肾小管上皮细胞转分化中整合素连接激酶表达及其与衰老的相关研究

The expression of integrin-linked kinase(ILK) in the transdifferentiation of tubular epithelial cell and its correlation with senescence

目的探讨整合素连接激酶(integrin-linked kinase,ILK)在肾小管上皮细胞转分化过程中的作用及其对衰老进程中细胞外基质(extracellular matrix,ECM)积聚增加的影响。方法分离培养3、24月龄 Wistar 雄性大鼠的原代肾小管上皮细胞,分别给予0、5、10、20～40μg/L 不同浓度的转化生长因子β(transforming growth factor β,TGF-β)刺激24h 后,用免疫细胞化学或免疫荧光方法检测细胞α-SMA、ILK、F-actin 表达;Western-blot 法检测两组细胞中纤维连接蛋白和 ILK 的表达水平;体外转录获得大鼠 ILK-c RNA 探针,检测两组细胞经刺激后 ILK mRNA 的变化。结果在0浓度 TGF-β刺激下,肾小管上皮细胞中 ILK 和 F-actin 表达水平24月龄组均比3月龄组高,但两组细胞α-SMA 表达水平均很低,且没有差别;随 TGF-β刺激浓度的增加,ILK、F-actin 和α-SMA 表达水平显著上调,其中 F-actin 与 ILK 表达增多相一致;FN 和 ILK 蛋白表达水平24月龄组分别为0.438±0.024和0.826±0.051,明显高于3月龄组的0.246±0.018和0.673±0.035(P<0.05)。结论随着肾小管上皮细胞转分化程度不断增高,ILK 表达增多,并可能通过调节 F-actin 的重排介导了转分化的过程;随增龄肾小管上皮细胞中 ILK 和 FN 表达增多,且 F-actin 表达随之增多,提示衰老肾脏的肾小管上皮细胞在应激状态下更易于发生转分化。

Objective To investigate the expression and function of integrin-linked kinase （ILK） in the transdifferentiation process of primary tubular epithelial cell（ TEC） and its effect on the accumulation of extracellular matrix （ECM） in the aging process. Methods Primary TEC was cultured from kidneys of male Wistar rats aged 3 and 24 months. Then the primary TEC was stimulated by TGF-β at the concentrations of 0, 5, 10 and 20-40μg/L for 24 h. The TEC expressions of a-SMA, ILK and F-actin were detected by immunocytochemistry or indirect immunofluorescence. FN or ILK protein expression levels were tested by Western-blot. DIG labelled cRNA probe of rat ILK in situ hybridization was obtained by transcription in vitro, with which ILK expression level and the location of TEC were detected. Results a SMA expression levels were very low in young or aging rat TEC on the base situation, no significant difference was found between the two groups. However, ILK and F-actin levels in 24 months group were higher than in 3 months group. Along with increasing concentration of TGF-β, a SMA, ILK and F-actin levels were increased. The expression of ILK was associated with F-actin expression. FN and ILK expression levels were significantly lower in 3 monthsrats TEC than in 24 monthsrats, P〈0.05. Conclusions （1） Along with the increased degree of transdifferentiation of primary TEC, the expression level of ILK is up-regulated, which mediates the process of transdifferntiation through regulating the rearrangement of F-actin. （2） ILK and F-actin expression levels increase along with the aging. ILK and FN expressions are up-regulated along with the aging. These suggest that the TEC of aging kidney is prone to transdifferentiation in the state of stress.