高糖刺激对人晶状体上皮细胞中KLF6基因表达的影响

摘要

背景 上皮-间充质转化(EMT)是后发性白内障的主要病理机制.锌指蛋白Kruppel样因子6( KLF6)在糖尿病肾病近曲小管中的表达增加,参与并促进转化生长因子-β(TGF-β)诱导的EMT.目的 观察高浓度葡萄糖刺激对人晶状体上皮细胞(LECs)中KLF6及其调控的靶基因转化生长因子β1(TGFB1)、转化生长因子βⅠ型受体(TGFBR1)、Ⅰ型胶原α1链(COLIAl)、热休克蛋白47(HSP47)等表达的影响.方法 用高浓度D-葡萄糖刺激人LECs系SRA01/04,运用荧光定量聚合酶链反应(PCR)和Western blot法检测KLF6mRNA及蛋白水平,同时运用荧光定量PCR检测KLF6下游靶基因TGFBI、TGFBR1、COLlA1、HSP47的mRNA 表达水平.结果 与对照组(5.5 mmol/L)比较,高浓度葡萄糖(22.2、44.4、66.6 mmol/L)刺激后的t-KLF6和wt-KLF6 mRNA水平明显上升,差异均有统计学意义(F=72.53、42.02,P＜0.01)；结合人体血糖范围,在进一步实验中选择22.2 mmol/L作为葡萄糖刺激浓度.各时间点t-KLF6和wt-KLF6 mRNA水平之间的差异有统计学意义(F=100.12、125.52,P＜0.01),两者mRNA水平均于处理后12h达到高峰,24 h则开始出现下降,48 h下降更为明显.Western blot结果显示,对照组(0 h)KLF6蛋白表达很弱,高糖处理24 h后的细胞表达KLF6蛋白增加,而处理48 h后的表达量进一步增加.随着KLF6表达的增加,TGFB1、TGFBR1、COLlA1、HSP47的mRNA水平在高糖刺激12h时明显上升,但24 h后开始下降,48 h时进一步下降至接近对照组水平(F=6.73、162.35、64.39、12.05,P＜0.05). 结论 高浓度葡萄糖可诱导人LECs上调KLF6基因的表达,并短暂促进了其下游与EMT密切相关的靶基因TGFB1、TGFBR1、COLlA1、HSP47等的转录表达.%Background Epithelial-mesenchymal transition (EMT) is a major contributor to the pathogenesis of posterior capsular opacification(PCO).Kruppel-like factor 6 (KLF6) is a zinc finger protein,which can be stimulated by high glucose in proximal tubule cells and involved in transforming growth factor beta (TGF-β)induced EMT of diabetic nephropathy. Objective This study was designed to investigate the effect of high glucose on the expression of KLF6 and its target genes( TGFB1,TGFBR1,COLIA1,HSP47) in human lens epithelial cells (LECs). MethodsHuman LECs(SRA01/04) were cultured and exposed to different concentration of glucose.The expressions of KLF6 mRNA and protein were analyzed by real time polymerase chain reaction( real time PCR) and western blot after treatment with high glucose.The expressions of KLF6 target genes were analyzed by real time PCR to evaluate the EMT of SRA01/04 cells. Results Compared with the control group(5.5 mmol/L),the relative mRNA levels of t-KLF6 and wt-KLF6 in SRA01/04 treated with high glucose(22.2,44.4,66.6 mmol/L) increased obviously (F =72.53,42.02,P＜0.01 ).Then,the concentration of 22.2 mmol/L was used in the next experiments.The relative mRNA levels of t-KLF6 and wt-KLF6 increased to the peaks after treatment with high glucose for 12 h,and began to decrease after 24 h until lower levels after 48 h ( F =100.12,125.52,P ＜ 0.01 ).Western blot showed that the expression of KLF6 protein was also upregulated by high glucose treatment.With the promotion of the expression of KLF6 gene,the relative mRNA levels of TGFB1,TGFBR1,COLlAl and HSP47 of treated cells also respectively increased after treatment for 12 h,and began to decrease after 24 h until nearly at the levels of the control groups after 48 h( F=6.73,162.35,64.39,12.05,P＜0.05 ). Conclusions It was concluded that high glucose induced the expression of KLF6 in human LECs,and KLF6 transiently stimulated the expression of target genes TGFB1,TGFBRl,COLlAl and HSP47 which were mainly involved in the mechanism of EMT.