转化生长因子β1诱导人肾小管上皮细胞转分化:Notch1受体阻断剂的影响

Transforming growth factor beta 1 induces the transdifferentiation of human renal tubular epithelial cells: the influence of Notch1 receptor specific inhibitor

背景:研究发现,在病理状态下各种细胞可通过转化生长因子β1的介导,促进肾小管上皮细胞-间充质细胞转分化为肌成纤维细胞,进而加速肾小管间质纤维化的进展。目的:验证Notch1受体特异性阻断剂γ-分泌酶抑制剂DAPT能否有效阻断、部分逆转或者完全逆转转化生长因子β1诱导的人肾小管上皮细胞转分化。方法:以体外培养的人近端肾小管上皮细胞株HK-2细胞为观察对象,建立转化生长因子β1诱导的肾小管上皮细胞-间充质细胞转分化体外模型,实验分为空白对照组、10μg/L转化生长因子β1组、转化生长因子β1(10μg/L)+γ-分泌酶抑制剂DAPT(5μmol/L)组、转化生长因子β1(10μg/L)+γ-分泌酶抑制剂DAPT(5μmol/L)部分延迟加入组、转化生长因子β1(10μg/L)+γ-分泌酶抑制剂DAPT(5μmol/L)延迟加入组。分别于12,24,48,72 h,在倒置相差显微镜下观察细胞形态变化;用免疫组织化学法和RT-PCR法检测α-平滑肌肌动蛋白、E-钙粘连素蛋白和mR NA表达变化。结果与结论:1在干预后在12,24,48,72 h,与空白对照组相比较,转化生长因子β1组α-平滑肌肌动蛋白蛋白和mR NA表达均明显增加(P<0.05),E-钙粘连素蛋白和mR NA表达逐渐减少(P<0.05)。2转化生长因子β1+γ-分泌酶抑制剂DAPT组α-平滑肌肌动蛋白的蛋白和mR NA、E-钙粘连素蛋白和mR NA的表达各时间段均接近空白对照组(P>0.05)。3γ-分泌酶抑制剂DAPT部分延迟加入组,α-平滑肌肌动蛋白的蛋白和mR NA的表达在12 h增加(P<0.05),之后其表达逐渐减少(P<0.05),E-钙粘连素蛋白表达在24 h开始减少(P<0.05),之后逐渐增加,E-钙粘连素mR NA各时间段都与空白对照组相接近,72 h时α-平滑肌肌动蛋白与E-钙粘连素的蛋白及mR NA表达均与空白对照组无显著差异(P>0.05)。4与空白对照组相比较,延迟加入组各时间点的α-平滑肌肌动蛋白的蛋白表达逐渐增加(P<0.05),E-钙粘连素蛋白的表达逐渐减少(P<0.05),但72 h时E-钙粘连素蛋白表达接近空白对照组。结果表明Notch1受体阻断剂γ-分泌酶抑制剂DAPT能够阻断、部分逆转转化生长因子β1所诱导的肾小管上皮细胞-间充质细胞转分化,但不能完全逆转其所诱导的肾小管上皮细胞-间充质细胞转分化。

BACKGROUND： In the pathological state, a variety of cells can be involved in the tubular epithelial-mesenchyma transition into myofibroblasts mediated by transforming growth factor-β1 （TGF-β1）, thereby accelerating the progress of renal tubular interstitial fibrosis. OBJECTIVE： To explore whether the y-secretase inhibitor DAPT, specific inhibitor of Notch1 receptor can effectively block, completely or partially reverse the tubular epithelial-mesenchymal transition induced by TGF-β1. METHODS： Normal human kidney epithelial cell lines （HK-2） cultured in vitro were used to establish in vitro model of tubular epithelial-mesenchymal transition, and then divided into blank control group, 10 μg/l_ TGF-β1 group, 10 μg/L TGF-β1＋5 pmol/L DAPT inhibited group, 10 μg/L TGF-β1＋5 μmol/L DAPT partially delayed group, 10 μg/L TGF-β1＋ 5μmol/l_ DAPT delayed group. After 12, 24, 48 and 72 hours, the HK-2 morphologic changes were observed by an inverted phase contrast microscope; the expressions of a-smooth muscle actin and E-cadherin at mRNA and protein levels were examined respectively by immunohistochemistry and RT-PCR. RESULTS AND CONCLUSION： （1） Compared with the blank control group, the mRNA and protein expressions of a-smooth muscle actin and E-cadherin were respectively increased （P 〈 0.05） and reduced （P 〈 0.05） significantly at 12, 24, 48 and 72 hours after intervention. （2） There was no difference in the mRNA and protein expression of a-smooth muscle actin and E-cadherin between the blank control group and TGF-β1＋DAPT inhibited group （P 〉 0.05）. （3） In the TGF-β1＋DAPT partially delayed group, the mRNA and protein expressions of a-smooth muscle actin were increased at 12 hours （P 〈 0.05）, and then gradually decreased （P 〈 0.05）; the expression of E-cadherin protein began to decrease at 24 hours （P 〈 0.05）, and then increased gradually; the mRNA expression of E-cadherin was similar in the TGF-β1＋DAPT partially delayed group and blank control group at different time points after intervention; the mRNA and protein expressions of a-smooth muscle actin and E-cadherin showed no difference from the blank control group at 72 hours after intervention （P 〉 0.05）. （4） Compared with the blank control group, the expressions of a-smooth muscle actin and E-cadherin were respectively increased （P 〈 0.05） and reduced （P 〈 0.05） significantly after intervention in the TGF-β1＋DAPT delayed group, but there was no difference in the expression of E-cadherin at 72 hours after intervention between the two groups. These findings indicate that DAPT can partially but not completely block and reverse the tubular epithelial-mesenchymal transition by TGF-β1.