Notch信號(hào)通路在RPE細(xì)胞間質(zhì)轉(zhuǎn)化及PVR形成中作用機(jī)制的實(shí)驗(yàn)研究.pdf

1、 分類號(hào)：R772.21 密級(jí)：不保密 UDC UDC：617 學(xué)校代碼：11065 博 士 學(xué) 位 論 文 Notch Notch 信號(hào)通路在 信號(hào)通路在 RPE RPE 細(xì)胞間質(zhì)轉(zhuǎn)化及 細(xì)胞間質(zhì)轉(zhuǎn)化及 PVR PVR 形成中 形成中 作用機(jī)制的實(shí)驗(yàn)

2、研究 用機(jī)制的實(shí)驗(yàn)研究 張靜靜 張靜靜 指 導(dǎo) 教 師史偉云 教授 學(xué) 科 專 業(yè) 名 稱 眼科學(xué) 論 文 提 交 日 期 2014-04-16 論 文 答 辯 日 期 2014-05-17 答辯委員會(huì)主席 Abstract Purpose To observe the function of Notch signaling in the transforming growth fa

3、ctor (TGF)-β1-mediated epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE), and the role in the pathogenesis of proliferative vitreoretinopathy (PVR). Methods In vitro cultivated the human

4、 RPE cells (ARPE-19) were treated with 10ng/ml TGF-β1 for 24, 48, and 72 h. The cell morphology was observed by inverted microscopy. The expression levels of ZO-1, α-SMA, Notch1 intracellular domain (NICD) and Hes-1 were

5、 evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence staining and Western blot. MTT assay was used to examine the effect of γ-secretase inhibitor DAPT and LY411575 on the

6、proliferation of ARPE-19 cells. To examine the effect of γ-secretase inhibitor DAPT and LY411575 on TGF-β1- induced EMT, ARPE-19 cells were pre-incubated for 60 minutes with 1μM LY411575 or 20μM DAPT before TGF-β1 treatm

7、ent, and the cell morphology change was observed and expression of ZO-1, α-SMA, NICD, Hes-1 were evaluated. Mouse proliferative vitreo-retinopathy (PVR) model was used for in vivo study. Subconfluent ARPE-19 cells were i

8、njected intravitreously with or without the γ-secretase inhibitor LY411575 to examine the effects of Notch signaling on PVR formation. Results The general morphology of the human RPE cells (ARPE-19) demonstrates an organ

9、ized epithelial morphology. TGF-β1 resulted in epithelial-mesenchymal transition (EMT), which developed a fibroblast-like morphology with decreased expression of ZO-1 and increased expression of α-SMA and vimentin, as we

10、ll as the increased Hes-1 and NICD expression. The growth of ARPE-19 cells was not affected by the γ-secretase inhibitor 1μM LY411575 for 1-2 days or 20μM DAPT for 1 day. LY411575 and DAPT significantly attenuated TGF-β1

11、-induced EMT in ARPE-19 cells by decreasing the mesenchymal markers vimentin and a-SMA and inhibiting Notch signal pathway Hes-1 and NICD expression. In vivo, PVR was induced by vitreal injections of ARPE-19 in C57BL/6J

12、mice, while LY411575 inhibited PVR formation that induced by RPE proliferation and EMT. Conclusions Notch signaling pathway plays a critical role in TGF-β1-induced EMT in human RPE cells and PVR, which provides a novel i