缓激肽对转化生长因子-β1(TGF-β1)诱导视网膜色素上皮细胞发生上皮间充质转化的影响  

Effects of bradykinin on TGF-β1-induced epithelialmesenchymal transformation in ARPE-19 cells

作　　者：梁秀玮 何梦梅 蔡雯婷[2] 张瑞玲[2] 金惠子[2] 范佳淇 于靖[1] LIANG Xiu-Wei;HE Meng-Mei;CAI Wen-Ting;ZHANG Rui-Ling;JIN Hui-Zi;FAN Jia-Qi;YU Jing(Nanchang University(LIANG Xiu-Wei,YU Jing),Nanchang 330031,Jiangxi Province,China;Department of Ophthalmology,the Tenth People’s Hospital,Tongji University,Shanghai 200072,China)

机构地区：[1]南昌大学,江西省南昌市330031 [2]同济大学附属上海市第十人民医院眼科,上海市200072

出　　处：《眼科新进展》2018年第11期1005-1009,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金面上项目(编号:81470648)~~

摘　　要：目的制作增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的上皮间充质转化(epithelial-mesenchymal transformation,EMT)细胞模型,研究缓激肽(bradykinin,BK)对视网膜色素上皮(retinal pigment epithelium,RPE)细胞发生EMT的影响,并探讨BK对PVR的影响机制。方法体外培养人RPE细胞株ARPE-19细胞,采用不同浓度转化生长因子-β1(transforming growth factor-β1,TGF-β1)分别作用于ARPE-19细胞24 h、48 h,于倒置显微镜下观察细胞形态变化;采用CCK-8检测细胞增殖情况,确定TGF-β1浓度及作用时间;利用Western blot和细胞免疫荧光检测EMT标志蛋白E-钙黏素、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和波形蛋白(Viminten)表达情况;细胞划痕实验、Transwell实验检测细胞迁移能力。同时采用Western blot检测TGF/Smad通路下游pSmad3和Smad7的表达。结果TGF-β1刺激ARPE-19细胞后可以成功诱导EMT体外细胞模型。当TGF-β1浓度为10μg·L^(-1)、作用时间为48 h时,细胞增殖最明显。BK在TGF-β1诱导的EMT中可以降低α-SMA、Viminten的表达,升高E-钙黏素的表达并且降低细胞迁移能力。这些影响能被BK-2受体拮抗剂HOE-140逆转。TGF-β1诱导ARPE-19细胞发生EMT时,pSmad3表达量升高;TGF-β1刺激前给予BK刺激,pSmad3表达量减少;加入BK前预敷HOE-140,然后给予TGF-β1刺激,BK作用减弱,pSmad3表达量升高。Smad7表达趋势与pSmad3表达趋势相反。结论10μg·L-1 TGF-β1可导致ARPE49细胞发生EMT。BK通过TGF-/Smad信号通路上调Smad7的表达、下调pSmad3的表达,从而逆转TGF-β1诱导的EMT。提示BK可能是一种新的、有效的治疗PVR的方法。Objective To establish the epithelial-mesenchymal transition(EMT)in vitro model of proliferative vitreoretinopathy(PVR),and to study the effects of bradykinin(BK)on EMT of retinal pigmental epithelium(RPE)and the relevant mechanism in PVR for providing a new target on treating PVR clinically.Methods Human PRE cell line ARPE-19 was treated with different concentrations of transforming growth factor-β1(TGF-β1)at 24 h and 48 h,respectively,and the cell morphology was observed.CCK-8 methods was applied to detect the cell proliferation and to determine the prominent concentration and application time of TGF-β1 on APRE-19 cells.The proteins expression of EMT marker such as E-cadherin,α-SMA and Viminten was determined through Western blot and cellular immunofluorescence.The cell migration was detected by cell wounding heal and transwell assays.Western blot was used to detect the phosphorylation levels of Smad 3 and Smad 7,which were downstream signals of TGF-β1/Smad signaling pathway.Results The in vitro ARPE-19 cell model was successfully established with stimulation of 10μg·L-1 of TGF-β1.BK could decrease the EMT markersα-SMA and Viminten protein levels while up-regulated the E-cadherin protein expression.Besides,BK also inhibited the cells migration ability.Conversely,the BK-2 receptor antagonist H0E-140 could reverse the BK-induced biological effects on ARPE-19 cells.Furthermore,BK suppressed p-Smad 3 and promoted p-Smad 7 expressions via TGF-β1/Smad pathway to reverse TGF-β1-mediated EMT.Conclusion TGF-β1 with 10μg·L-1 can facilitate ARPE-19 cells’EMT process while BK treatment acts as negatively in TGF-β1-induced EMT.BK also regulates the TGF-β1/Smad signaling pathway with upregulation of Smad 7 expression and downregulation of Smad 3 expression,thus circumventing the TGF-β1-induced EMT.This study suggests that BK can be a new therapeutic method for PVR.

关 键 词：缓激肽 转化生长因子-Β1 视网膜色素上皮细胞 上皮间充质转化 

分 类 号：R774[医药卫生—眼科]