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目的观察整合素连接激酶(ILK)在单侧输尿管梗阻小鼠(UUO)肾脏中的表达,并探讨其在肾小管上皮细胞转化中的作用。方法将10周龄健康雄性 CD-1小鼠随机分为假手术组(C,n=20)和 UUO 组(n=40),分别于术后1、3、7、14 d 处死小鼠。采用 Masson 染色观察肾间质纤维化程度;用免疫组织化学(SP 法)的方法观察 ILK、E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)的表达;Western blot 半定量分析梗阻侧肾组织 ILK 的蛋白水平;用即时荧光逆转录聚合酶链反应(RT-PCR)检测 ILK、E-cadherin 和α-SMA mRNA 的表达。结果免疫组织化学显示 ILK 在假手术组只有微量表达,主要位于肾小球脏层上皮细胞,UUO 术后1 d ILK 蛋白表达显著增多(t=16.5,P<0.01),7 d 达高峰。术后3 d 肾小管上皮细胞及肾间质α-SMA 阳性细胞数显著增多(t=21.0,P<0.01),肾小管上皮细胞表达 E-cadherin 明显减少(t=5.6,P<0.01)。根据免疫组织化学结果分析,术后1～7 d,ILK 蛋白表达与α-SMA 蛋白表达呈正相关(R=0.88,P<0.01),与 E-cadherin表达呈负相关(R=-0.87,P<0.01)。Western blot 结果显示 ILK 蛋白表达于术后1 d 明显增加,7 d达高峰,14 d 表达不再增加(P>0.05)。RT-PCR 结果显示术后1 d ILK mRNA 表达增多(t=141.6,P<0.01),E-cadherin mRNA 表达于术后3 d 显著减少(P<0.01),α-SMA mRNA 表达于术后3 d 开始增多(P<0.01)。结论 UUO 模型早期整合素连接激酶表达增高,可能通过介导肾小管上皮细胞-间充质转化而参与肾脏纤维化的发生发展。
Objective To investigate the expression of integrin - linked kinase (ILK) in the kidney of unilateral ureteral obstruction mice (UUO) and its role in renal tubular epithelial cell transformation. Methods Healthy male CD-1 mice of 10 weeks old were randomly divided into sham operation group (C, n = 20) and UUO group (n = 40). Mice were sacrificed at 1, 3, 7 and 14 days after operation. Masson staining was used to observe the degree of renal interstitial fibrosis. The expression of ILK, E-cadherin and α-smooth muscle actin (α-SMA) was observed by immunohistochemistry (SP method) The protein level of ILK in obstructed kidney was semi-quantitively analyzed by Western blotting. The expressions of ILK, E-cadherin and α-SMA mRNA were detected by real-time fluorescence reverse transcription polymerase chain reaction (RT- Results Immunohistochemistry showed that ILK was only slightly expressed in the sham-operated group, mainly located in the glomerular visceral epithelial cells. The expression of ILK protein was significantly increased at 1 day after UUO (t = 16.5, P <0.01), reaching the peak on the 7th day. The number of α-SMA positive cells in renal tubular epithelial cells and renal interstitium was significantly increased 3 days after operation (t = 21.0, P <0.01), while the expression of E-cadherin in renal tubular epithelial cells was significantly decreased (t = 5.6, P <0.01). According to the results of immunohistochemistry, the expression of ILK protein was positively correlated with the expression of α-SMA (R = 0.88, P <0.01), and negatively correlated with the expression of E-cadherin from 1 to 7 days after operation (R = -0.87, P <0.01). Western blot results showed that the expression of ILK protein increased significantly on the 1st day after operation, reaching its peak on the 7th day and no longer increasing on the 14th day (P> 0.05). The mRNA expression of E-cadherin decreased significantly on the 3rd postoperative day (P <0.01), and the expression of α-SMA mRNA increased on the 1st day after operation (P <0.01) d began to increase (P <0.01). Conclusion UUO model early integrin ligation kinase expression increased, may be involved in renal tubular epithelial cells - mesenchymal transition and development of renal fibrosis.