本文選題：Furin + 胰腺癌��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：研究目的:本研究旨在探討前體蛋白加工酶Furin對(duì)人胰腺癌細(xì)胞生長(zhǎng)、轉(zhuǎn)移、侵襲能力等生物學(xué)行為的影響,并初步探索其對(duì)腫瘤細(xì)胞上皮間質(zhì)轉(zhuǎn)化的影響及可能存在的分子調(diào)節(jié)機(jī)制,為進(jìn)一步的臨床應(yīng)用研究完善理論基礎(chǔ)。研究方法:通過(guò)Realtime PCR、Western Blot技術(shù)檢測(cè)Furin在四種人胰腺癌細(xì)胞中m RNA和蛋白質(zhì)的表達(dá)差異。構(gòu)建sh-Furin干擾質(zhì)粒,通過(guò)慢病毒包裝感染Furin高表達(dá)的胰腺癌細(xì)胞,篩選出持續(xù)低表達(dá)Furin的穩(wěn)轉(zhuǎn)細(xì)胞株,Realtime PCR及Western Blot技術(shù)驗(yàn)證干擾效率。構(gòu)建Flag-Furin過(guò)表達(dá)質(zhì)粒,瞬時(shí)轉(zhuǎn)染Furin低表達(dá)的細(xì)胞株,同樣在m RNA和蛋白質(zhì)水平檢測(cè)過(guò)表達(dá)效率。通過(guò)CCK-8法和細(xì)胞克隆形成實(shí)驗(yàn)檢測(cè)Furin對(duì)人胰腺癌細(xì)胞生長(zhǎng)的影響,Transwell migration實(shí)驗(yàn)和劃痕實(shí)驗(yàn)檢測(cè)Furin對(duì)人胰腺癌細(xì)胞遷移能力的影響,Transwell invasion實(shí)驗(yàn)檢測(cè)Furin對(duì)人胰腺癌細(xì)胞侵襲能力的影響,并通過(guò)Western Blot技術(shù)檢測(cè)Furin對(duì)侵襲相關(guān)蛋白的表達(dá)量的改變。通過(guò)上述實(shí)驗(yàn),初步探索Furin對(duì)人胰腺癌細(xì)胞生物學(xué)行為的影響。Western Blot技術(shù)檢測(cè)上皮表型N-cadherin、Vimentin及間質(zhì)表型E-cadherin蛋白水平的變化,研究Furin是否促進(jìn)人胰腺癌細(xì)胞的上皮間質(zhì)轉(zhuǎn)化。同時(shí)檢測(cè)Hippo-YAP通路相關(guān)蛋白的改變,初步探明Furin對(duì)上皮間質(zhì)轉(zhuǎn)化作用的分子調(diào)控機(jī)制。研究結(jié)果:成功構(gòu)建Furin特異性sh-Furin干擾質(zhì)粒和Flag-Furin過(guò)表達(dá)質(zhì)粒。Realtime PCR、Western Blot結(jié)果顯示在Pa Tu8988和PANC1細(xì)胞株中Furin表達(dá)量相對(duì)較低,而在Bx PC3和SW1990細(xì)胞株中表達(dá)量相對(duì)較高。Sh-Furin慢病毒包裝并感染Bx PC3和SW1990細(xì)胞株,Flag-Furin瞬時(shí)轉(zhuǎn)染Pa Tu8988和PANC1細(xì)胞株,Realtime PCR、Western Blot檢測(cè)結(jié)果驗(yàn)證sh-Furin在胰腺癌細(xì)胞中良好且穩(wěn)定的干擾效率,Flag-Furin具有過(guò)表達(dá)效率。在Bx PC3和SW1990細(xì)胞株中下調(diào)Furin的表達(dá)后,癌細(xì)胞的增殖、克隆形成、遷移和侵襲能力顯著減弱,細(xì)胞中的上皮表型E-cadherin表達(dá)水平增加,間質(zhì)表型N-cadherin和Vimetin表達(dá)水平下降。同時(shí)Western Blot檢測(cè)出細(xì)胞中總的YAP、p-Mob1表達(dá)量明顯下降,p-YAP和總的Mob1的表達(dá)量顯著增加。在Pa Tu8988和PANC1細(xì)胞株中過(guò)表達(dá)Furin后,細(xì)胞的的增殖、克隆形成、遷移和侵襲能力增強(qiáng),而Western Blot檢測(cè)上皮間質(zhì)轉(zhuǎn)化和Hippo-YAP通路相應(yīng)蛋白的表達(dá)水平改變與干擾實(shí)驗(yàn)結(jié)果相反。研究結(jié)論:Furin能夠促進(jìn)胰腺癌細(xì)胞的增殖、遷移、侵襲能力和上皮間質(zhì)轉(zhuǎn)化,可能通過(guò)Hippo-YAP通路進(jìn)行調(diào)節(jié)。
[Abstract]:Objective: to investigate the effects of precursor protein processing enzyme (Furin) on the growth, metastasis and invasion of human pancreatic cancer cells. The effects on epithelial mesenchymal transformation of tumor cells and the possible molecular regulatory mechanisms were explored, which is the theoretical basis for further clinical application. Methods: the expression of m RNA and protein in four kinds of human pancreatic cancer cells was detected by Realtime PCR Western Blot. Sh-Furin interference plasmids were constructed and infected with Furin high expression pancreatic cancer cells by lentivirus packaging. Stable transformed cell lines with low expression of Furin were screened to verify the interference efficiency by using real time PCR and Western Blot techniques. The overexpression plasmid of Flag-Furin was constructed, and the expression efficiency was also detected at the