miR-328对高糖诱导人脐静脉内皮细胞间质转化调控作用的研究

摘要

Objective To investigate the regulation of miR-328 on endothelial-mesenchymal transition (EndMT) induced by high glucose in human umbilical vein endothelial cell (HUVECs) . Methods The recombinant lentiviruses of miR-328 and antagomiR-328 were prepared . HUVECs were cultured and divided into seven groups :normal glucose control ,mannitol ,high glucose ,miR-328 positive , miR-328 negative ,high glucose + antagomiR-328 ,and high glucose + antagomiR-328 negative group . Double immunofluorescent staining was used to determine expression of EndMT markers .The miR-328 expression was examined by RT-PCR .The expressions of typeⅠ /Ⅲ collagen were tested by western blot . Results Positive staining for CD31 and α-SMA in HUVECs were found in high glucose group . The expression of miR-328 was up-regulated [2.800 ± 0.175 )vs 1 ,P< 0.05 ] in high glucose group than in normal glucose group .The expression of type Ⅰ /Ⅲ collagen were increased in high glucose group[(0.414 ± 0.014) vs 0.501 ± 0.014)] and miR-328 positive group [(0.403 ± 0.016) vs (0.474 ± 0.016)] than in control group[(0.175 ± 0.025) vs (0.185 ± 0.014) ,P<0.05] .The expression of typeⅠ /Ⅲ collagen was lower in antagomiR-328 group [(0.227 ± 0.012) vs (0.222 ± 0.015)] than in high glucose group (P<0.05) . Conclusion High glucose induced EndMT in HUVECs ,increased the expression of miR-328.Overexpression of miR-328 induced EndMT in HUVECs ,while down-regulation of miR-328 by antagomiR-328 suppressed EndMT .%目的 探讨miR-328对高糖诱导人脐静脉内皮细胞(HUVECs)间质转化的调控作用.方法 构建miR-328和antagomiR-328慢病毒,培养HUVECs分为正常对照组(NC)、甘露醇对照组(M T)、高浓度葡萄糖组(HG),miR-328、miR-328阴性组、高浓度葡萄糖+antagomiR-328组、高浓度葡萄糖+antagomiR-328阴性组共7组.免疫荧光鉴定HUVECs间质转化;qRT-PCR检测miR-328;Western blot检测Ⅰ 、Ⅲ型胶原蛋白.结果 高糖处理HUVECs呈CD31、α-SMA双阳性;HG组较NC组miR-328表达增高[(2.800±0.175)vs 1,P<0.05];HG组及miR-328组Ⅰ 、Ⅲ型胶原蛋白较NC组表达增加[(0.414±0.014)v s(0.403±0.016)v s(0.175±0.025),(0.501±0.014)v s(0.474±0.016)v s(0.185±0.014),P<0.05];antagomiR-328组Ⅰ 、Ⅲ型胶原蛋白较HG组表达减少[(0.227±0.012)vs(0.414±0.014),(0.222±0.015)vs(0.501±0.014),P<0.05].结论 高糖诱导HUVECs间质转化,miR-328表达增加;过表达miR-328诱导HUVECs间质转化,并具有相应分泌功能,而过表达antagomiR-328可抑制这一现象.