本文選題：自噬 + 自噬溶酶體��； 參考：《新疆醫(yī)科大學》2017年碩士論文

【摘要】：目的:研究Survivin對腫瘤細胞自噬溶酶體形成過程相關(guān)基因的表達調(diào)控。方法:在實驗室建立自噬誘導模型,使用IKKβ的抑制劑TPCA-1以及Survivin的過表達載體質(zhì)粒對自噬模型進行干預。通過透射電鏡,激光共聚焦顯微鏡觀察自噬體和自噬進展狀態(tài),qPCR和Western blot等分子生物學方法對自噬溶酶體形成階段相關(guān)基因的表達變化進行檢測,使用流式細胞儀技術(shù)檢測細胞凋亡情況。結(jié)果:通過雷帕霉素誘導ECA109細胞和KYSE450細胞后,透射電鏡下可見更多的自噬體,激光共聚焦顯微鏡下可見細胞表達LC3增加,Western blot檢測顯示RAPA誘導后,LC3-Ⅰ、LC3-Ⅱ蛋白表達量有逐漸升高的趨勢;激光共聚焦顯微鏡采圖并對相應結(jié)果計數(shù),過表達Survivin后KYSE450細胞自噬模型中顯示紅色熒光的細胞數(shù)目降低(P0.05),含有紅色熒光顆粒的細胞數(shù)目降低(P0.05);加入TPCA-1干預后,ECA109細胞自噬模型中紅色、綠色熒光顆粒數(shù)和含有紅色熒光顆粒的細胞數(shù)目減少(P0.05),KYSE450細胞自噬模型中顯示紅色熒光的細胞數(shù)目、含有紅色熒光顆粒的細胞數(shù)目均減少(P0.05);qPCR檢測自噬溶酶體形成過程相關(guān)基因發(fā)現(xiàn),Survivin過表達載體質(zhì)粒轉(zhuǎn)染后,可以在基因轉(zhuǎn)錄和蛋白水平檢測到Survivin表達量升高(P0.05);過表達Survivin時,Rab7、TAK1、LAMP1 mRNA表達量減少(P0.05);TPCA-1干預后,在ECA109細胞中Rab7 mRNA的表達量升高(P0.05),LAMP1 mRNA的表達量降低(P0.05);在KYSE450細胞中IKKβmRNA的表達量降低(P0.05),Rab7 mRNA的表達量降低(P0.05),TAK1 mRNA的表達量降低(P0.05);在蛋白水平,過表達Survivin時自噬體形成過程相關(guān)蛋白的表達沒有顯著變化;流式細胞儀檢測凋亡,顯示TPCA-1干預后,ECA109細胞48h的凋亡增加。結(jié)論:雷帕霉素誘導可成功建立自噬模型;Survivin和TPCA-1對細胞自噬有抑制作用;在自噬溶酶體形成過程中,Survivin的過表達在基因轉(zhuǎn)錄水平可能對Rab7、TAK1、LAMP1 mRNA的表達有抑制作用;TPCA-1可促進ECA109細胞自噬模型的48h凋亡。
[Abstract]:Aim: to investigate the regulation of Survivin on the expression of genes associated with autophagy formation in tumor cells. Methods: a model of autophagy was established in laboratory. The model was treated with TPCA-1, an inhibitor of IKK 尾, and the overexpression vector plasmid of Survivin. By means of transmission electron microscope (TEM) and laser confocal microscopy (LSCM), the changes of gene expression in autophagy and autophagy progression were detected by QPCR and Western blot. Apoptosis was detected by flow cytometry. Results: after inducing ECA109 cells and KYSE450 cells by rapamycin, more autophagy was observed under transmission electron microscope. The increase of LC3 expression in cells was observed under laser confocal microscope. Western blot analysis showed that the expression of LC3- 鈪，

本文編號：1909417