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

【摘要】：目的:研究Survivin對(duì)腫瘤細(xì)胞自噬溶酶體形成過程相關(guān)基因的表達(dá)調(diào)控。方法:在實(shí)驗(yàn)室建立自噬誘導(dǎo)模型,使用IKKβ的抑制劑TPCA-1以及Survivin的過表達(dá)載體質(zhì)粒對(duì)自噬模型進(jìn)行干預(yù)。通過透射電鏡,激光共聚焦顯微鏡觀察自噬體和自噬進(jìn)展?fàn)顟B(tài),qPCR和Western blot等分子生物學(xué)方法對(duì)自噬溶酶體形成階段相關(guān)基因的表達(dá)變化進(jìn)行檢測(cè),使用流式細(xì)胞儀技術(shù)檢測(cè)細(xì)胞凋亡情況。結(jié)果:通過雷帕霉素誘導(dǎo)ECA109細(xì)胞和KYSE450細(xì)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(xì)胞表達(dá)LC3增加,Western blot檢測(cè)顯示RAPA誘導(dǎo)后,LC3-Ⅰ、LC3-Ⅱ蛋白表達(dá)量有逐漸升高的趨勢(shì);激光共聚焦顯微鏡采圖并對(duì)相應(yīng)結(jié)果計(jì)數(shù),過表達(dá)Survivin后KYSE450細(xì)胞自噬模型中顯示紅色熒光的細(xì)胞數(shù)目降低(P0.05),含有紅色熒光顆粒的細(xì)胞數(shù)目降低(P0.05);加入TPCA-1干預(yù)后,ECA109細(xì)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(xì)胞數(shù)目減少(P0.05),KYSE450細(xì)胞自噬模型中顯示紅色熒光的細(xì)胞數(shù)目、含有紅色熒光顆粒的細(xì)胞數(shù)目均減少(P0.05);qPCR檢測(cè)自噬溶酶體形成過程相關(guān)基因發(fā)現(xiàn),Survivin過表達(dá)載體質(zhì)粒轉(zhuǎn)染后,可以在基因轉(zhuǎn)錄和蛋白水平檢測(cè)到Survivin表達(dá)量升高(P0.05);過表達(dá)Survivin時(shí),Rab7、TAK1、LAMP1 mRNA表達(dá)量減少(P0.05);TPCA-1干預(yù)后,在ECA109細(xì)胞中Rab7 mRNA的表達(dá)量升高(P0.05),LAMP1 mRNA的表達(dá)量降低(P0.05);在KYSE450細(xì)胞中IKKβmRNA的表達(dá)量降低(P0.05),Rab7 mRNA的表達(dá)量降低(P0.05),TAK1 mRNA的表達(dá)量降低(P0.05);在蛋白水平,過表達(dá)Survivin時(shí)自噬體形成過程相關(guān)蛋白的表達(dá)沒有顯著變化;流式細(xì)胞儀檢測(cè)凋亡,顯示TPCA-1干預(yù)后,ECA109細(xì)胞48h的凋亡增加。結(jié)論:雷帕霉素誘導(dǎo)可成功建立自噬模型;Survivin和TPCA-1對(duì)細(xì)胞自噬有抑制作用;在自噬溶酶體形成過程中,Survivin的過表達(dá)在基因轉(zhuǎn)錄水平可能對(duì)Rab7、TAK1、LAMP1 mRNA的表達(dá)有抑制作用;TPCA-1可促進(jìn)ECA109細(xì)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(hào)：1909417