转化生长因子-β刺激人近端肾小管上皮细胞结缔组织生长因子的表达  

Expression of connective tissue growth factor induced by transforming growth factor-beta in human renal epithelial cells

作　　者：陈楠[1] 赵青[1] 王伟铭[1] 卢健[2] 

机构地区：[1]上海第二医科大学附属瑞金医院肾脏科,上海200025 [2]上海第二医科大学生化教研室

出　　处：《肾脏病与透析肾移植杂志》2005年第2期117-121,共5页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：国家自然科学基金(NO:30270613);上海市卫生局医学领先专业(NO:983009);上海市教委发展基金(NO:02BK12);上海市科委重点学科

摘　　要：目的:探讨转化生长因子-β(TGF-β)对人近端肾小管上皮细胞结缔组织生长因子(CTGF)表达的影响。方法:应用逆转录-聚合酶链反应(RTP-CR)和蛋白印迹技术,观察TGF-β1对人近端肾小管上皮细胞(HK-2)α平滑肌肌动蛋白(α-SMA)和CTGFmRNA及其蛋白质表达的影响,同时观察肾小管上皮细胞形态改变。结果:HK2细胞有基础量的CTGFmRNA和蛋白质分子表达,在TGF-β1刺激下,其表达量显著增加,经不同浓度TGF-β1干预24h后,CTGFmRNA表达量明显升高,且呈剂量依赖性。1ng/mlTGF-β1刺激时,CTGFmRNA表达量开始显著增加,5ng/ml时达到高峰,10ng/ml时略有下降,CTGFmRNA表达量分别为对照组的2.40倍,3.34倍和2.73倍(P<0.05)。CTGFmRNA表达量呈时间依赖效应。TGF-β1(5ng/ml)干预30min后,CTGFmRNA表达量开始呈现增加趋势,为对照组的1.48倍(P>0.05),2h后出现显著差异,为对照组的2.14倍(P<0.05);CTGFmRNA表达量在24h达到高峰,持续至36h,分别为对照组的3.19倍和2.75倍(P<0.005);0.5ng/mlTGF-β1刺激时,CTGF蛋白表达量略有增加,5ng/ml时达到高峰,10ng/ml时略有下降。在TGF-β1(5ng/ml)干预下,HK2细胞由立方形铺路石样逐渐转变为长梭形长条样,免疫荧光检测见梭形细胞胞浆中有大量αSMA表达。大部分细胞转分化的时间(72h)明显晚于CTGFmRNA?Objective: To investigate the effect of transforming growth factor-β(TGF-β) on th e expression of connective tissue growth factor (CTGF) in human renal proximal t ubular epithelial cell line IK-2. Methodology:The mRNA and protein expression of CTGF in HK-2 cells was measured by rever se transcriptase-polymerase chain reaction (RT-PCR) and western blot, resp ectively. The effect of TGF-β 1 on the phenotypic transformation of HK-2 cel ls was observed by light microscopy, and the α-smooth muscle actin (α-SMA) e xpression in HK-2 cells was detected by immunofluorescence. Results:A basal level of CTGF mRNA and protein expression was detected in HK-2 cells. T he mRNA expression of CTGF in HK-2 cells was begun to increase by stimulati ng with 1 ng/ml TGF-β 1 (increased 2.4 times) and reached the peak at the co ncentration of 5 ng/ml (increased 3.34 times).The TGF-β 1 enhanced their expr ession in a time-dependent manner, with the maximal response at a concentration of 5 ng/ml and the best stimulating time course at 12~24 hour. At 24 hour, the mRNA expression of CTGF was increased by 3.19 times. In untreated cells, they s howed a typical cubical cobblestone phenotype and had negligible α-SMA, wherea s a more elongated fibroblastic appearance and α-SMA staining was noticeably i ncreased in the cells treated with TGF-β 1. However, the time course of HK-2 cells phenotypic transformation (72 hours after treatment with 5 ng/ml TGF-β 1 ) was much latter than that of CTGF mRNA and protein expression beginning to increase and reaching its peak (30 minutes and 24 hours after treatment with 5 ng/ml TGF-β 1, respectively). Conclusion:These results demonstrate that TGF-βdirectly induces CTGF expression in cultur ed human proximal tubular epithelial cells without myofibroblastic transdifferen tiation.

关 键 词：人近端肾小管上皮细胞 结缔组织生长因子 转化生长因子-β(TGF-β) mRNA表达量 TGF-β1 逆转录一聚合酶链反应 α-平滑肌肌动蛋白 HK-2细胞 CTGF Α-SMA表达 蛋白质表达 蛋白印迹技术 免疫荧光检测 剂量依赖性 细胞转分化 

分 类 号：R692[医药卫生—泌尿科学] R737.31[医药卫生—外科学]