level of m RNA and protein by transient transfection of Furin low expression cell lines. The effects of Furin on the growth of human pancreatic cancer cells were detected by CCK-8 assay and cell clone formation assay. The effects of Furin on the migration ability of human pancreatic cancer cells were detected by transwell migration assay and scratch test. The effect of Furin on the invasion of human pancreatic cancer cells was detected by Transwell invasion assay. Western Blot technique was used to detect the expression of invasion related protein in Furin. The effects of Furin on the biological behavior of human pancreatic cancer cells were studied. Western Blot technique was used to detect the changes of phenotype N-cadherin vimentin and interstitial phenotype E-cadherin protein in human pancreatic cancer cells, and to investigate whether Furin could promote the transformation of human pancreatic cancer cells. At the same time, the changes of Hippo-YAP pathway related proteins were detected, and the molecular regulation mechanism of Furin on epithelial interstitial transformation was preliminarily investigated. Results: the successful construction of Furin specific sh-Furin interference plasmid and Flag-Furin overexpression plasmid. The results showed that Furin expression was relatively low in Pa Tu8988 and PANC1 cell lines. However, the expression level of BX PC3 and SW1990 cells was relatively high. Sh-Furin lentivirus was packaged and infected with Bx PC3 and SW1990 cell lines. Flag-Furin transient transfection of Tu8988 and PANC1 cell lines confirmed the good and stable interference of sh-Furin in pancreatic cancer cells. The efficiency of Flag-Furin was higher than that of Flag-Furin. After down-regulating the expression of Furin in BX PC3 and SW1990 cell lines, the proliferation, clone formation, migration and invasion of cancer cells decreased significantly, the expression of E-cadherin in epithelial cells increased, and the expression of N-cadherin and Vimetin in interstitial cells decreased. At the same time, Western Blot showed that the total expression of YAPP p-Mob1 decreased significantly, and the expression of Mob1 and YAPP-YAP increased significantly. After overexpression of Furin in Pa Tu8988 and PANC1 cell lines, the cell proliferation, clone formation, migration and invasion were enhanced. However, the expression level of Western Blot in the epithelial interstitial transformation and Hippo-YAP pathway was different from the results of interference assay. Conclusion the results suggest that the cell proliferation, migration, invasion and epithelial mesenchymal transformation may be regulated by Hippo-YAP pathway.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Significance and relationship between Yes-associated protein and survivin expression in gastric carcinoma and precancerous lesions[J];World Journal of Gastroenterology;2009年32期

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本文編號(hào)：1867622