氧化应激条件下miR-21对人眼小梁网细胞胞外基质蛋白表达的影响

发布时间：2018-04-30 20:37

  本文选题：miR- + 转化生长因子　； 参考：《眼科新进展》2017年01期

【摘要】：目的探讨氧化应激条件下miR-21对人眼小梁网细胞(human trabecular meshwork cells,HTMCs)胞外基质蛋白表达的影响。方法用不同浓度H_2O_2(0μmol·L~(-1)、200μmol·L~(-1)、400μmol·L~(-1)、600μmol·L~(-1))刺激HTMCs 1 h,MTT法检测其对HTMCs活力的影响,从而确定后续实验所需H_2O_2浓度。随后将细胞分成正常组和H_2O_2组,Real-time PCR检测miR-21的表达,Western blot检测胞外基质蛋白(纤维连接蛋白和胶原蛋白I)的表达。然后将细胞分成5组:H_2O_2组(只用H_2O_2处理)、miR-21干预组(H_2O_2+miR-21模拟物)、miR-21对照组(H_2O_2+miR-21对照模拟物)、miR-21抑制组(H_2O_2+miR-21抑制剂)和miR-21抑制对照组(H_2O_2+miR-21抑制剂对照物),Real-time PCR检测miR-21、转化生长因子(transforming growth factor,TGF)-β2和PTEN mRNA表达,Western blot检测胞外基质蛋白、TGF-β2和PTEN蛋白表达。最后将细胞分成3组:H_2O_2组(只用H_2O_2处理)、PTEN干扰组(H_2O_2+PTEN siRNA)和干扰对照组(H_2O_2+control siRNA),检测PTEN、胞外基质蛋白和TGF-β2蛋白水平表达。结果当H_2O_2浓度≥400μmol·L~(-1)时,可显著抑制HTMCs的活性,后续实验选择此浓度。与正常组相比,H_2O_2组中miR-21、纤维连接蛋白和胶原蛋白I的表达均增加,差异均有统计学意义(均为P0.05)。检测氧化应激条件下miR-21对胞外基质蛋白、TGF-β2和PTEN表达的影响,发现与H_2O_2组相比,miR-21对照组和miR-21抑制对照组中miR-21、纤维连接蛋白与胶原蛋白I蛋白水平表达以及TGF-β2和PTEN mRNA及蛋白水平表达差异均无统计学意义(均为P0.05);与miR-21对照组相比,miR-21干预组miR-21、纤维连接蛋白和胶原蛋白I蛋白水平及TGF-β2 mRNA和蛋白水平表达均增加,PTEN蛋白水平表达降低,差异均有统计学意义(均为P0.05),而PTEN mRNA表达差异均无统计学意义(均为P0.05);与miR-21抑制对照组相比,miR-21抑制组miR-21、纤维连接蛋白和胶原蛋白I蛋白水平及TGF-β2 mRNA和蛋白水平表达均降低,PTEN蛋白水平表达增加,差异均有统计学意义(均为P0.05),而PTEN mRNA水平差异无统计学意义(P0.05)。采用Western blot检测氧化应激条件下PTEN对TGF-β2和胞外基质蛋白表达的影响,发现与H_2O_2组相比,干扰对照组PTEN、TGF-β2、纤维连接蛋白和胶原蛋白I的表达差异均无统计学意义(均为P0.05);与干扰对照组相比,PTEN干扰组PTEN的表达下调,TGF-β2、纤维连接蛋白和胶原蛋白I的表达均升高,差异均有统计学意义(均为P0.05)。结论氧化应激条件下miR-21可增加HTMCs胞外基质产物,这可能与其靶向沉默PTEN基因,调节TGF-β2的表达相关。
[Abstract]:Objective to investigate the effect of miR-21 on the expression of extracellular matrix protein in human trabecular meshwork cells under oxidative stress. Methods the effects of different concentrations of H_2O_2(0 渭 mol L ~ (1) on the activity of HTMCs were determined by HTMCs 1 ~ (-1) stimulation with different concentrations of H_2O_2(0 渭 mol / L ~ (mol) mol / L ~ (1) ~ (-) ~ (400) 渭 mol / L ~ (+) ~ (-) Then the cells were divided into normal group and H_2O_2 group. The expression of miR-21 was detected by real-time PCR. The expression of extracellular matrix protein (fibronectin and collagen I) was detected by Western blot. Then, the cells were divided into 5 groups: h _ 2O _ 2 group (treated only with H_2O_2) and miR-21 control group (miR-21 control group) were divided into two groups: H2O2 miR-21 control group and miR-21 inhibitory control group. H2O2 miR-21 inhibitor control group was used to detect miR-21 by real-time PCR, and the control group with H2O2 miR-21 was used to detect miR-21, and the control group with H2O2 miR-21 was divided into two groups: H2O2 miR-21 inhibitor group and miR-21 inhibitory control group (miR-21 inhibitory control group). The miR-21 was detected by real-time PCR in H2O2 miR-21 control group and the control group was transformed into a control group. The expression of TGF- 尾 2 and PTEN mRNA was detected by Western blot. The expression of TGF- 尾 2 and PTEN protein was detected by Western blot. At last, the cells were divided into three groups: H_2O_2 group (treated with H_2O_2 alone) and the control group (H2O2 control siRNAs). The expression of PTEN, extracellular matrix protein (ECM) and TGF- 尾 2 protein were detected. Results when the concentration of H_2O_2 was 鈮，

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