整合素连接激酶介导大鼠肾小球系膜细胞生物学行为的变化

Effect of Integrin-Linked Kinase in Biological Behavior Changes of Glomerular Mesangial Cells

目的探讨整合素连接激酶(ILK)对大鼠肾小球系膜细胞形态、黏附、迁移、增殖、分化、细胞外基质(ECM)组装等细胞生物学行为的影响,进一步揭示ILK在肾脏的病理、生理意义。方法体外分离、培养SD大鼠肾小球系膜细胞,应用基因转染技术,将携带小鼠ILKcDNA全长的腺病毒感染系膜细胞,同时设立空白对照组、空载体组。采用Westernblot方法检测ILK、纤维连接蛋白(FN)、平滑肌肌动蛋白(α-SMA)的蛋白质表达水平;应用细胞生物学方法观察细胞伸展、黏附率;四甲基偶氮唑蓝(MTT)法检测细胞增殖率。结果Westernblot显示ILK基因转染细胞组ILK、FN、α-SMA蛋白质表达水平均显著高于其他二组(Pa<0.01),而空白对照与空载体组比较无差异(P>0.05);空白对照、空载体、ILK基因转染细胞组ILK蛋白质半定量值分别为0.42±0.06、0.49±0.07和2.79±0.16;FN蛋白质半定量值分别0.46±0.07、0.50±0.07和2.53±0.19;α-SMA蛋白质半定量值分别0.52±0.09、0.58±0.07和2.61±0.18。空白对照、空载体、ILK基因转染细胞组细胞伸展率分别为:(69±4)%、(63±8)%、(80±3)%(Pa<0.01);黏附率为(42±4)%、(38±4)%、(75±5)%(P<0.01)。细胞增殖率也以ILK基因转染细胞组最高,与其他二组比较有显著差异(Pa<0.01),空白对照组、空载体组、ILK基因转染细胞的MTT值分别为0.60±0.07、0.58±0.06、1.16±0.10。结论高表达ILK的肾小球系膜细胞,其表型、黏附、迁移、增殖等细胞生物学行为发生改变,细胞对ECM的重塑能力增强。因此ILK可能在肾脏疾病的进展中起重要作用。

Objective To investigate the effects of integrin - linked kinase （ILK） on the cytobiology action of mesangial cell such as cellular morphology, adhesion, migration, proliferation, differentiation, and extracellular matrix（ECM） abnormal deposited,and to further probe into its mechanism of ILK in the pathological and physiological functions of kidney disease. Methods Used the gene transfer technique in SD rats mesangial cell culture, the recombinant mouse full - length ILK cDNA expression adenovirus vector was transfected into the cultured mesangial cell as the high - level - ILK group, at the same time, the carrier - blanked and the normal control groups as control. The protein levels were tested by Western blot for expression of ILK, fibronectin（ FN ）, smooth muscle actin protein（ α- SMA ） ; the ratio of the cell adhesion and stretch were observed by cytobiology;the cell proliferation were assessed by 3 - （4,5 - dimethyhhiazol -2 - yl） -2,5 - dipitenyl - tetrazolium bromide（MTT） colorimetric assay. Results By Western blot, the protein expression level of ILK, FN, α- SMA of high - level - ILK, were higher than these of the others（Pa 〈0.01 ）, but there were no difference between the control and carrier .- without; the protein semiquantity of II,K in groups control, carrier - withouted and high - level - ILK, respectively were 0.42± 0.06, 0.49 ± 0.07 and 2.79 ± 0.16 ; FN were 0. 46 ±0.07, 0.50 ±0.07, and 2.53 ±0.19;then ± SMA were 0.52 ±0.19, 0.58±0.07, 2.61 ±0. 18;the stretch ratio were （69 .±4 ） %, （63 ± 8 ） % , （80 ± 3 ） % （ Pa 〈 0.01 ） ; the adhere ratio were （42 ± 4 ） % , （38 ± 4 ） % , （75± 5 ） % （ Pa 〈 0.01 ）. The proliferative index of tI,K - high - expressed was the highest, and were higher than the others （ P 〈 0.01 ） ; the MTY index of the three were 0.60 ± 0.07, 0. 58 ±0.06, 1. 16 ±0. 1 0. Conclusions The mesangial cell of high ILK - expressed biological action changes in thcir phaenotype, adhesion, translocation, proliferation, the deposited of ECM. The study suggested that ILK may have important effect on the progression of renal disease.