ILK在白蛋白诱导人HK-2细胞转分化中的表达和意义

Expression and Significance of Integrin-linked Kinase in the Renal Tubular Epithelial to Mesenchymal Transition Induced by Albumin

目的探讨整合素连接激酶(ILK)在白蛋白诱导人肾小管上皮细胞转分化(TMET)过程中的表达及其意义。方法将体外培养的人近端肾小管上皮细胞株HK-2细胞随机分为4组:1)空白对照组。仅加入DMEM/F12培养基培养细胞。2)白蛋白诱导组。加入含人纯化白蛋白的培养液,白蛋白终浓度为5g·L-1(以下白蛋白终浓度为此浓度)。3)转化生长因子-β1(TGF-β1)中和抗体对照组。加入含TGF-β1中和抗体的培养液,TGF-β1中和抗体终浓度为5μg·L-1(以下TGF-β1中和抗体终浓度为此浓度)。4)TGF-β1拮抗组。在加入人纯化白蛋白15min前加入TGF-β1中和抗体。观察4组细胞形态的变化。采用逆转录-聚合酶链反应(RT-PCR)法检测TGF-β1、ILK、纤维连接蛋白(FN)、E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)mRNA表达水平。结果空白对照组0、12、24和48h时TGF-β1、ILK、E-cadherin、α-SMA和FN mRNA表达水平与TGF-β1中和抗体对照组比较差异均无统计学意义(均P>0.05)。白蛋白诱导组、TGF-β1拮抗组12、24和48h时TGF-β1、ILK、E-cadherin、α-SMA和FN mRNA表达水平均明显高于同组0h时、空白对照组和TGF-β1中和抗体对照组(均P<0.05);TGF-β1拮抗组12、24和48h时TGF-β1、ILK和FN mRNA表达水平均显著低于白蛋白诱导组(均P<0.01),24、48h时E-cadherin、α-SMA mRNA表达水平均显著低于白蛋白诱导组(均P<0.01)。相关性分析结果显示,ILK mRNA表达与TGF-β1、α-SMA和FN mRNA表达均呈正相关(r=0.944、0.974和0.989,均P<0.01)。结论白蛋白可以诱导HK-2细胞发生转分化,ILK在其中发挥促进作用。

Objective To investigate the expression and significance of integrin-linked kinase （ILK） in albumin-induced tubular epithelial-myofibroblast transdifferentiation（TMET）. Methods Cultured human proximal tubular epithelial cells（HK-2 cells） were randomly into four groups. Blank control group only received DMEM/F12 culture medium; Albumin induction group was treated with purified human albumin（5 g · L-1） ;TGF-β1 neutralizing antibody control group was treated with transforming growth factor beta-1（TGF-β1,5 μg · L-1） and neutralizing anti-TGF-β1 antibody（5 μg · L）;TGF-131 antagonist group was challenged with neutralizing anti-TGF-131antibody（5μg · L -1） 15 minutes before treatment with purified human albumin（5 g · L ）. Mor- phological changes in HK-2 cells were observed,and mRNA expression ofTGF-β1 ,ILK,fibronectin（FN）, E-cadherin and smooth muscle actin（a-SMA） was detected by RT-PCR. Results There were no significant differences in the expression of TGF-β1, ILK, E-cadherin,-SMA and FN mRNA between blank control group and TGF-β1 neutralizing antibody control group during the 0,12,24 and 48 hours of culture（P〈0.05）. Furthermore,the levels of TGF-β1,ILK,E-cad- herin,a-SMA and FN mRNA expression in albumin induction group and TGF-β1 antagonist group during 12,24 and 48 hours of culture were significantly higher than those in albumin induc- tion group at the beginning of culture,TGF-β1 antagonist group at the beginning of culture,blank control group,and TGF-β1 neutralizing antibody control group（P〈0.05）. Compared with albu- min induction groupTGF-β1 antagonist group decreased the expression of TGF-β1 ,ILK and FN mRNA during 12,24 and 48 hours of culture,and reduce the expression of E-cadherin and a-SMA mRNA at 24 and 48 hours in culture（P〈0.01）. Pearson correlation analysis showed that the ex- pression of ILK mRNA was positively correlated with the expression of TGF-β1,FN and a-SMA mRNA（r=0. 944,0. 974 and 0.989,respectively;P〈0.01）.Conelusion ILK plays a promoting role in albumin-induced HK-2 cell transdifferentiation.