转染维A酸受体α在肾小管上皮细胞缺氧损伤后转分化中的作用

Effect of overexpression of retinoic acid receptor α on epithelial - to - mesenchymal transition induced by hypoxia in renal tubular epithelial cells

目的探讨转染维A酸受体α（RARα）在肾小管上皮细胞（NRK-52E细胞）缺氧损伤后转分化中的作用。方法构建过表达RARa慢病毒载体和阴性病毒载体，将NRK-52E细胞分为4组：正常对照组、缺氧模型组、转染组、阴性病毒对照组。转染组、阴性病毒对照组基因干扰72 h后，用2 mg/L嘌呤霉素筛选，再进行缺氧处理。正常对照组不做任何处理。缺氧48 h后采用光镜观察细胞形态变化，采用实时荧光定量PCR法和Western blot法检测NRK-52E细胞的RARα、E-钙黏蛋白（E-cadherin）、α-平滑肌肌动蛋白（α-SMA）的mRNA和蛋白表达。结果1．光镜下缺氧模型组与阴性病毒对照组NRK-52E细胞均出现萎缩，呈长梭型，类似成纤维细胞形态改变；而转染组NRK-52E细胞萎缩不明显，细胞呈多方形，保留上皮细胞形态。2.与正常对照组比较，缺氧模型组NRK-52E细胞RARα、E-cadherin的mRNA和蛋白表达均显著降低（mRNA：0.58±0.12比1.00±0.00，0.11±0.00比1.00±0.00，t＝－0.63、767.30，均P〈0.05；蛋白：0.63±0.12比1.62±0.16，0.44±0.22比1.27±0.08，t＝8.61、6.19，均P〈0.05），而α-SMA的mRNA和蛋白表达均显著升高（3.47±0.83比1.00±0.00，1.39±0.16比0.64±0.10，t＝－5.01、－6.91，均P〈0.05）。3.缺氧干预后，转染组NRK-52E细胞RARα和E-cadherin的mRNA和蛋白表达均较缺氧模型组（mRNA：4.69±1.34比0.58±0.12，0.23±0.00比0.11±0.00，q＝9.13、25.48，均P〈0.05；蛋白：1.39±0.19比0.63±0.12，0.87±0.09比0.44±0.22，q＝7.92、4.30，均P〈0.05）和阴性病毒对照组显著升高（mRNA：4.69±1.34比0.55±0.21，0.23±0.00比0.12±0.01，q＝9.20、23.35，均P〈0.05；蛋白：1.39±0.19比0.65±0.18，0.87±0.09比0.39±0.21，q＝7.71、4.80，均P〈0.05），而α-SMA的mRNA和蛋白表达均较缺氧模型组（1.52±0.34比3.47±0.83，0.82±0.13比1.39±0.10，q＝4.88、7.50，均P〈0.05）和阴性病毒对照组显著降低（1.52±0.34比4.05±0.81，0.82±0.13比1.17±0.10，q＝6.33、4.61，均P〈0.05）。与缺氧模型组比较，阴性病毒对照组NRK-52E细胞RARα、E-cadherin、α-SMA的mRNA和蛋白表达差异均无统计学意义（均P〉0.05）。 结论转染RARα可以减轻肾小管上皮细胞缺氧损伤后的转分化。

Objective To explore the effect of overexpression of retinoic acid receptor α（RARv） on epithelial - to -mesenchymal transition （EMT） induced by hypoxia in renal tubular epithelial cells（ NRK -52E）. Methods The RARer lentivirus vector and negative control lentivirus vector were synthetised. The NRK - 52E cells were divided into 4 groups：the normal control group, the hypoxia model group, the transfection group and the negative control group. Puromycin（2 mg/L） was added in transfection group and negative control group for screening after gene interference for 72 h. Then the 2 groups were subjected to hypoxia/reoxygenation, but the normal control group had no treatment. The change of cellular morphology was observed by using light microscope;the mRNA and protein expressions of RARα, E- cadherin,α -smooth muscle actin（ α- SMA） in NRK- 52E ceils were detected by adopting reverse transcription - polymerase chain reaction （ RT - PCR） and Western blot after hypoxia for 48 h. Results （ 1 ） Light microscope revealed that cells in both hypoxia model group and negative control group cells became atrophic and elongated, which were consistent with the morphology of myofibroblasts. But cells in transfection group cells were cubic, forming an epithelial monolayer. （2） Compared with the normal control group, the mRNA and protein expressions of RARα and E - cadherin in hypoxia model group were dramatically reduced （mRNA：0.58 ± 0. 12 vs. 1.00 ± 0.00,0.11 ± 0.00 vs. 1. 00 ± 0. 00, t = - 0. 63 ,767. 30 , all P 〈 0.05 ; protein ：0.63 ± 0.12 vs. 1.62 ± 0. 16,0.44 ± 0. 22 vs. 1.27 ± 0.08, t = 8.61,6.19 ,aU P 〈 0.05 ） , but the mRNA and protein expressions of α- SMA were higher（ 3.47 ± 0. 83 vs. 1. 00 ± 0. 00,1.39 ±0.16 vs. 0. 64 ±0.10,t = -5.01, -6.91 ,all P 〈0.05）. （3）The mRNA and protein expressions of RARcL and E -cadherin in the transfection group were significantly increased, compared with hypoxia model group （mRNA： 4.69 ±1.34 vs.0. 58 ±0.12,0.23 ±0.00 vs.0. 11 ±0.00,q=9.13,25.48,all P 〈0.05;protein： 1.39 ±0. 19 vs. 0. 63 ± 0.12,0.87 ± 0.09 vs. 0. 44 ± 0.22, q = 7.92,4.30, all P 〈 0.05 ） and negative control group （ mRNA ：4.69 ± 1.34 vs.0.55 ±0.21,0.23 ±0.00 vs.0. 12 ±0. 01,q =9.20,23.35,all P 〈0.05 ;protein： 1.39 ±0.19 vs. 0.65 ±0. 18,0.87 ±0.09 vs. 0. 39 ±0.21 ,q = 7.71,4.80, all P 〈 0.05 ）. Conversely, the mRNA and protein levels of ct - SMA were obviously lower in transfection group（ 1.52 ± 0.34 vs. 3.47 ± 0.83,4.05 ± 0.81,0.82 ± 0.13 vs. 1.39 ± 0. 10,1.17 ±0.10, q = 4.88,6.33,7.50,4.61, all P 〈 0.05 ）. The difference in mRNA and protein expressions of RAR Retinoic acid receptor α ; Renal tubular epithelial cells ; Epithelial - to - mesenchymal transition ; Transfection; Hypoxia, E -cadherin, Retinoic acid receptor α ; Renal tubular epithelial cells ; Epithelial - to - mesenchymal transition ; Transfection; Hypoxia-SMA between the hypoxia group and the negative control group had no statistical significance （all P 〉 0.05）. Conclusion Overexpression of RAR Retinoic acid receptor α ; Renal tubular epithelial cells ; Epithelial - to - mesenchymal transition ; Transfection; Hypoxia could alleviate EMT of renal tubular epithelial cells induced by hypoxia.