本文選題：miR- + 轉(zhuǎn)化生長因子��； 參考：《眼科新進(jìn)展》2017年01期

【摘要】：目的探討氧化應(yīng)激條件下miR-21對人眼小梁網(wǎng)細(xì)胞(human trabecular meshwork cells,HTMCs)胞外基質(zhì)蛋白表達(dá)的影響。方法用不同濃度H_2O_2(0μmol·L~(-1)、200μmol·L~(-1)、400μmol·L~(-1)、600μmol·L~(-1))刺激HTMCs 1 h,MTT法檢測其對HTMCs活力的影響,從而確定后續(xù)實(shí)驗(yàn)所需H_2O_2濃度。隨后將細(xì)胞分成正常組和H_2O_2組,Real-time PCR檢測miR-21的表達(dá),Western blot檢測胞外基質(zhì)蛋白(纖維連接蛋白和膠原蛋白I)的表達(dá)。然后將細(xì)胞分成5組:H_2O_2組(只用H_2O_2處理)、miR-21干預(yù)組(H_2O_2+miR-21模擬物)、miR-21對照組(H_2O_2+miR-21對照模擬物)、miR-21抑制組(H_2O_2+miR-21抑制劑)和miR-21抑制對照組(H_2O_2+miR-21抑制劑對照物),Real-time PCR檢測miR-21、轉(zhuǎn)化生長因子(transforming growth factor,TGF)-β2和PTEN mRNA表達(dá),Western blot檢測胞外基質(zhì)蛋白、TGF-β2和PTEN蛋白表達(dá)。最后將細(xì)胞分成3組:H_2O_2組(只用H_2O_2處理)、PTEN干擾組(H_2O_2+PTEN siRNA)和干擾對照組(H_2O_2+control siRNA),檢測PTEN、胞外基質(zhì)蛋白和TGF-β2蛋白水平表達(dá)。結(jié)果當(dāng)H_2O_2濃度≥400μmol·L~(-1)時,可顯著抑制HTMCs的活性,后續(xù)實(shí)驗(yàn)選擇此濃度。與正常組相比,H_2O_2組中miR-21、纖維連接蛋白和膠原蛋白I的表達(dá)均增加,差異均有統(tǒng)計學(xué)意義(均為P0.05)。檢測氧化應(yīng)激條件下miR-21對胞外基質(zhì)蛋白、TGF-β2和PTEN表達(dá)的影響,發(fā)現(xiàn)與H_2O_2組相比,miR-21對照組和miR-21抑制對照組中miR-21、纖維連接蛋白與膠原蛋白I蛋白水平表達(dá)以及TGF-β2和PTEN mRNA及蛋白水平表達(dá)差異均無統(tǒng)計學(xué)意義(均為P0.05);與miR-21對照組相比,miR-21干預(yù)組miR-21、纖維連接蛋白和膠原蛋白I蛋白水平及TGF-β2 mRNA和蛋白水平表達(dá)均增加,PTEN蛋白水平表達(dá)降低,差異均有統(tǒng)計學(xué)意義(均為P0.05),而PTEN mRNA表達(dá)差異均無統(tǒng)計學(xué)意義(均為P0.05);與miR-21抑制對照組相比,miR-21抑制組miR-21、纖維連接蛋白和膠原蛋白I蛋白水平及TGF-β2 mRNA和蛋白水平表達(dá)均降低,PTEN蛋白水平表達(dá)增加,差異均有統(tǒng)計學(xué)意義(均為P0.05),而PTEN mRNA水平差異無統(tǒng)計學(xué)意義(P0.05)。采用Western blot檢測氧化應(yīng)激條件下PTEN對TGF-β2和胞外基質(zhì)蛋白表達(dá)的影響,發(fā)現(xiàn)與H_2O_2組相比,干擾對照組PTEN、TGF-β2、纖維連接蛋白和膠原蛋白I的表達(dá)差異均無統(tǒng)計學(xué)意義(均為P0.05);與干擾對照組相比,PTEN干擾組PTEN的表達(dá)下調(diào),TGF-β2、纖維連接蛋白和膠原蛋白I的表達(dá)均升高,差異均有統(tǒng)計學(xué)意義(均為P0.05)。結(jié)論氧化應(yīng)激條件下miR-21可增加HTMCs胞外基質(zhì)產(chǎn)物,這可能與其靶向沉默PTEN基因,調(diào)節(jié)TGF-β2的表達(dá)相關(guān)。
[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，

本文編號：1826128