D-半乳糖对大鼠骨髓间充质干细胞拟衰老作用的实验研究

目的 探讨D-半乳糖（D-galactose，D-gal）诱导SD大鼠骨髓间充质干细胞（marrow mesenchymal stemcells，MSCs）衰老后的形态学及生物学变化情况。方法取3只2月龄SD大鼠MSCs分离培养至第3代，一部分置于含8g／LD-gal的培养液中继续培养至第6代作为诱导组；另一部分在原培养液中继续培养至第6代作为对照组，并作表型鉴定。采用流式细胞仪检测对照组加入8g／LD-gal后4d内细胞周期的分布变化。两组MSCs培养7d并绘制生长曲线，在光镜和透射电镜下观察其形态和结构变化，β-半乳糖苷酶染色、单细胞凝胶电泳检测DNA损伤方法检测其衰老后的生物学行为变化。结果诱导组MSCs镜下呈典型的衰老细胞形态；对照组MSCs形态均匀，长势良好。流式细胞仪检测表明加入D-gal诱导后90％MSCs停留在G0／G1期，而S期和G2／M期MSCs趋于消失，并随诱导时间延长趋势越明显；对照组MSCs各期比例正常。MSCs生长曲线示诱导组生长速度较对照组明显减慢。诱导组约85％呈β-半乳糖苷酶阳性染色，对照组染色为阴性。单细胞凝胶电泳示诱导组MSCs DNA有拖尾现象，对照组MSCs无DNA损伤。结论 D-gal对SD大鼠MSCs的老化具有诱导作用，初步推断8g／L为最适浓度，为研究MSCs衰老提供了较好的模型。

STUDY ON CELL SENESCENCE INDUCED BY D-GALACTOSE IN CULTURED RAT MESENCHYMAL STEM CELLS

OBJECTIVE: To study morphological and biological senescence changes induced by D-galactose in the cultured rat mesenchymal stem cells. METHODS: After 3rd generations cultured in the DMEM-F12, MSCs were changed into DMEM-F12 medium containing 8 g/L D-galatose and cultured to the 6th generations as the inducement group. The comparison were the 6th generations which was cultured in the DMEM-F12 medium all along, and then indentified by surface wave. Using flow cytometer to check the comparisons cell cycle change after swing in with 8 g/L D-galatose within the 4 days. In the first 7 days to draw the growth curve to the two groups. Optical and electronic microscope were used to identify the influences of characteristic morphological of mesenchymal stem cells of the two groups, the influences of biological markers were identified by single cell gel electrophoresis and beta-galactose dye. Results After treatment with D-galactose, the mesenchymal stem cells displayed morphological and biological changes in the cell senescence with the senescent characteristic morphological markers; 85% of the cells were X-gal dye masculine, and the singal cell gel electrophoresis showed DNA damnification. The flow cytometry showed that 90% of the cells stayed in G0/G1, but the cells in S and G2/M almost disappeared. However, the cells in the control group had no such DNA damages. CONCLUSION: D-galactose can induce senescence of the mesenchymal stem cells, and 8 g/L is the best concentration to do so. This study has provided a good model for the research of the mesenchymal stem cells senescence.