补肾活血方诱导大鼠骨髓间充质干细胞成骨分化的实验研究

Experimental study on Bushen Huoxue recipe induced osteogenic differentiation of bone marrow mesenchymal stem cells in rats

目的观察补肾活血方对大鼠骨髓间充质干细胞(BMSCs)向成骨细胞分化的情况,从而为其治疗骨质疏松症提供细胞生物学研究依据。方法 SPF健康雌性SD大鼠,采用密度梯度离心法分离骨髓间质干细胞,差速贴壁法进行纯化,细胞培养至第3代(P3),应用流式细胞术鉴定BMSCs纯度并分选纯化细胞,纯化过的细胞经补肾活血方诱导1天,3天和7天,在不同时间点进行碱性磷酸酶(ALP)染色鉴定成骨细胞,经茜素红染色鉴定钙结节。结果 P3含有CD44的细胞数占总数的96. 3%,CD29的细胞数占总数的77. 1%,说明培养的细胞中骨髓间充质干细胞的纯度较高,经补肾活血方诱导后可见细胞由梭型变成铺路石样,体积增大,细胞成簇状生长。经碱性磷酸酶鉴定可见胞浆着蓝色,有典型成骨细胞的表达。经茜素红染色,可见成簇的细胞呈红色钙结节样。结论补肾活血方的作用靶点可能是通过促进BMSCs向成骨的分化,从而起到防治骨质疏松的作用。

Objective To observe the differentiation of bone marrow mesenchymal stem cells （BMSCs） into osteoblasts in rats, and to provide a basis for the treatment of osteoporosis. Methods Female SD rats, SPF health using density gradient centrifugation separation of bone marrow mesenchymal stem ceils, differential sticking wall method to purify, cell culture to third - generation （ P3）, application of flow cytometry and purity identification of BMSCs cells separation and purification, purification of the cells by kidney invigorate the circulation induced by 1 day, 3 and 7 days, at different time points for alkaline phosphatase （ALP） staining identified osteoblast, alizarin red staining identified the formation of calcium nodules. Results P3 contains cells number accounts for 96.3% of the total number of CD44, CD29 77.1% of the total number of cells of high purity, visible after inducing by Bushen Huoxue recipe the circulation of the induced cells changed from fusiform to paving stone, sample, size, into clusters of cell growth. The cytoplasmic blue was identified by alkaline phosphatase, and the expression of typical osteoblasts was found. The cells in the clusters were red with red calcium nodules. Conclusion The action target of invigorating kidney and activating blood may be to promote the differentiation of BMSCs to osteoblasts, thus preventing osteoporosis.