糖尿病大鼠来源骨髓间充质干细胞体外培养观察及其生物学特性

Observation on the culture in vitro of bone marrow mesenchymal stem derived from diabetic rats and its biological characteristics

目的:研究糖尿病是否会导致在体外培养条件下自体骨髓间充质干细胞的增殖、分泌和抗凋亡能力的改变。方法:利用链脲佐菌素(STZ)诱导制作成年大鼠糖尿病模型(n=6),正常大鼠作为对照组(n=6),分别于体外利用全骨髓贴壁法来扩增和纯化骨髓间充质干细胞后,取生长良好的第2代细胞作为本实验的研究对象。采用Cell Count kit-8(CCK-8)分别绘制两组细胞的生长曲线来评价增殖能力;采用酶联免疫吸附试验(ELISA)检测血管内皮生长因子(VEGF)和胰岛素样生长因子-1(IGF-1)的分泌量;应用Hoechst33342染色和膜联蛋白V/PI双染流式细胞术观察细胞在低氧无血清条件下的抗凋亡能力。结果:来源于糖尿病大鼠的骨髓间充质干细胞的增殖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。VEGF和IGF-1的分泌量较正常大鼠来源的明显降低,其中VEGF的分泌量为(80.7±13.1)pg/mL和(67.9±12.3)pg/mL(P<0.05),IGF-1的分泌量分别为(374.8±41.2)pg/mL和(281.8±26.8)pg/mL(P<0.01)。在低氧无血清条件下,糖尿病来源的干细胞抗凋亡能力显著性降低,两组早期细胞凋亡指数分别为28.7±3.4和37.1±2.42(P<0.01)。结论:糖尿病大鼠来源的骨髓间充质干细胞可以成功地在体外进行增殖培养,但是其在增殖能力、分泌、抗凋亡能力等一系列生物学性状上有不同程度的损害。

Objective：To study the effect of diabetic inner environment on the biological properties of marrow mesenchymal stem cells derived from bone（BMSCs）.Methods：BMSCs were harvested from the normal and diabetic ratsinduced by streptozotocin（STZ）.Cell proliferation was evaluated using cell counting kit-8（CCK-8） assay.The secretory volume of vascular endothelial growth factor（VEGF） and insulin-like growth factor（IGF-1） were measured by enzyme-linked immunosorbent assay（ELISA）and the anti-apoptosis capacity of the cell under the hypoxia and serum deprivation（hypoxia/ SD）was detected by Hoechst33342 staining and annexin V/PI dual-color flow cytometry.Results：BMSCs from the diabetic rats induced by STZ could be successfully harvested and expanded in vitro.However they showed more spread-out and flattened appearance and larger size.The proliferative ability of diabetic rats derived from BMSCs were significantly decreased compared with the normal rats（P0.01）.In addition,similar results were also presented in the cytokines（VEGF and IGF-1） release（80.7± 13.1 vs 67.9±12.3 pg/mL P 0.05,374.8±41.2 vs 281.8±26.8 pg/mL P 0.01,respectively）and the ability of anti-apoptosis under hypoxia/SD（P 0.01）.Conclusion：BMSCs derived from diabtetic rats can be successfully cultured in vitro.The ability of series of a biological properties in proliferation,paracrine and anti-apoptosis have been impaired in different degrees.