瘦素对人肾小管上皮细胞表型转化及纤维连接蛋白表达的影响

Effect of Leptin on the Transdifferentiation and FN Expressionof Human Renal Tubular Epithelial Cell Line

目的:通过观察瘦素(leptin)刺激对人肾小管上皮细胞(HK-2)表型转化和纤维连接蛋白(FN)表达的影响,探讨瘦素对HK-2细胞转分化的作用。方法:将体外培养的HK-2细胞分为对照组和不同浓度瘦素作用组。倒置显微镜下观察HK-2细胞形态学变化;实时荧光定量RT-PCR法检测HK-2细胞α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、E-钙黏蛋白(E-cadherin)和FN mRNA的表达水平;免疫细胞化学法检测HK-2细胞表达α-SMA的阳性细胞百分数;酶联免疫吸附法(ELISA)检测HK-2细胞培养液上清中FN的表达。结果:分别用50、100、200 ng/ml瘦素作用HK-2细胞48 h后,(1)HK-2细胞逐渐由椭圆形变成长梭形,类似肌成纤维细胞的形态;(2)RT-PCR结果表明,瘦素作用可以下调E-cadherin mRNA的表达,同时上调α-SMA、FN mRNA的表达。(3)免疫细胞化学结果表明,对照组HK-2细胞几乎不表达α-SMA,随着瘦素作用浓度的增加,α-SMA阳性的HK-2细胞百分数逐渐增多;(4)ELISA结果表明,对照组HK-2细胞有基础水平的FN分泌,各瘦素作用组上清液中FN的表达水平较对照组显著增加,且呈一定的剂量依赖关系。结论:瘦素可诱导肾小管上皮细胞转分化为肌成纤维细胞,分泌细胞外基质,从而可能参与肾间质纤维化的发生发展。

Objective ：To examine effect of leptin on transdifferentiation of human tubular epithelial cells （ HK - 2） and se- cretion of fibronectin（ FN）, and to analyze the role of leptin in renal interstitial fibrosis. Methods：Cultured HK -2 cells were divided into four groups： control group and leptin -treated groups （50, 100,200 ng/ml for 48h, respectively）. The cell morphological changes were traced with inverted microscope; The expression of α- smooth muscle actin （α- SMA） and E- cadherin at both transcrip- tional and protein levels were detected by real -time quantitative reverse transcription polymerase chain reaction（ RT- PCR） and immunohistochemical method; The expression of FN mRNA were detected by RT - PCR, while FN secreted into the culture supernatant was assessed by enzyme - linked immunosorbent assay （ELISA）. Results： （ 1 ） Leptin stimulated the oval - to - fusiform transdifferentiation in morphology of HK- 2 cells. （2）At transcriptional level, compared to the control group, after leptin（50,100,200 ng/ml） treatment for 48h,the expression of E - cadherin mRNA downregulated, while the expression of α - SMA and FN mRNA was upregulated. （3）At protein level, the percentage of α- SMA positive cells increased dramatically as the dose of leptin increased. （4） The expression of FN increased significantly in different leptin - treated groups. Conclusion： Leptin can promote the transdifferentiation of human renal tubular epithelial cells and play an important role in renal interstitial fibrosis.