蛋白酪氨酸激酶活性在人肾小管上皮细胞转分化中的作用

Effects of activity of PTKS on human renal tubular epithelial-myofibroblast transdifferentiation in vitro

目的 探讨蛋白酪氨酸激酶 (PTKs)活性在人肾小管上皮细胞 (HKC)转分化中的作用。方法 将传代的HKC细胞分为 :(1)无血清对照组 (C) ;(2 )酸性成纤维细胞生长因子 (aFGF)组 (A) ;(3) genistein组 (G) ,加入 80ng/ml重组人aFGF条件下 ,分别加入不同浓度的 genistein ;培养 72h。应用免疫组化检测HKC细胞表达α SMA(α smoothmuscleactin ,α SMA)的变化 ;对转分化过程中PTKs的活性用流式细胞仪加以分析。结果 aFGF(A)组加入 80ng/mlaFGF后a 平滑肌动蛋白 (a SMA)表达增强 ,细胞a SMA阳性率 (6 0 .6 0± 2 .70 ) %较对照组 (C) (3.35± 1.4 5 ) %有非常显著性差异 (P <0 .0 1)。当G4,5组介入genistein浓度 4 8、96 μg/ml时 ,细胞α SMA阳性率为 (3.90± 2 .0 9) % ,(3.98± 1.75 ) % ,较aFGF组 (A)明显降低 (P <0 .0 1)。PTKs的活性随加入aFGF的浓度增加而增高 ,当aFGF的浓度为 2 0、4 0、80ng/ml时 ,分别比对照组 (C)PTKs的活性增加 16 %、17%、2 4 .6 % ,当介入genistein剂量为 6、12、2 4、4 8μg/ml时 ,分别比aFGF组 (A)PTKs的活性下降 4 5 .6 %、5 3.5 %、6 6 %、81.9% ,干预因素作用下HKC细胞PTKs活性与genistein剂量呈负相关 (r =- 0 .987,P <0 .0 5 )。

Objective To investigate the possible roles of protein tyrosine kinases(PTKS) in transdifferentiation of cultured human renal tubular epithelial cell line(HKC).Methods The HKC cells were divided into three groups:(serum-free (negative contro1);(2)aFGF(positve control);(3)aFGF-inhibition(treated with aFGF+genistein).Then cells were cultured for 72h.The expression α-SMA (α-smooth muscle actin) in HKC was examined by immunohistochemistry method.Flow cytometry was adopted to measure the activity of PTKS.Results The percentage of a-SMA +HKC cells in the positive controls was significantly higher than that in negative controls[(60.60±2.70)%vs (3.35±1.45)%,P<0.01];while the percentage of α-SMA +HKC cells in aFGF +genistein group at genistein dose of 48,96 μg/ml(3.90±2.09)%,(3.98±1.75)% was dramatically lower than that in positive controls(P<0.01).In the course of epithelial cell transdifferent to myofibroblast,PTKs was activited in aFGF dose-dependent manner.This represented 16%,17%,246% increase in the activity of PTKs(aFGF 20,40,80ng/ml vs negative controls).On the contrary,inhibition of the activity of PTKs was achieved in aFGF+genistein group at genistein dose of 6,12,24,48μg/m1,(45.6%,53.5%,66%,81.9%decrease of PTKs of positive group respectively.)Conclusion The increase of the activity of PTKs may be related to the transdifferention of HKC cells.Genistein may be a good candidate with promising future in intervention progress of inters titial fibrosis.