摘要
目的探讨微小RNA（microRNA，miR）92a抑制剂对高糖诱导的内皮细胞损伤的作用。方法分离原代人脐静脉内皮细胞（HUVECs），脂质体转染miR？92a抑制剂或阴性对照，采用5 mmol/L正常浓度葡萄糖或30 mmol/L葡萄糖刺激HUVECs 48 h后，分为4组：空白组（5 mmol/L葡萄糖），高糖组（30 mmol/L葡萄糖），阴性对照＋高糖组（转染阴性对照＋30 mmol/L葡萄糖处理），miR？92a抑制剂＋高糖组（转染miR？92a抑制剂＋30 mmol/L葡萄糖处理）。处理后，用荧光实时定量PCR法分析miR？92a水平，Hoechst染色观察细胞核改变，MTS观察HUVECs活力，Western blotting检测caspase？3和cleaved caspase？3蛋白表达。2组间比较采用t检验，多组间比较采用单因素方差分析。结果高糖组（30 mmol/L）HUVECs与空白对照组相比，miR？92a水平增加31%（1.31±0.37比1，t=1.93，P〈0.05）。细胞数目减少，排列不规则，轮廓模糊。细胞核染色质浓缩凝集、边缘化，凋亡小体增加。细胞活力下降19%（81%±2%比1，t=-29.85，P〈0.01），cleaved caspase？3/caspase？3增加24%（0.31±0.07比0.25±0.02， t=2.18，P〈0.05）。miR？92a抑制剂＋高糖组HUVECs与高糖组相比，形态损伤减轻，凋亡小体减少，细胞活力增加9%（88%±11%比81%±2%，t=1.82，P〈0.05），cleaved caspase？3/caspase？3减少26%（0.23±0.03比0.31±0.07，t=-2.65，P〈0.05）。结论 miR？92a抑制剂可缓解高糖诱导的内皮细胞形态损伤、活力抑制，减少caspase？3激活。miR？92a具有成为防治糖尿病血管并发症靶点的临床价值。
Objective To investigate the effect and mechanism of miR？92a in glucose？induced endothelial cells dysfunction. Method Primary human umbilical vein endothelial cells were stimulated by different concentration （5 mmol/L and 30 mmol/L）of glucose. The effect of the miR？92a inhibitor was observed by using lipofection transfection. The cells were divided into 4 groups：blank control group （Con）, high glucose group （HG）, transfecting negative control group （NC） and transfecting miR？92a inhibitor group （IN）. The level of miR？92a was detected by qRT？PCR and florescence observation. Hoechst staining was used to investigate the change of nucleus. MTS was applied to observe cell activity. The level of caspase？3 and cleaved caspase？3 protein level were analyzed by Western blotting. Results Compared with the Con group, HUVECs treated with high concentration of glucose have induced significant morphologic damage and apoptosis. The miR？92a level of HG group was increased for 31%（1.31±0.37 vs 1, t=1.93,P〈0.05） and cell activity was decreased for 19%（（81%±2%） vs 1, t=-29.85,P〈0.01） ,while caspase？3/cleaved caspase？3 protein level was increased for 24%（0.31±0.07 vs 0.25±0.02,t=2.18,P〈0.05）. In contrast, compared with HG group, there were significant alleviation on morphologic damage and apoptosis in IN group. Cell activity of IN group was increased for 9%than that in HG group （（88%± 11%） vs （81%± 2%）,t=2.18,P〈0.05） and cleaved caspase？3/caspase？3 was reduced for 26%（0.23 ± 0.03 vs 0.31 ± 0.07,t=-2.65,P〈0.05）.nbsp;Conclusion miR？92a inhibitor may release high？glucose？induced endothelial damage and reduce apoptosis pathway activation. miR ？ 92a maybe the potential treatment target of diabetes vascular complications.
出处
《中华糖尿病杂志》
CAS
CSCD
2016年第5期299-303,共5页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
国家自然科学基金项目（81471050、81270917）
