ultrasound pyrolysis, and centrifuged (12 000 r/min at 4 ℃) for 10 minutes. The supernatant was incubated in buffer at 95 ℃ for 10 minutes, and preserved at -80 ℃. The concentration of the total protein was determined by colorimetric method, and the quantitative sample of total protein was loaded per well, separated in 8% SDS-PAGE for 1.5-2.0 hours, and transferred to membranes. The membranes were incubated with primary antibodies (diluted 1:500) at 4 ℃ overnight, were rinsed three times with PBS-T

(10 minutes once) on the second day, and subsequently incubated with secondary antibodies (labeled by horseradish peroxidase, 1:2 000) at 37 ℃ for 1 hour. Then the membranes were washed with PBS-T for three times

(5 minutes/time), and followed by rinsing three times with PBS for 15 minutes. Finnally, the membranes were colored in chemiluminescence reagent for 1 minute or so, then developed, fixed and exposed in the cassette. The software Quantity one 4.6 was used to measure absorbance value of every zone, and β-actin served as internal control.

Enzyme-linked immunosorbent assay (ELISA) assay

The supernatant was extracted 7 days after transfection, and ELISA was used to detect the concentration of hIGF-1 in the secretion^[23-26].

Main outcome measures

Human bone marrow mesenchymal stem cells morphology was observed, and the mRNA and protein expressions of hCOX2, hAggrecanase-1, hIGF-1 were detected.

Statistical analysis

Results were denoted as mean±SD, and the independent-sample t-test of software SPSS19.0 was used for statistical analysis. A value of P < 0.05 was regarded as significant difference.

全文链接：

讨论

The complex causes of arthritis are mainly related to inflammation and autoimmune reactions, infections, metabolic disorders, trauma, degenerative changes and other factors. And inflammatory factors play an important role in the process of the onset of arthritis. It has researched that, in the synovial membrane of arthritis patients, the COX-2 expression is obviously increased. Under the catalysis of COX and a series of enzymes, the starting factor triggers inflammation reaction, decomposites membrane phospholipids into arachidonic acid, prompts synthesis of the proinflammatory mediators and inflammatory factors, which cause chronic inflammation and cause the damage of the joints^[27-28]. In rheumatoid arthritis, synovial pannus is aggressive and destructive, and associated with the damage and destruction of joints in rheumatoid arthritis. Experiments have confirmed that COX-2 promotes the formation of blood vessels in the synovial membrane. COX-2 inhibitors have been widely used throughout the world, and the inhibition of the expression and synthesis for the gene COX-2 is an important part of treating arthritis.

Aggrecan is an important factor to regulate the cartilage extracellular matrix and to maintain the normal biochemistry and structural stability of the articular cartilage. Experiments have shown that the increase of aggrecanase expression causes the degradation of aggrecan, so aggrecanase is one of the major genetic causes of articular cartilage damage^[29]. The members of aggrecanase family called ADAMTS include ADAMTS-1, 4, 5, 8, 9, 15. But only ADAMTS-4 (Aggrecanase-1) and ADAMTS-5 (Aggrecanase-2) have been recognized playing a key role in the loss of aggrecan and degradation of cartilage matrix, but mainly dominated by Aggrecanase-1^[30].

IGF-1 is a kind of important cytokines in the human body. Chondrocytes under the action of IGF-1 can proliferate and synthesize cartilage matrix proteoglycan and matrix collagen. Under physiological conditions, in juvenile period, IGF-1 through stimulating proliferation of chondrocytes shapes the bones into linear growth. In adulthood, IGF-1 by stimulating chondrocytes matrix protein synthesis, inhibits the cartilage cell decay and death. Chondrocytes can secrete IGF-1, but they are also adjusted by the IGF-1. IGF-1 is the first identified as a growth factor with secretion regulation to cartilage, which can stimulate the synthesis of DNA and proteoglycan, promote cell differentiation and proliferation, and play a key role for cartilage matrix synthesis and stability^[31]. IGF-1 exerts a regulatory effect on bone metabolism, which can promote proliferation and differentiation of bone cells, stimulate osteoblasts to synthesize non-collagen proteins such as osteocalcin and type 1 collagen, promote the formation of bone matrix and fracture, play an important role to maintain the capacity of bone matrix. Direct application of IGF-1 to repair damaged joints still has limitations, mainly: (1) short half-life of bioactive growth factor, easy dilution when applied topically, difficult to keep the effective concentration, need to be repeated or increase the dose; (2) extraction and genetic recombination is expensive.

Nowadays, using viral vector to import seed cell is a hot spot in arthritis gene therapy. Lentiviral vector system is widely used in all kinds of gene therapy experiments. It can infect not only dividing cells and non-dividing cells, but also can infect cells difficult to transfect with high transfection efficiency, such as lymphocyte stem cells and multiple primary cells^[32]. The exogenous gene imported into the host cell will be integrated into the host cell DNA, resulting in the long-term stable expression of the target gene, and the lentiviral vector system will not cause cell damage and immune response^[33]. Single gene and dual-gene transfection into bone marrow mesenchymal stem cells for COX-2, Aggrecanase-1, IGF-1 have been studied more^[34], but three joint gene transfection is rarely reported, and most research on RNA interference only focuses on one target. In this research, using the lentiviral system, the silencing genes hCOX-2 and hAggrecanase-1 as well as the over-expressing gene hIGF-1 were combinedly transfected into bone marrow mesenchymal stem cells, which is good for the treatment of arthritis.

