Smad2与Smad3参与TGF-β诱导的后发性白内障发生的研究

发布时间：2018-07-08 12:53

本文选题：TGF-β2 + Smad2　；参考：《天津医科大学》2014年博士论文

【摘要】：目的后发性白内障(posterior capsular opacification, PCO)作为白内障术后主要并发症,可导致患者视力再次下降。多种细胞因子参与了后发性白内障的发生发展过程,其中TGF-β2的调节作用尤为重要,是重要的Smad通路介导发生的。本研究旨在深入研究选择性过表达Smad2或Smad3对后发性白内障的发生分别所起的作用,研究TGF-β2/Smad2和TGF-β2/Smad3信号传导通路在晶状体上皮细胞生长、凋亡、迁移、上皮间质转化及细胞外基质的调节作用,从而达到探索对后发性白内障基因治疗的目的。方法以pcDNA3.1为质粒载体,分别构建Smad2和Smad3过表达载粒,转化大肠杆菌,酶切、测序进行鉴定。体外培养人晶状体上皮细胞HLE B-3,通过瞬时转染方法分别过量表达Smad2或Smad3,并加入1ng/ml TGF-β2以选择性激活TGF-β2/Smad2或TGF-β2/Smad3信号传导通路。通过MTT方法和流式细胞仪技术检测TGF-β2/Smad2和TGF-β2/Smad3信号传导通路对HLE B-3的生长、凋亡的影响,通过Transwell小室检测细胞的迁移能力。酶联免疫吸附试验检测细胞培养液上清的可溶性细胞外基质表达情况。Western blot, Real-time PCR和免疫荧光染色检测细胞外基质、上皮间质转化相关蛋白的表达。结果 1.成功构建Smad2、Smad3真核表达载体,进行酶切、测序分析,确定为所需序列。 2. Smad3过表达并激活TGF-β2/Smad3信号传导通路可抑制HLE B-3细胞的生长,与对照组相比有明显意义(P0.05)。选择性激活TGF-β2/Smad3信号通路使细胞的凋亡率较激活TGF-β2/Smad2信号通路组明显增加(P0.05)。酶联免疫吸附试验发现Smad3过表达组细胞培养液上清表达可溶性纤连蛋白,一型胶原含量较对照组及Smad2过表达明显增加(P0.05)。Western Blot及Real-time PCR检测细胞内纤连蛋白,一型胶原、Lumican表达较对照组及Smad2过表达有不同程度的增加(P0.05)。 3. Smad2过表达并激活TGF-β2/Smad2信号传导通路可见迁移的细胞数量较pcDNA3对照组增加,并促进上皮间质转化的发生,Western Blot及Real-timePCR发现上皮细胞标记物E-cadherin表达明显低于对照组,Western Blot、 Real-time PCR及免疫荧光染色发现间质细胞标记物a-SMA的表达明显高于Smad3过表达及pcDNA3对照组(P0.05)。结论 Smad2和Smad3在PCO发生过程中共同起着作用,但对细胞的生物学影响不尽相同。Smad2在介导EMT发生及细胞迁移能力增加起着重要的作用,Smad3参与诱导细胞凋亡和细胞外基质的积聚。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R776.1

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