[1]梁秀玮,何梦梅,蔡雯婷,等.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[J].眼科新进展,2018,38(11):1005-1009.[doi:10.13389/j.cnki.rao.2018.0237]
　LIANG Xiu-Wei,HE Meng-Mei,CAI Wen-Ting,et al.Effects of bradykinin on TGF-β1-induced epithelial-mesenchymal transformation in ARPE-19 cells[J].Recent Advances in Ophthalmology,2018,38(11):1005-1009.[doi:10.13389/j.cnki.rao.2018.0237]
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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/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
38卷
期数:
2018年11期
页码:
1005-1009
栏目:
实验研究
出版日期:
2018-11-05

文章信息/Info

Title:
Effects of bradykinin on TGF-β1-induced epithelial-mesenchymal transformation in ARPE-19 cells
作者:
梁秀玮; 何梦梅; 蔡雯婷; 张瑞玲; 金惠子; 范佳淇; 于靖
330031　江西省南昌市，南昌大学(梁秀玮，于靖）；200072　上海市，同济大学附属上海市第十人民医院眼科（何梦梅，蔡雯婷，张瑞玲，金惠子，范佳淇）
Author(s):
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(HE Meng-Mei，CAI Wen-Ting，ZHANG Rui-Ling，JIN Hui-Zi，FAN Jia-Qi)，Shanghai 200072，China
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
Keywords:
Bradykinin; TGF-β1; Retinal pigmental epithelium; epithelial-mesenchymal transformation
分类号:
R774
DOI:
10.13389/j.cnki.rao.2018.0237
文献标志码:
A
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10 μg·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　10 μg·L-1 TGF-β1可导致ARPE-19细胞发生EMT。BK通过TGF-/Smad信号通路上调Smad 7的表达、下调pSmad 3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。
Abstract:
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 10 μg·L-1 of TGF-β1.BK could decrease the EMT markers α-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 10 μg·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.

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备注/Memo

备注/Memo:
国家自然科学基金面上项目（编号：81470648）
更新日期/Last Update: 2018-10-30