The results of RT-PCR and Western blot assay showed that hCOX-2 and hAggrecanase-1 were both inhibited in gene and protein levels. According to RT-PCR, hIGF-1 was overexpressed. ELISA further confirmed that hIGF-1 gene was transfected into human bone marrow mesenchymal stem cells cultured in vitro, and a relatively high level of hIGF-1 protein produced. It proved that the three genes expressed correspondingly and effectively in human bone marrow mesenchymal stem cells after transfection, and the expression was statistically different from that in the blank group and the negative control group. It signified that the virus vectors were indeed integrated into the bone marrow mesenchymal stem cells DNA, and expressed the objective product.

To sum up, in this experiment, the successful transfection of COX-2, Aggrecanase-1 and IGF-1 provides a new way for arthritis treatment at the level of cytokine and some experimental basis for further in vivo experiments and systemic treatment of arthritis, and brings new hope for gene therapy of arthritis.

骨髓间充质干细胞中环氧化酶2及聚蛋白多糖酶1沉默与胰岛素样生长因子1过表达

Lentivirus-mediated cyclooxygenase 2 and aggrecanase 1 silencing and insulin-like growth factor 1 overexpression in human bone marrow mesenchymal stem cells

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表（结果） 4

参考文献

相关文章 15

引言

材料方法

讨论

文章快阅

延伸阅读

编辑推荐

Metrics

本文评价

[1]	蒲锐, 陈子扬, 袁凌燕. 不同细胞来源外泌体保护心脏的特点与效应[J]. 中国组织工程研究, 2021, 25(在线): 1-.
[2]	林清凡, 解一新, 陈婉清, 叶振忠, 陈幼芳. 人胎盘源间充质干细胞条件培养液可上调缺氧状态下BeWo细胞活力和紧密连接因子的表达[J]. 中国组织工程研究, 2021, 25(在线): 4970-4975.
[3]	黄登承, 王志科, 曹学伟. 体外冲击波疗法治疗中老年膝骨关节炎短期疗效对比的荟萃分析[J]. 中国组织工程研究, 2021, 25(9): 1471-1476.
[4]	肖国庆, 刘选泽, 严钰皓, 钟喜红. 后交叉韧带替代型假体全膝关节置换术后屈曲受限的影响因素[J]. 中国组织工程研究, 2021, 25(9): 1362-1367.
[5]	彭智浩, 冯宗权, 邹勇根, 牛国庆, 吴峰. 活动平台单髁置换后下肢力线与外侧间室骨关节炎进展的关系[J]. 中国组织工程研究, 2021, 25(9): 1368-1374.
[6]	黄泽晓, 杨妹, 林诗炜, 何和与. 血清n-3多不饱和脂肪酸水平与全膝关节置换早期股四头肌肌力变化的相关性[J]. 中国组织工程研究, 2021, 25(9): 1375-1380.
[7]	李嘉程, 梁学振, 刘金豹, 许波, 李刚. 骨性关节炎mRNA差异表达谱及竞争性内源RNA调控的网络分析[J]. 中国组织工程研究, 2021, 25(8): 1212-1217.
[8]	刘翔翔, 黄云梅, 陈文列, 林如辉, 卢小冬, 李钻芳, 许亚晔, 黄美雅, 李西海. 早期骨关节炎模型大鼠半月板白区细胞的超微结构变化[J]. 中国组织工程研究, 2021, 25(8): 1237-1242.
[9]	张秀梅, 翟运开, 赵杰, 赵萌. 类器官模型国内外数据库近10年文献研究热点分析[J]. 中国组织工程研究, 2021, 25(8): 1249-1255.
[10]	王正东, 黄娜, 陈婧娴, 郑作兵, 胡鑫宇, 李梅, 苏晓, 苏学森, 颜南. 丁酸钠抑制氟中毒可诱导小胶质细胞活化及炎症因子表达增多[J]. 中国组织工程研究, 2021, 25(7): 1075-1080.
[11]	汪显耀, 关亚琳, 刘忠山. 提高间充质干细胞治疗难愈性创面的策略[J]. 中国组织工程研究, 2021, 25(7): 1081-1087.
[12]	万然, 史旭, 刘京松, 王岩松. 间充质干细胞分泌组治疗脊髓损伤的研究进展[J]. 中国组织工程研究, 2021, 25(7): 1088-1095.
[13]	廖成成, 安家兴, 谭张雪, 王倩, 刘建国. 口腔鳞状细胞癌干细胞的治疗靶点及应用前景[J]. 中国组织工程研究, 2021, 25(7): 1096-1103.
[14]	谢文佳, 夏天娇, 周卿云, 刘羽佳, 顾小萍. 小胶质细胞介导神经元损伤在神经退行性疾病中的作用[J]. 中国组织工程研究, 2021, 25(7): 1109-1115.
[15]	李珊珊, 郭笑霄, 尤冉, 杨秀芬, 赵露, 陈曦, 王艳玲. 感光细胞替代治疗视网膜变性疾病[J]. 中国组织工程研究, 2021, 25(7): 1116-1121.