Effect of human amniotic membrane mesenchymal stem cell transplantation on ovariectomized osteoporosis

doi:10.3969/j.issn.2095-4344.2017.01.017

Abstract

Abstract:

BACKGROUND: There are rare studies directly exploring the use of human amniotic mesenchymal stem cells to promote differentiation and maturation of osteocytes. Therefore, human amniotic mesenchymal stem cell transplantation is likely to open new horizons for the treatment of osteoporosiss.
OBJECTIVE: To explore the effect of human amniotic mesenchymal stem cell transplantation on bone metabolism index and bone microstructure in ovariectomized osteoporosis rats and the relevant mechanisms.
METHODS: Thirty-six female Wistar rats without pregnancy were randomly divided into sham and model group. Twenty-three rats in the model group were ovariectomized, and thirteen rats in the sham group received no treatment. Three months after ovariectomy, three rats from each group were executed. ELISA was used to detect serum levels of tartrate-resistant acid phosphatase (TRACP) and bone alkaline phosphatase (BALP). Hematoxylin-eosin staining was performed to observe the pathological changes of the bone. After successful modeling, the remaining rats in the model group were randomized into control and treatment groups, respectively given tail vein injection of sodium chloride and human amniotic mesenchymal stem cells, twice a week, for consecutive 6 weeks. Six weeks after cell transplantation, ELISA was used to detect serum levels of TRACP and BALP; RT-PCR and western blot assay were used to detect expressions of Runx2 and Osterix mRNA and protein; and hematoxylin-eosin staining was used to observe bone microstructure changes.
RESULTS AND CONCLUSION: At 3 months after ovariectomy, the rats in the model group exhibited significantly decreased BALP and remarkably increased TRACP as well as sparse number of bone trabeculae some of which were even ruptured shown by hematoxylin-eosin staining. However, the bone trabeculae of rats in the sham group still remained intact. These results indicated the successful modeling in the model group. At 6 weeks after cell transplantation, significantly increased BALP, Runx2, Osterix and significantly decreased TRACP were observed in the treatment group compared with the control group (P < 0.05). Additionally, there a wide-range formation of new bone trabeculae in the treatment group, and damage to the trabecular structure was milder in the treatment group than the control group. In summary, human amniotic mesenchymal stem cell transplantation exerts therapeutic effects on ovariectomized osteoporosis, probably through promotion of Runx2 and Osterix.

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

Key words: Stem Cells, Mesenchymal Stem Cells, Osteoporosis, Tissue Engineering

CLC Number:

R394.2

Lei Ming, Liu Chun-ying, Liu Xiao-feng, Sun Lin, Zheng Wen-kui . Effect of human amniotic membrane mesenchymal stem cell transplantation on ovariectomized osteoporosis[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 92-97.

Figures/Tables 2

2.1 实验动物数量分析选择雌性未孕的Wistar大鼠36只，随机分组，模型组23只，假手术组13只。术后3个月模型组取3只大鼠处死，判断造模情况，组剩余20只大鼠随机分为对照组和治疗组，每组10只。全部进入结果分析，无脱失。 2.2 造模结果判定造模术后3个月，对随机选取的模型组和假手术组大鼠进行血清骨碱性磷酸酶和抗酒石酸酸性磷酸酶水平检测及苏木精-伊红染色结果显示，模型组大鼠摘除双侧卵巢造模成功。结果显示模型组大鼠血清骨碱性磷酸酶水平明显低于假手术组大鼠，而抗酒石酸酸性磷酸酶水平较假手术组明显升高，详见表1。苏木精-伊红染色的结果表明，假手术组大鼠的股骨骨小梁与模型组大鼠相比稠密、数目较多，模型组股骨小梁结构不如假手术组大鼠完整，出现骨断裂现象，见图1，因此判定造模成功。 2.3 抗酒石酸酸性磷酸酶和骨碱性磷酸酶水平变化移植人羊膜间充质干细胞6周后，与对照组相比，治疗组的骨碱性磷酸酶水平明显升高，抗酒石酸酸性磷酸酶水平明显降低，差异均有显著性意义(P < 0.05)，结果详见表2及图2。 2.4 Runx2、Osterix基因的表达水平通过RT-PCR检测Runx2、Osterix mRNA的表达水平结果可知，治疗组的Runx2、Osterix mRNA均高于对照组(P < 0.05)，由此可推测人羊膜间充质干细胞可能在治疗骨质疏松上起到一定的作用，见图3。 2.5 Runx2、Osterix蛋白表达水平通过蛋白印迹法检测Runx2、Osterix蛋白的表达水平结果可知，治疗组的Runx2、Osterix蛋白表达水平均高于对照组(P < 0.05)，由此可推测人羊膜间充质干细胞可能在治疗骨质疏松上起到一定的作用，此结果与Runx2、Osterix的mRNA表达水平一致，见图4。 2.6 股骨结构的病理变化通过苏木精-伊红染色，对比治疗组和对照组的股骨微观结构。对照组的骨结构破坏与治疗组相比较严重，治疗组的股骨有新生的骨小梁形成，且骨小梁结构完整、数目较多，可见成骨细胞数目多于对照组，提示人羊膜间充质干细胞对骨小梁起到一定的保护作用，见图5。"

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