發(fā)布時(shí)間：2018-06-19 15:32

  本文選題：Wnt3a + 晶狀體上皮細(xì)胞��； 參考：《天津醫(yī)科大學(xué)》2012年博士論文

【摘要】：目的： 后囊下混濁(posterior capsular opacification, PCO)是白內(nèi)障摘除術(shù)后最常見的并發(fā)癥,常常導(dǎo)致術(shù)后視力下降。晶狀體上皮細(xì)胞上皮間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)和增殖在PCO的發(fā)生和發(fā)展中起重要作用。Wnt配體蛋白參與調(diào)控細(xì)胞增殖、遷移及分化等眾多生理過程,對(duì)人晶狀體上皮細(xì)胞中Wnt3a介導(dǎo)的經(jīng)典wnt信號(hào)通路目前研究甚少。本研究將wnt3a與經(jīng)典wnt信號(hào)通路相關(guān)聯(lián),通過構(gòu)建wnt3a過表達(dá)載體應(yīng)用轉(zhuǎn)染技術(shù)使人晶狀體上皮細(xì)胞中過表達(dá)wnt3a配體蛋白,激活經(jīng)典wnt信號(hào)通路,探討其對(duì)晶狀體上皮細(xì)胞EMT和增殖能力的影響,初步明確wnt在晶狀體上皮細(xì)胞發(fā)生PCO的作用機(jī)制。同時(shí),力求從中找到新的靶點(diǎn)。 方法： 1.構(gòu)建Wnt3a過表達(dá)載體,轉(zhuǎn)化大腸桿菌,酶切、測(cè)序進(jìn)行鑒定。 2.應(yīng)用Lipo2000轉(zhuǎn)染技術(shù)轉(zhuǎn)染W(wǎng)nt3a過表達(dá)載體,人晶狀體上皮細(xì)胞HLE B-3細(xì)胞和SRA01/04細(xì)胞過表達(dá)Wnt3a蛋白,用Western Blotting技術(shù)驗(yàn)證其表達(dá)。 3.用趨化運(yùn)動(dòng)實(shí)驗(yàn)和劃痕實(shí)驗(yàn)檢測(cè)Wnt3a過表達(dá)對(duì)晶狀體上皮細(xì)胞的遷移運(yùn)動(dòng)能力影響。 4.采用MTT和流式細(xì)胞儀檢測(cè)Wnt3a過表達(dá)對(duì)細(xì)胞增殖能力的影響。 5.用Western Blotting檢測(cè)Wnt3a過表達(dá)后其下游信號(hào)分子β-catenin的活化程 度。用免疫熒光的方法檢測(cè)β-catenin在細(xì)胞中的定位情況,觀察在Wnt3a 刺激后,它們的分布變化,進(jìn)一步明確Wnt/β-catenin信號(hào)通路的激活狀態(tài)。 6.應(yīng)用Western Blotting方法檢測(cè)Wnt3a過表達(dá)對(duì)晶狀體上皮細(xì)胞EMT標(biāo)志蛋白和相關(guān)核內(nèi)靶基因表達(dá)的影響,用免疫熒光的方法檢測(cè)上皮和間充質(zhì)標(biāo)記蛋白在細(xì)胞中的定位情況,進(jìn)一步明確Wnt/β-catenin信號(hào)通路在調(diào)控晶狀體上皮細(xì)胞形成PCO中的分子機(jī)制。 結(jié)果： 1.成功構(gòu)建Wnt3a-pcDNA3真核表達(dá)載體,基因測(cè)序與Genebank一致。 2.正常人晶狀體上皮細(xì)胞系僅有少量Wnt3a表達(dá)。Wnt3a-pcDNA3瞬時(shí)轉(zhuǎn)染后獲得Wnt3a過表達(dá)晶狀體上皮細(xì)胞系。 3. wnt3a過表達(dá)上調(diào)HLE B-3細(xì)胞內(nèi)β-catenin的蛋白表達(dá),并促使HLE B-3細(xì)胞和SRA01/04細(xì)胞內(nèi)的β-catenin細(xì)胞定位由胞漿轉(zhuǎn)入胞核。 4.與對(duì)照組相比,Wnt3a過表達(dá)的HLE B-3細(xì)胞和SRA01/04細(xì)胞的遷移運(yùn)動(dòng)能力比對(duì)照組細(xì)胞明顯增強(qiáng)(P0.01)。 5. Wnt3a過表達(dá)促進(jìn)HLE B-3細(xì)胞和SRA01/04細(xì)胞的增殖(P0.01),細(xì)胞周期分析發(fā)現(xiàn)在S期細(xì)胞的比例升高。 6.與對(duì)照組相比,Wnt3a過表達(dá)的HLE B-3細(xì)胞和SRA01/04細(xì)胞的上皮表型E-cadherin表達(dá)降低,間質(zhì)表型fibronectin表達(dá)升高；并且經(jīng)免疫熒光實(shí)驗(yàn)證實(shí),Wnt3a過表達(dá)的HLE B-3細(xì)胞和SRA01/04細(xì)胞發(fā)生EMT。 7. Wnt3a過表達(dá)使HLE-B3細(xì)胞和SRA01/04細(xì)胞中Wnt/β-catenin信號(hào)通路靶蛋白Cyclin D1和c-Myc表達(dá)上調(diào)。 結(jié)論： 1. Wnt3a-pcDNA3真核表達(dá)載體能有效上調(diào)人晶狀體上皮細(xì)胞Wnt3a蛋白的表達(dá),使Wnt/β-catenin信號(hào)通路活化。 2. Wnt3a過表達(dá)能夠增強(qiáng)晶狀體上皮細(xì)胞的運(yùn)動(dòng)遷移能力。 3. Cyclin D1和c-Myc是Wnt/p-catenin信號(hào)通路的下游信號(hào)分子,Wnt3a通過激活Wnt/β-catenin信號(hào)通路,下游靶蛋白Cyclin D1和c-Myc表達(dá)上調(diào),促進(jìn)晶狀體上皮細(xì)胞增殖。 4. Wnt3a過表達(dá)誘導(dǎo)晶狀體上皮細(xì)胞發(fā)生上皮間充質(zhì)轉(zhuǎn)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 .
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R776.1

【共引文獻(xiàn)】

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本文編號(hào)：2040354