DNA去甲基化对大鼠骨髓间充质干细胞端粒酶反转录酶的调节

Effect of DNA demethylation on telomerase reverse transcriptase expression in rat bone marrow mesenchymal stem cells

背景:DNA去甲基化是一种重要的表观遗传修饰,对肿瘤细胞的端粒酶具有重要调节作用,而对骨髓间充质干细胞端粒酶活性有何影响尚不清楚。目的:观察DNA去甲基化对骨髓间充质干细胞增殖及端粒酶反转录酶蛋白表达的影响。方法:全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞;按照下列分组加入5-杂氮胞苷,使各组5-杂氮胞苷终浓度分别为0,3,6,12,24μmol/L。加入5-杂氮胞苷后第1,2,3,5,7天进行指标检测。结果与结论:与对照组相比,5-杂氮胞苷干预24h,各浓度组均显著促进细胞增殖活性(P<0.05);干预48h,6,12,24μmol/L组显著促进细胞增殖活性(P<0.05);干预72h,12,24μmol/L组显著抑制细胞增殖活性(P<0.05);干预120,168h,对照组与各浓度组间差异均无显著性意义(P>0.05)。5-杂氮胞苷干预48h,6,12,24μmol/L组端粒酶反转录酶蛋白IA值较对照组显著增加(P<0.05)。提示在一定浓度范围及一定作用时间内,5-杂氮胞苷可以促进骨髓间充质干细胞增殖与端粒酶反转录酶蛋白的表达。

BACKGROUND：DNA demethylation is an important epigenetic modification,which plays a vital role in telomerase regualtion of tumor cells.It is not clear to effect of DNA demethylation on telomerase activity in bone marrow mesenchymal stem cells.OBJECTIVE：To observe the effect of DNA demethylation on proliferation and telomerase reverse transcriptase expression in bone marrow mesenchymal stem cells.METHODS：Bone marrow mesenchymal stem cells were isolated by using the method of adhesive culture of whole bone marrow.In the third passage bone marrow mesenchymal stem cells,5-azacytidine was added to 0,3,6,12,24 μmol/L.CD44,CD45 and telomerase reverse transcriptase were dectcted by immunocytochemistry,and effect of DNA demethylation on proliferation was detected by MTT method at 1,2,3,5,7 days after 5-azacytidine intervention.RESULTS AND CONCLUSION：Compared with the control group,after 24-hour intervention of 5-azacytidine,5-azacytidine with 3,6,12,24 μmol/L increased significantly cell proliferation without dose-dependence（P〈0.05）.After 48-hour intervention of 5-azacytidine,5-azacytidine with 6,12,24 μmol/L increased significantly cell proliferation without dose-dependence（P〈0.05）.After 72-hour intervention of 5-azacytidine,5-azacytidine with 12,24 μmol/L decreased significantly cell proliferation without dose-dependence（P〈0.05）.After 5-day and 7-day intervention of 5-azacytidine,5-azacytidine has no effect on cell proliferation（P〉0.05）.Compared with the control group,after 48-hour intervention of 5-azacytidine,5-azacytidine with 6,12,24 μmol/L increased significantly telomerase reverse transcriptase expression in bone marrow mesenchymal stem cells without dose-dependence（P〈0.05）.In certain concerntration and time,5-azacytidine can increase proliferation and expression of telomerase reverse transcriptase in bone marrow mesenchymal stem cells.