miR-141及成骨基因Dlx5、Msx2、Runx2在去卵巢骨质疏松模型小鼠BMSCs成骨分化中的表达

Expression of miR-141 and Osteogenic Gene Dlx5, Msx2 and Runx2 in Osteogenic Differentiation of BMSCs in Ovariectomized Osteoporosis Model Mice

目的:观察去卵巢骨质疏松模型组与假手术组小鼠骨髓间充质干细胞成骨分化中miR-141及成骨基因Dlx5、Msx2、Runx2的表达差异和变化趋势,探讨绝经后骨质疏松症的发生机制。方法:选用20只4周龄清洁级C57雌性小鼠,按照随机数字表法分为模型组和假手术组,每组各10只。模型组采用卵巢切除法建立小鼠绝经后骨质疏松症模型,假手术组保留卵巢,只切除卵巢周围脂肪。造模2个月后模型组小鼠经鉴定建模成功,此时采用全骨髓法分离培养2组小鼠BMSCs,进行细胞形态学观察,运用流式细胞技术进行细胞表型鉴定。对BMSCs成骨诱导分化,分别于第3天、8天、13天时采用碱性磷酸酶染色、定量和茜素红染色,观察BMSCs成骨能力。成骨诱导第3天、8天、13天时运用q-PCR检测miR-141及成骨基因Dlx5、Msx2、Runx2的表达,Western blot检测成骨基因蛋白的表达。结果:BMSCs经流式细胞表型鉴定CD29、CD44均阳性表达,CD34、CD45均阴性表达。2组BMSCs成骨诱导后,ALP表达量逐渐提高,第8天达到高峰,随后逐渐下降,呈"Λ"型变化;模型组较假手术组染色浅,活性检测结果与染色深浅程度一致。成骨诱导分化第3天、8天、13天时,模型组ALP表达量均低于假手术组(P<0.05),2组细胞进行茜素红染色,第8天时钙化结节出现,在第13天时,钙化结节数增多;成骨诱导第3天、第8天时,2组钙化结节数无明显差异,第13天时,与假手术组比较,模型组钙化结节数较少,成骨分化明显减缓。2组BMSCs成骨诱导后,成骨基因与蛋白的表达:成骨诱导第3天、8天、13天时,组内比较显示,Dlx5、Runx2表达均呈持续上升,miR-141、Msx2持续下降(P<0.05),与第3天时比较,第8天、13天的各项指标均有明显差异(P<0.05),组间比较显示,模型组miR-141、Msx2表达高于假手术组,Dlx5、Runx2表达低于假手术组,差异有统计学意义(P<0.05)。结论:卵巢切除影响BMSCs的成骨分化;BMSCs成骨诱导分化中,miR-141、Msx2是成骨负性调节因子,Dlx5、Runx2是正性调节因子。

Objective: To observe the expression difference and change trend of mi R-141 and osteogenic gene Dlx5, Msx2 and Runx2 in osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) in the ovariectomy model group and the sham operation group, and investigate the mechanism of postmenopausal osteoporosis. Methods: Twenty 4-week-old clean C57 female mice were selected as the model group and the sham operation group according to the random number table method, and ten cases in each group. The mouse model of postmenopausal osteoporosis in the model group was established by ovariectomy, and the ovaries were preserved and only the fat around the ovaries were removed in the sham operation group. The mice in the model group were identified and successfully modeled two months after modeling, then BMSCs of mice in the two groups were isolated and cultured through whole marrow culture method. The morphology of the cells was observed and the cell phenotype was identified by flow cytometry. The osteogenic differentiation of BMSCs was observed by alkaline phosphatase staining, quantification and alizarin red staining on the 3 rd,8 th and 13 th day, respectively to observe the osteogenic ability of BMSCs. The expression of mi R-141 and osteogenic genes Dlx5,Msx2 and Runx2 were detected by q-PCR on the 3 rd, 8 th and 13 th day after osteogenic induction of BMSCs. The protein expression of osteogenic gene was detected by Western blot. Results: Positive expression of CD29 and CD44 and negative expression of CD34 and CD45 in BMSCs were found by flow cytometry. After osteogenic induction of BMSCs in both groups, the expression of ALP gradually increased, reaching a peak on the 8 th day, and then gradually decreased, showing a " ∧" type change. The model group was lighter than the sham operation group, and the result of activity detection was consistent with the degree of staining. The expression of ALP in model group was lower than that of the sham operation group after osteogenic differentiation on the 3 rd, 8 th and 13 th day(P<0.05). When the two groups of cells were stained with alizarin red, calcified nodules appeared on the 8 th day, and the number of calcified nodules increased on the 13 th day. There was no significant difference in the number of calcified nodules between the model group and the sham operation group on the 3 rd and 8 th day of osteogenic induction. On the 13 th day, compared with the sham operation group, the number of calcified nodules in the model group was less, and osteogenic differentiation slowed down significantly. On the 3 rd, 8 th and 13 th day of osteogenic induction, the expression of Dlx5 and Runx2 were increased continuously, and mi R-141 and Msx2 were decreased continuously(P<0.05). Compared with the 3 rd day, the indexes of the 8 th day and the 13 th day were significantly different(P<0.05). Comparison of between groups showed that the expression of mi R-141 and Msx2 in the model group was higher, and the expression of Dlx5 and Runx2 was lower than those in the sham operation group(P<0.05). Conclusion: The ability of osteogenic differentiation in BMSCs is affected by ovariectomy. Mi R-141 and Msx2 are negative regulators, and Dlx5 and Runx2 are positive regulators in osteogenic differentiation of BMSCs.