摘要
目的探讨信号传导与转录激活基因3（STAT3）对白细胞介素6（IL-6）诱导人腹膜间皮细胞（HPMC）上皮间质转分化（EMT）的作用。方法体外培养HPMC并分组：（1）50μg/LIL-6刺激HPMC不同时间，按刺激时间24、48、72h分组；（2）不同浓度IL-6刺激HPMC24h，按IL-6浓度50、100μg/L分组；（3）应用慢病毒介导的RNA干扰技术建立稳定沉默STAT3基因的HPMC，分成空白对照组、IL-6组、空载体组、空载体＋IL-6组、慢病毒感染组、慢病毒感染＋IL-6组，其中IL-6刺激均以50μg/L终浓度刺激24h。实时荧光定量PCR检测上皮钙黏素（E—cadherin）、仅平滑肌肌动蛋白（仅．SMA）及血管内皮生长因子（VEGF）mRNA的表达；Western印迹检测上述分子的蛋白表达及通路蛋白Janus激酶2（JAK2）、STAT3磷酸化水平；细胞免疫荧光观察E-cadherin、d—SMA的表达和分布。结果与对照组比较，不同IL-6浓度组和不同作用时间组细胞E-cadherin蛋白和mRNA表达均下降（均P〈0．05），α-SMA、VEGF的蛋白和mRNA表达均升高（均P〈0．05），通路蛋白磷酸化（P）-JAK2／JAK2比值和P-STAT3／STAT3比值均升高（均P〈0．05），且均呈剂量和时间依赖性。沉默STAT3基因后，与空载体＋IL-6组比较，慢病毒感染＋IL-6组α-SMA、VEGF蛋白表达均下降（均P〈0．05），E-cadherin蛋白表达上升（P〈0．05）。结论IL-6通过激活STAT3通路而刺激HPMC分泌VEGF并诱导EMT的发生。沉默STAT3基因可抑制人腹膜间皮细胞EMT的发生。
Objective To investigate the role of STAT3 transcription factor in IL-6 inducing epithelial mesenchymal transition （EMT） of human peritoneal mesothelial cells （HPMCs）. Methods HPMCs were cultured in vitro and grouped. （1） According to the stimulation time with 50 μg/L IL-6, HPMCs were divided into 24, 48, 72 h groups. （2） HPMCs were grouped 50, 100 μg/L according to IL-6 concentration. （3） HPMCs were respectively divided into control group, IL-6 group, empty vector group, empty vector＋IL-6 group, virus infecting group and virus infecting＋IL-6 group, as lenti-virus vector mediating RNA interference targeting STAT3 was applied. The mRNA expressions of E- cadherin, α- smooth muscle actin （α- SMA） and vascular endothelial growth factor （VEGF） were detected by real time PCR; their protein expressions and the phosphorylation of JAK2 and STAT3 were detected by Western blotting; the expressions and distribution of E-cadherin and α-SMA were observed by immunofluorescence. Results Compared with those in control group, the expression of E-cadherin decreased remarkably （P 〈 0.05）, while the expressions of VEGF and α-SMA and the ratio of phosphorylated （p）-JAK2/JAK2 and p-STAT3/STAT3 increased significantly in IL-6 concentration groups and stimulation time groups （all P 〈 0.05）, which had been dose and time dependent. Compared with empty vector＋IL-6 group, virus infecting＋IL-6 group had decreased expressions of VEGF and α-SMA, while increased expressions of E-cadherin （all P 〈 0.05）. Conclusions IL-6 can promote VEGF and α-SMA gene expression and prevent E-cadherin gene expression by STAT3, which involves in EMT of peritoneum fibrosis. While STAT3 gene is knocked-down, EMT is inhibited in HPMCs.
出处
《中华肾脏病杂志》
CSCD
北大核心
2017年第9期711-717,共7页
Chinese Journal of Nephrology
基金
国家自然科学基金（81400763）
