Influence of inhibiting the expression of integerin-linked kinase on the expression of connexin 43 in rat mesangial cells
- VernacularTitle:抑制整合素连接激酶(ILK)表达对大鼠肾小球系膜细胞细胞间隙连接蛋白43表达的影响
- Author:
Qiang MA
;
Guangyan CAI
;
Xiangmei CHEN
- Publication Type:Journal Article
- Keywords:
integerins;
connexin43;
mesangial cells
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of the expression of integerin-linked kinase (ILK) on connexin 43 (Cx 43) in rat mesangial cells (RMCs). Methods RMCs were divided into three different groups (6 for each group): RMC group, ILK-con siRNA group and ILK-siRNA group. ILK siRNA was synthesized, and then transfected into RMCs by LipofectAMIN 2000. RMCs were transiently transfected with ILK-con siRNA, ILK-siRNA and lysed 24h later, and mRNA was then extracted and detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis using ILK and Cx43-specific primers, and an aliquot of protein from each sample was subjected to western blot analysis using ILK and Cx43 antibodies. Cells were seeded into 96-well plates (2?103 cells/well) and then transfected with ILK-con siRNA and ILK-siRNA. After incubation for 24, 48 and 72 hours, respectively, 20?l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5mg/ml) was added into each well and incubated for 4 hours. Subsequently, 150?l of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and the absorption at 492nm was measured. Results ILK mRNA and protein levels decreased by 30%-50% after being transiently transfected with ILK-siRNA, while Cx 43 mRNA and protein levels increased by 30%-40% and 60%-70%, respectively. The viabilities in ILK-siRNA infected RMCs at 24, 48 and 72 hours were significantly higher than that in ILK-con siRNA infected RMCs. Conclusion Inhibition of ILK pathway will up-regulate the expression of Cx 43 and the viability of RMCs, implying that the regulation of connexin 43 might possibly be achieved via ILK pathway.
