【作者】 张伟；

【导师】 张浩； 易斌；

【作者基本信息】 中南大学 ， 临床医学， 2014， 硕士

【摘要】 目的：通过观察维生素D受体激动剂活性维生素D(1,25-(OH)-2D3, VitD3)及其类似物埃罗骨化醇(BXL-628)对高糖诱导人肾小管上皮细胞(HK-2) RhoA活化的影响,研究其对HK-2细胞间充质转分化及细胞外基质的作用,并探讨其可能机制。方法：体外培养人肾小管上皮细胞HK-2,(1)使用高糖培基(30mmol/L)刺激HK-2细胞。使用Western Blot观察0min、30min、1h、2h、4h、8h、12h活性RhoA蛋白的表达情况；(2)分别使用Western Blot及RealTime PCR观察Oh、12h、24h、48h、72h时α-平滑肌肌动蛋白(α-SMA)和E钙粘素(E-cadherin)蛋白及mRNA的表达；(3)将HK-2细胞设正常对照组(葡萄糖5.6mmol/mL)高糖(葡萄糖30mmol/mL)组、VitD3组(高糖+VitD3,10-7mol/L)组、BXL-628组(高糖+BXL-628,10-5nmol/L),使用RealTime PCR、 Western Blot方法观察各组细胞48h α-SMA、E-cadherin蛋白及mRNA的表达；采用Elisa酶联免疫法及RealTime PCR方法检测各组细胞48h Ⅰ型胶原蛋白(Collagen-Ⅰ, col-Ⅰ)、纤维连接蛋白(Fibronectin,Fn)蛋白及nRNA的表达。(4)实验分组同第(3),分别使用Western Blot免疫印迹法及免疫荧光技术检测各组细胞2h活性RhoA蛋白的表达情况。结果：(1)高糖刺激下HK-2细胞活性RhoA蛋白表达随时间逐渐上调,30min时表达较0min即有增加(P>0.05),1h时表达增加即有统计学差异,2h达高峰(P<0.01)。(2)高糖刺激HK-2细胞12h以上a-SMA蛋白及mRNA表达可增高,12h点较0h比表达增加具有统计学意义(P<0.05),48h a-SMA蛋白及mRNA表达达最高(P<0.01)；高糖刺激下,E-cadherin表达随时间逐渐下调,12h表达下调较0h组有统计学差异(P<0.05),48h下调最明显(P<0.01)：(3)VitD3组、BXL-628组a-SMA蛋白及mRNA的表达较高糖组均明显下调(P<0.01),且蛋白水平两组较正常对照组无统计学差异(P>0.05),而mRNA水平较正常对照组下调(P<0.05)；VitD3组及BXL-628组E-cadherin蛋白及mRNA水平均较高糖组明显上调(P<0.05),蛋白水平两组较正常对照组无统计学差异(P>0.05),而mRNA水平两组较正常对照组均有上调(P<0.05); ELISA及RealTime PCR结果显示,VitD3组及BXL-628组col-Ⅰ、Fn蛋白及mRNA的表达较高糖组明显下调(P<0.01)。(4)对比正常对照组,高糖组活性RhoA蛋白表达明显上调(P<0.01),而VitD3组及BXL-628组活性RhoA蛋白的表达较高糖组明显下调(P<0.01),且两组较正常对照组RhoA蛋白的表达无统计学差异(P>0.05),免疫荧光定性结果同样提示,VitD3组及BXL-628组活性RhoA蛋白的表达较高糖组明显减弱。结论：维生素D受体激动剂VitD3及其类似物BXL-628可通过影响RhoA的活化,抑制高糖诱导HK-2细胞间充质转分化及细胞外基质表达的增加；提示维生素D受体激动剂可能通过影响RhoA/ROCK信号通路,延缓肾间质纤维化进程。更多还原

【Abstract】 Objectives:To investigate the role of Vitamin D Receptor Agonist1,25-(OH)2D3and its analogue BXL-628in high glucose-induced activation of RhoA and epithelial-mesenchymal transition in human renal proximal tubular cell.Methods:HK-2cells were cultured with low glucose DMEM/F12and passed to the second generation cells; then HK-2cells were stimulated by high glucose (30mmol/mL) for0min,30min,1h,2h,4h,8h, and12h to determin the activation of RhoA protein by Western Blot. Meanwhile, HK-2cells were stimulated by high glucose (30mmol/mL) for0h,12h,24h,48h, and72h to determin the expression of a-SMA, E-cadherin at both mRNA and protein level by Wetstern Blot and Real Time PCR. According to the results above, the cells were divided into four groups:Normal Control Group (glucose5.5mM/L); High glucose Group (glucose30mM/L); VitD3group(High glucose+1,25-(OH)2D3); BXL-628Group (High glucose+BXL-628); The48h-treated cells were harvested to determine the expression of a-SMA, E-cadherin at mRNA and protein level by Western Blot analysis and Real-Time PCR.,the expression of COL-I, and Fn were determined by ELISA and Real-Time PCR.The2h-treated cells were harvested to determine the expression of active RhoA protein by Western Blot and immunofluorescence.Results:Active RhoA protein was increased by high glucose treatment, compared with0min the expression showed an significantly increase at lh (P<0.01), and came to a heat at2h (P<0.01). And the expression of a-SMA was significantly up regulated at24h by high glucose treatment and48h treatment showed the utmost increase (P<0.01), while the expression of E-cadherin was decreased by high glucose treatment at both protein and mRNA level. Western Blot analysis and Real-Time PCR results showed1,25-(OH)2D3and BXL-628had significantly decreased the expression of a-SMA, COL I and Fibronectin at both protein and mRNA level(P<0.01), meanwhile increased E-cadherin expression compared with high glucose group(P<0.01).Both Western Blot and immunofluorescence results showed Both1,25-(OH)2D3and BXL-628treatment decreased the expression of active RhoA protein at2h compared with high glucose group.Conclusion:High glucose could induce the activation of RhoA/ROCK pathway and mediate epithelial-mesenchymal transition in human renal proximal tubular cell. Vitamin D receptor agonist1,25-(OH)2D3and its analogue BXL-628could attenuate high glucose-induced epithelial-mesenchymal transition in human renal proximal tubular cell by suppressing RhoA/ROCK signaling pathway.更多还原