本文選題：TGF-β2 + Smad2　； 參考：《天津醫(yī)科大學(xué)》2014年博士論文

【摘要】：目的 后發(fā)性白內(nèi)障(posterior capsular opacification, PCO)作為白內(nèi)障術(shù)后主要并發(fā)癥,可導(dǎo)致患者視力再次下降。多種細胞因子參與了后發(fā)性白內(nèi)障的發(fā)生發(fā)展過程,其中TGF-β2的調(diào)節(jié)作用尤為重要,是重要的Smad通路介導(dǎo)發(fā)生的。本研究旨在深入研究選擇性過表達Smad2或Smad3對后發(fā)性白內(nèi)障的發(fā)生分別所起的作用,研究TGF-β2/Smad2和TGF-β2/Smad3信號傳導(dǎo)通路在晶狀體上皮細胞生長、凋亡、遷移、上皮間質(zhì)轉(zhuǎn)化及細胞外基質(zhì)的調(diào)節(jié)作用,從而達到探索對后發(fā)性白內(nèi)障基因治療的目的。 方法 以pcDNA3.1為質(zhì)粒載體,分別構(gòu)建Smad2和Smad3過表達載粒,轉(zhuǎn)化大腸桿菌,酶切、測序進行鑒定。體外培養(yǎng)人晶狀體上皮細胞HLE B-3,通過瞬時轉(zhuǎn)染方法分別過量表達Smad2或Smad3,并加入1ng/ml TGF-β2以選擇性激活TGF-β2/Smad2或TGF-β2/Smad3信號傳導(dǎo)通路。通過MTT方法和流式細胞儀技術(shù)檢測TGF-β2/Smad2和TGF-β2/Smad3信號傳導(dǎo)通路對HLE B-3的生長、凋亡的影響,通過Transwell小室檢測細胞的遷移能力。酶聯(lián)免疫吸附試驗檢測細胞培養(yǎng)液上清的可溶性細胞外基質(zhì)表達情況。Western blot, Real-time PCR和免疫熒光染色檢測細胞外基質(zhì)、上皮間質(zhì)轉(zhuǎn)化相關(guān)蛋白的表達。 結(jié)果 1.成功構(gòu)建Smad2、Smad3真核表達載體,進行酶切、測序分析,確定為所需序列。 2. Smad3過表達并激活TGF-β2/Smad3信號傳導(dǎo)通路可抑制HLE B-3細胞的生長,與對照組相比有明顯意義(P0.05)。選擇性激活TGF-β2/Smad3信號通路使細胞的凋亡率較激活TGF-β2/Smad2信號通路組明顯增加(P0.05)。酶聯(lián)免疫吸附試驗發(fā)現(xiàn)Smad3過表達組細胞培養(yǎng)液上清表達可溶性纖連蛋白,一型膠原含量較對照組及Smad2過表達明顯增加(P0.05)。Western Blot及Real-time PCR檢測細胞內(nèi)纖連蛋白,一型膠原、Lumican表達較對照組及Smad2過表達有不同程度的增加(P0.05)。 3. Smad2過表達并激活TGF-β2/Smad2信號傳導(dǎo)通路可見遷移的細胞數(shù)量較pcDNA3對照組增加,并促進上皮間質(zhì)轉(zhuǎn)化的發(fā)生,Western Blot及Real-timePCR發(fā)現(xiàn)上皮細胞標記物E-cadherin表達明顯低于對照組,Western Blot、 Real-time PCR及免疫熒光染色發(fā)現(xiàn)間質(zhì)細胞標記物a-SMA的表達明顯高于Smad3過表達及pcDNA3對照組(P0.05)。 結(jié)論 Smad2和Smad3在PCO發(fā)生過程中共同起著作用,但對細胞的生物學(xué)影響不盡相同。Smad2在介導(dǎo)EMT發(fā)生及細胞遷移能力增加起著重要的作用,Smad3參與誘導(dǎo)細胞凋亡和細胞外基質(zhì)的積聚。
[Abstract]:Objective posterior cataract (posterior capsular opacification, PCO), as a major complication after cataract surgery, can lead to another decline in visual acuity. A variety of cytokines are involved in the development of post-cataract, and the regulation of TGF- 尾 _ 2 is especially important, which is mediated by Smad pathway. The purpose of this study was to investigate the effects of selective overexpression of Smad2 or Smad3 on the occurrence of post-cataract, and to investigate the growth, apoptosis and migration of TGF- 尾 _ 2 / Smad2 and TGF- 尾 _ 2 / Smad3 signaling pathways in lens epithelial cells. Epithelial interstitial transformation and the regulation of extracellular matrix, so as to explore the gene therapy of posterior cataract. Methods using pcDNA3.1 as plasmid vector, Smad2 and Smad3 were constructed respectively, transformed into Escherichia coli, digested and sequenced. Human lens epithelial cells HLE B-3 were overexpressed by transient transfection, and 1ng/ml TGF- 尾 2 was added to activate the signal transduction pathway of TGF- 尾 2 / Smad2 or TGF- 尾 2 / Smad3 selectively. The effects of TGF- 尾 2 / Smad2 and TGF- 尾 2 / Smad3 signal transduction pathway on the growth and apoptosis of HLE B-3 were detected by MTT assay and flow cytometry. Enzyme linked immunosorbent assay (Elisa) was used to detect the expression of soluble extracellular matrix in supernatant. Western blot, Real-time PCR and immunofluorescence staining were used to detect the expression of extracellular matrix and proteins associated with epithelial interstitial transformation. Result 1. The eukaryotic expression vector of Smad2 and Smad3 was successfully constructed, digested by enzyme and sequenced and identified as the desired sequence. 2. Overexpression of Smad3 and activation of TGF- 尾 _ 2 / Smad3 signaling pathway inhibited the growth of HLE B-3 cells, which had significant significance compared with the control group (P0.05). Selective activation of the TGF- 尾 _ 2 / Smad3 signaling pathway significantly increased the cell apoptosis rate compared with that of the TGF- 尾 _ 2 / Smad2 signaling pathway group (P0.05). The results of enzyme-linked immunosorbent assay (Elisa) showed that soluble fibronectin was expressed in the supernatant of Smad3 overexpression group, and the level of type 1 collagen was significantly higher than that of control group and Smad2 (P0.05). Western blot and Real-time PCR were used to detect intracellular fibronectin. The expression of Lumican was significantly higher than that of control group and Smad2 (P0.05). Over expression of Smad2 and activation of TGF- 尾 2 / Smad2 signal transduction pathway showed that the number of migrating cells was higher than that of pcDNA3 control group. The expression of E-cadherin was significantly lower than that of control group. The expression of a-SMA was significantly higher than that of Smad3 and pcDNA3 (P0.05). Conclusion Smad2 and Smad3 play an important role in the pathogenesis of PCO. However, Smad2 plays an important role in inducing apoptosis and extracellular matrix accumulation in EMT and cell migration.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R776.1

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本文編號：2107496