JAK2／STAT3信号通路在IL-6介导肾小管上皮细胞转分化中的作用

目的：研究Janus激酶（JAK2）和信号转导因子和转录活化因子（STAT3）途径的激活在IL-6介导人近端肾小管上皮细胞转分化中的作用。方法：（1）细胞培养及分组：待生长状况良好的HK-2细胞株60%-80%细胞贴壁后,无血清培养基同步24 h,然后根据分组给予相应的刺激。（2）指标检测：采用荧光定量PCR技术检测0 h、24 h、48 h7、2 h不同时间点α-SMA mRNA、E-cadherin mRNA的表达。采用Western blot方法检测25 ng/ml浓度的IL-6刺激24 h、48 h、72 h后α-SMA、E-cadherin蛋白的表达;检测0、5、10、25、50 ng/ml浓度的IL-6刺激30 min,以及IL-6（25 ng/ml）刺激0、15 min、30 min、60 min、120 min后P-STAT3蛋白的表达水平;检测AG490预处理细胞4 h,IL-6（25 ng/ml）分别刺激30 min后及48 h后P-STAT3、P-JAK2和α-SMA、E-cadherin蛋白的表达水平。结果：与刺激前比较,IL-6以时间依赖方式上调HK-2细胞α-SMA mRNA、蛋白的表达,下调E-cadherin mRNA、蛋白的表达;IL-6以时间和剂量依赖方式上调HK-2细胞P-STAT3的表达,高峰发生在30 min;AG490部分抑制STAT3、JAK2的磷酸化,部分抑制IL-6诱导的α-SMA蛋白表达的上调、部分抑制IL-6诱导的E-cadherin蛋白表达的下调。结论：HK-2细胞中,JAK2/STAT3信号通路的激活可能参与了IL-6介导的肾小管上皮细胞转分化的发生。

Role of Jak2/Stat3 Pathway in IL-6-Induced Epithelial-Myofibroblast Transdifferentiation

Objective：To study the role of JAK2/STAT3 in IL - 6 stimulated epithelial - myofibroblast transdifferentiation in human proximal tubular epithelial cells. Methods： HK- 2 cells were cultured in RPMI 1640 medium containing 10% fetal bovine .serum for 24h in free .serum medium after 60 - 80% cells pasted on the wall. IL - 6 at designated concentration was added into the HK -2 cell for designated periods. The expression of α- SMA mRNA, E- cadherin mRNA at 0 h,24 h,48 h and 72 h was detected with real- time PCR assay. The expression of α- SMA and E - cadherin was assessed by Western blot after the induction of IL - 6 at 25 ng/ml concentrations for a period of 24 h,48 h,72 h. The expression of P - STAT3 was detected with Western blot after the induction of IL - 6 respectively at 25 ng/ml concentration for a period of 0, 15, 30, 60, 120 min and at the concentrations of 0, 5, 10, 25, 50 ng/ml for 30 min. The cells were pre-incubated with 100 pM AG490 for 4 h and cells and then stimulated with 25 ng/ml IL- 6 for 30 vain and 48 h respeetivdy, The expression of P- STAT3, P - JAK2, α- SMA and E- cadherin was repectively determined by Western blot. Results： IL - 6 induced the expression of α- SMA mRNA and down - regulated the expression of E- cadherin rnRNA in time - dependent manner. IL - 6 promoted the expression of α- SMA and reduced the expression of E - cadherin. IL - 6 rapidly induced the phosphorylation of STAT3 in a dose- and - time - dependent manner with a peak expression at 30 min. 100 tdVI AG490, a specific JAK2 inhibitor, partially inhibited STAT3, JAK2 activation and α- SMA production, meanwhile enhanced E- cadherin production. Conclusion： It is indicated that the JAK2/STAT3 pathway partially mediates IL - 6 - induced epithelial - myofibroblast transdifferentiation in HK - 2 cells. It is implicated that J K2/STAT3 signaling pathway might play a role in the development of tubulointerstitial fibrosis.