骨质疏松大鼠骨髓间充质干细胞的培养方法及生物学特性鉴定

Culture and characteristic identification of bone mesenchymal stem cells in ovariectomy rats

目的:探索骨质疏松大鼠(OVX)骨髓间充质干细胞(BMSCs)的培养方法,进行鉴定并观察其生物学特性。方法:通过去卵巢法构建SD大鼠骨质疏松模型,在OVX组大鼠和假手术组(Sham)大鼠,分别冲取不同部位(肱骨、股骨和胫骨)的骨髓,贴壁法分离纯化BMSCs,并通过MTT法检测其增殖活性,流式细胞仪检测其表面抗原;对BMSCs进行成骨、成脂诱导,并与Sham组BMSCs对比,观察OVX组BMSCs成骨、成脂肪分化的差别。结果:在OVX大鼠,无法培养出股骨中的BMSCs,可培养出胫骨和肱骨的BMSCs,但前者细胞量少,后者量多;与Sham组相比较,成骨诱导后OVX组BMSCs的碱性磷酸酶活性和骨钙素分泌均较低;成脂诱导后,OVX组BMSCs生成的脂滴数目较多。结论:通过选取肱骨骨髓,加大细胞培养密度,可以成功培养出OVX大鼠的BMSCs,细胞量明显多于其他部位骨髓,可满足实验的样本量要求;大鼠BMSCs(OVX)具有增殖速率低、成骨能力弱、成脂能力强的生物学特性。

PURPOSE：To investigate the cultivation method of BMSCs in ovariectomy rats, and observe the biological characteristics. METHODS： The animal model of osteoporosis was established by performing ovariectomy on SD rats. Bone marrow mesenchymal stern cells （BMSCs） in humerus, temur, tibia of OVX rats and Sham rats were isolated from the rats of ovariectomized （OVX） group and sham group respectively by means of adherence. The proliferation was tested by MTT, and the expression of antigens on cell surface was detected by flow cytometly. BMSCs in humerus was subject to osteogenic induction, and adipogenie induction; then, the difference in differentiation between the BMSCs （OVX） and BMSCs （Sham） was investigated. The data was statistically analyzed with SPSSI6.0 software package. RESULTS： BMSCs in femur and tibia were not successfully cultured; BMSCs in humerus were cultured. Compared with BMSCs （Sham）, the ALP activity and OCN secretion in BMSCs （OVX） was low. The number of lipid droplets in BMSCs （OVX） was more than that in BMSCs（Sham） after adipogenic induction. CONCLUSION： BMSCs（OVX） can be suceessfully cultured by selecting humerus marrow and increasing cell culture density; BMSCs （OVX） were characterized by low proliferation rate, weak osteogenesis ability and greater adipogenic ability. Supported by National Natural Science Foundation of China （30901698,10972142）,Key Basic Research Foundation of Science and Technology Commission of Shanghai Municipality（12JC1405700）, Collaborative Foundation of Medical and Engineering Science of Shanghai Jiao Tong University （YG2012MS40）, and Research Fund of Science and Technology Commission of Shanghai Municipality（08411961600）.