不同剂量健腰密骨片对骨形成和小鼠骨髓间充质干细胞BMP-7、OC表达的影响

Effects of different doses of Jianyao Migu Tablets on bone formation and the expression of BMP-7 and OC in mouse bone marrow mesenchymal stem cells

目的:研究健腰密骨片对骨形成及骨髓间充质干细胞(BMSCs)骨形态发生蛋白(BMP-7)、骨钙素(OC)表达的影响。方法:选取1月龄昆明小鼠60只,雌雄各半,按体质量随机分为健腰密骨片高、中、低剂量组,仙灵骨葆组,生理盐水组,灌胃8周后取材。用Micro-CT扫描分析L4腰椎,免疫组化染色观察不同剂量健腰密骨片对椎体BMP-7、OC表达的影响;分离培养获得各组小鼠原代BMSCs,利用碱性磷酸酶(ALP)染色、实时荧光定量RT-PCR法检测BMSCs成骨分化过程中BMP-7、OC的表达情况。结果:与生理盐水组比较,健腰密骨片高剂量组能够改善小鼠椎体三维结构,明显升高小鼠椎体骨密度(P<0.05),增加椎体骨小梁数目和骨小梁厚度增加(P<0.05),降低骨小梁分离度(P<0.05),促进椎体BMP-7、OC蛋白的表达;健腰密骨片高剂量组能够明显促进BMSCs中ALP的表达,上调成骨基因BMP-7、OC m RNA的表达量,其中OC m RNA升高显著(P<0.05)。结论:健腰密骨片高剂量组能够明显提高小鼠椎体骨密度,促进BMSCs成骨分化,上调椎体和BMSCs成骨分化过程中BMP-7、OC表达,从而促进骨形成。

Objective: To investigate the influence of the Jianyao Migu Tablets on the bone formation of the wild-type mice and the expression of BMP-7, OC in bone marrow mesenchymal stem cells(BMSCs). Methods: Sixty 1-month-old Kunming mice, male and female in half, were randomly divided into five groups including high-dose group, middle-dose group, low-dose group, the Xianling Gubao group and the saline group. All the mice were treated intragastrically and sacrificed after 8 weeks. The fourth lumbar vertebra(L4) was scanned by Micro-CT. The expression of BMP-7 and OC in different doses of Jianyao Migu Tablets groups were observed by immunohistochemical staining. BMSCs were isolated and cultured in each group. Alkaline phosphatase(ALP) staining was performed to observe the osteogenic differentiation. Real-time quantitative PCR analysis was performed to observe the expression of BMP-7 and OC. Results: Compared with the saline group, high-dose group improved three-dimensional structure of mice vertebral and increased vertebral bone mineral density significantly(P<0.05), increased the vertebral trabecular number and trabecular thickness significantly(P<0.05), and decreased the trabecular separation significantly(P<0.05), promoted the m RNA and protein expression of BMP-7 and OC. High-dose of Jianyao Migu Tablets could promote the expression of ALP in BMSCs and increase the m RNA expression of BMP-7 and OC, OC m RNA expression was significally increased(P<0.05). Conclusion: High-dose of Jianyao Migu Tablets can increase vertebral bone mineral density and promote BMSCs osteogenic differentiation, increase the expression of BMP-7 and OC, so as to promote the bone formation.