Wnt信号诱导人晶状体上皮细胞上皮间充质转化和增殖的研究

发布时间：2018-06-19 15:32

本文选题：Wnt3a + 晶状体上皮细胞　；参考：《天津医科大学》2012年博士论文

【摘要】：目的：后囊下混浊(posterior capsular opacification, PCO)是白内障摘除术后最常见的并发症,常常导致术后视力下降。晶状体上皮细胞上皮间充质转化(epithelial-mesenchymal transition, EMT)和增殖在PCO的发生和发展中起重要作用。Wnt配体蛋白参与调控细胞增殖、迁移及分化等众多生理过程,对人晶状体上皮细胞中Wnt3a介导的经典wnt信号通路目前研究甚少。本研究将wnt3a与经典wnt信号通路相关联,通过构建wnt3a过表达载体应用转染技术使人晶状体上皮细胞中过表达wnt3a配体蛋白,激活经典wnt信号通路,探讨其对晶状体上皮细胞EMT和增殖能力的影响,初步明确wnt在晶状体上皮细胞发生PCO的作用机制。同时,力求从中找到新的靶点。方法： 1.构建Wnt3a过表达载体,转化大肠杆菌,酶切、测序进行鉴定。 2.应用Lipo2000转染技术转染Wnt3a过表达载体,人晶状体上皮细胞HLE B-3细胞和SRA01/04细胞过表达Wnt3a蛋白,用Western Blotting技术验证其表达。 3.用趋化运动实验和划痕实验检测Wnt3a过表达对晶状体上皮细胞的迁移运动能力影响。 4.采用MTT和流式细胞仪检测Wnt3a过表达对细胞增殖能力的影响。 5.用Western Blotting检测Wnt3a过表达后其下游信号分子β-catenin的活化程度。用免疫荧光的方法检测β-catenin在细胞中的定位情况,观察在Wnt3a 刺激后,它们的分布变化,进一步明确Wnt/β-catenin信号通路的激活状态。 6.应用Western Blotting方法检测Wnt3a过表达对晶状体上皮细胞EMT标志蛋白和相关核内靶基因表达的影响,用免疫荧光的方法检测上皮和间充质标记蛋白在细胞中的定位情况,进一步明确Wnt/β-catenin信号通路在调控晶状体上皮细胞形成PCO中的分子机制。结果： 1.成功构建Wnt3a-pcDNA3真核表达载体,基因测序与Genebank一致。 2.正常人晶状体上皮细胞系仅有少量Wnt3a表达。Wnt3a-pcDNA3瞬时转染后获得Wnt3a过表达晶状体上皮细胞系。 3. wnt3a过表达上调HLE B-3细胞内β-catenin的蛋白表达,并促使HLE B-3细胞和SRA01/04细胞内的β-catenin细胞定位由胞浆转入胞核。 4.与对照组相比,Wnt3a过表达的HLE B-3细胞和SRA01/04细胞的迁移运动能力比对照组细胞明显增强(P0.01)。 5. Wnt3a过表达促进HLE B-3细胞和SRA01/04细胞的增殖(P0.01),细胞周期分析发现在S期细胞的比例升高。 6.与对照组相比,Wnt3a过表达的HLE B-3细胞和SRA01/04细胞的上皮表型E-cadherin表达降低,间质表型fibronectin表达升高；并且经免疫荧光实验证实,Wnt3a过表达的HLE B-3细胞和SRA01/04细胞发生EMT。 7. Wnt3a过表达使HLE-B3细胞和SRA01/04细胞中Wnt/β-catenin信号通路靶蛋白Cyclin D1和c-Myc表达上调。结论： 1. Wnt3a-pcDNA3真核表达载体能有效上调人晶状体上皮细胞Wnt3a蛋白的表达,使Wnt/β-catenin信号通路活化。 2. Wnt3a过表达能够增强晶状体上皮细胞的运动迁移能力。 3. Cyclin D1和c-Myc是Wnt/p-catenin信号通路的下游信号分子,Wnt3a通过激活Wnt/β-catenin信号通路,下游靶蛋白Cyclin D1和c-Myc表达上调,促进晶状体上皮细胞增殖。 4. Wnt3a过表达诱导晶状体上皮细胞发生上皮间充质转化。
[Abstract]:Purpose :

The posterior capsular opacity ( PCO ) is the most common complication after cataract extraction , which often leads to the decrease of visual acuity . The epithelial - mesenchymal transition ( EMT ) and proliferation play an important role in the pathogenesis and development of PCO . Wnt ligand protein plays an important role in regulating the proliferation , migration and differentiation of human lens epithelial cells .

