链脲佐菌素诱导糖尿病大鼠骨髓间充质干细胞体外培养观察及其增殖能力检测

To Study the Culture of Bone Marrow Mesenchymal Stem Cells in Vitro and Their Proliferation Ability of Diabetic Rats Induced by STZ

摘要: 目的：研究糖尿病模型大鼠不同时间段体外培养条件下骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)的增殖能力的改变，初步探讨糖尿病模型骨质疏松形成与BMSCs生物学特性改变的相关性。方法：利用链脲佐菌素(Streptozotocin, STZ)加高糖高脂饲料诱导制作大鼠糖尿病模型，正常大鼠作为对照组。分别于注射STZ液4、8、12周后，体外利用全骨髓贴壁法来扩增和纯化BMSCs，取生长良好的第3代细胞作为本实验的研究对象。流式细胞仪检测细胞表面标志物CD34、CD29的表达。采用 Cell Count kit-8 (CCK-8)分别绘制细胞的生长曲线来评价增殖能力。结果：培养的细胞CD29表达为阳性，CD34表达为阴性。注射STZ液4、8、12周后，来源于糖尿病大鼠的BMSCs的增殖均较正常的大鼠显著性减慢(P < 0.01)，在形态学上则表现为较正常的细胞更扁平。随着时间的增加(注射STZ12周后)，体外培养的BMSCs几乎没有增殖，培养第7天的OD值与第1天比较，P > 0.05。结论：糖尿病大鼠来源的BMSCs可以成功地在体外进行增殖培养，但是随着时间的增加，其在增殖能力生物学性状上有不同程度的损害。糖尿病大鼠BMSCs 的增殖影响可能具有一定的时间依赖性，可能与骨质疏松的形成有一定的关系。

Abstract: Objective: To explore the proliferation ability of bone marrow mesenchymal stem cells (BMSCs) by diabetic model rats during different time periods, and make a preliminary discussion of relationship between osteoporosis formed by diabetic and change by biological characteristics of the BMSCs. Methods: The diabetic rats were induced by STZ (Streptozotocin) combined with high glucose and high-fat diet, and normal rats were as control group. The whole bone marrow adherence method was used to amplify and purify BMSCs injected with STZ after 4, 8 and 12 weeks, and the third well-grown generation cells were selected. The expression of cell surface markers CD34 and CD29 was detected with Flow cytometry. The cell proliferation ratio index was tested with Cell Count kit-8 (CCK-8). Results: The expression of CD29 in cultured cells was positive, and the expression of CD34 was negative. The proliferation of BMSCs of diabetic rats related to the injection time was significantly slower than that of normal rats (P < 0.01), and the morphology was more flat than normal cells in morphology. With the increase of time (STZ12 weeks after injection), in vitro BMSCs had almost no proliferation, compared with first days of training od seventh days, P > 0.05. Conclusion: BMSCs derived from diabetic rats can be successfully cultured in vitro, but with the increase of time, it has different degrees of damage in the biological characteristics of proliferation ability. The proliferation of BMSCs in diabetic rats may be time-dependent, which may be related to the formation of osteoporosis.