Method :

1 . Construction of Wnt3a overexpression vector , transformation of E . coli , enzyme digestion and sequencing .

2 . Wnt3a overexpression vector , human lens epithelial cell HLE B - 3 cells and SRA01 / 04 cells were transfected with Lipo2000 transfection technique . Wnt3a protein was overexpressed in human lens epithelial cells . Western Blotting technique was used to verify the expression of Wnt3a protein .

3 . The effect of Wnt3a overexpression on the migration and movement of lens epithelial cells was investigated by means of chemoattractant and scratch test .

4 . MTT and flow cytometry were used to detect the effect of Wnt3a overexpression on cell proliferation .

5 . Western Blotting was used to detect the activation history of 尾 - catenin in downstream signaling molecule after overexpression of Wnt3a .

The localization of 尾 - catenin in cells was measured by immunofluorescence and Wnt3a was observed .

After stimulation , their distribution changes further clarify the activation status of Wnt / 尾 - catenin signaling pathways .

6 . Western Blotting was used to detect the influence of Wnt3a overexpression on the expression of EMT marker protein and related nuclear target genes in lens epithelial cells . The localization of epithelial and mesenchymal marker proteins in the cells was detected by immunofluorescence , and the molecular mechanism of Wnt / 尾 - catenin signaling pathway in regulating the formation of PCO in lens epithelial cells was further clarified .

Results :

1 . Wnt3a - pcDNA3 eukaryotic expression vector was constructed successfully , and the gene sequencing was consistent with Genebank .

2 . Only a small amount of Wnt3a was expressed in normal human lens epithelial cells . Wnt3a - pcDNA3 was transiently transfected to obtain Wnt3a overexpression lens epithelial cell line .

3 . The overexpression of wnt3a up - regulates the expression of 尾 - catenin in HLE B - 3 cells and promotes the localization of 尾 - catenin in HLE B - 3 cells and SRA01 / 04 cells from cytoplasm to nucleus .

4 . Compared with the control group , the migration ability of Wnt3a overexpression HLE B - 3 cells and SRA01 / 04 cells was significantly higher than that in the control group ( P0.01 ) .

5 . Wnt3a overexpression promotes proliferation of HLE B - 3 cells and SRA01 / 04 cells ( P0.01 ) . Cell cycle analysis shows that the proportion of cells in S phase is increased .

6 . In comparison with the control group , the expression of E - cadherin in the epithelial phenotype of Wnt3a overexpression HLE B - 3 cells and SRA01 / 04 cells decreased , and the expression of fibronectin was increased .
It was confirmed by immunofluorescence that HLE B - 3 cells and SRA01 / 04 cells that were overexpressed in Wnt3a were EMT .

7 . Wnt / 尾 - catenin signaling pathway target protein Cyclin D1 and c - Myc in HLE - B3 cells and SRA01 / 04 cells were up - regulated by Wnt3a overexpression .

Conclusion :

1.Wnt3a - pcDNA3 eukaryotic expression vector can effectively regulate the expression of Wnt3a protein in human lens epithelial cells and activate Wnt / 尾 - catenin signaling pathway .

2 . Wnt3a overexpression can enhance the movement and migration of lens epithelial cells .

3 . Cyclin D1 and c - Myc are the downstream signaling molecules of Wnt / p - catenin signaling pathway . Wnt3a is up - regulated by activating Wnt / 尾 - catenin signaling pathway , downstream target protein Cyclin D1 and c - Myc , and promotes proliferation of lens epithelial cells .

4 . Wnt3a overexpression induces epithelial mesenchymal transition in lens epithelial cells .
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R776.1

【共引文献】