本文選題：ING4基因 + 腺病毒載體��； 參考：《蘇州大學(xué)》2015年碩士論文

【摘要】：目的:研究重組腺病毒介導(dǎo)的ING-4(Inhibitor of tumor growth factor 4)基因感染人MCF-7乳腺癌細(xì)胞后對后者所起的生長抑制作用及化療增敏作用。方法:將搭載有ING-4基因的重組腺病毒載體Ad-ING4感染人MCF-7乳腺癌細(xì)胞,用熒光顯微鏡觀察感染后的MCF-7細(xì)胞形態(tài)學(xué)變化;RT-PCR和Western-Blot法檢測ING-4基因在MCF-7細(xì)胞中的轉(zhuǎn)錄和表達(dá);CCK法分別測定ADM、5-Fu、CTX對病毒感染前后的MCF-7乳腺癌細(xì)胞的半數(shù)抑制濃度IC50,以觀察其化療藥物增敏現(xiàn)象。用流式細(xì)胞技術(shù)檢測ING-4基因?qū)CF-7細(xì)胞的促凋亡作用;半定量RT-PCR法檢測ING-4基因表達(dá)對MCF-7細(xì)胞中相關(guān)凋亡基因表達(dá)的影響。結(jié)果:搭載有ING-4基因的重組腺病毒載體Ad-ING4成功感染人MCF-7乳腺癌細(xì)胞,并且隨著感染作用時間的延長,于熒光顯微鏡下可見Ad-ING4作用下的MCF-7乳腺癌細(xì)胞變圓、脫落、皺縮、聚集等現(xiàn)象愈加明顯,細(xì)胞貼壁數(shù)量明顯減少;重組腺病毒載體感染MCF-7細(xì)胞后ING-4成功轉(zhuǎn)染入細(xì)胞內(nèi),RT-PCR法檢測出其于MCF-7細(xì)胞內(nèi)獲得表達(dá);蛋白印跡法表明在感染后的MCF-7細(xì)胞內(nèi)存在ING-4基因相關(guān)蛋白的表達(dá);CCK-8試驗表明在有ING-4基因表達(dá)的情況下,MCF-7細(xì)胞對化療藥物的敏感性有所增強;Ad-ING4與化療藥物聯(lián)合使用時對MCF-7細(xì)胞的增殖抑制作用更為明顯;RT-PCR檢測表明Ad-ING4的轉(zhuǎn)染使MCF-7細(xì)胞的Bax表達(dá)上升,Bcl-2與Survivin的表達(dá)下降,其機制可能與ING4降低Bcl-2/Bax比值和抑制Survivin的表達(dá)有關(guān)。結(jié)論:MCF-7細(xì)胞在轉(zhuǎn)染ING4基因后,其細(xì)胞增殖受到了明顯抑制,且對化療藥物的敏感度更高,更易凋亡,該現(xiàn)象可能是通過改變Bax,Bcl-2及Survivin表達(dá)水平來實現(xiàn)的。
[Abstract]:Aim: to study the growth inhibition and chemosensitization of recombinant adenovirus-mediated ING-4 (inhibitor of tumor growth factor 4) gene in human MCF-7 breast cancer cells. Methods: the recombinant adenovirus vector Ad-ING4 was transfected into human MCF-7 breast cancer cells. Morphological changes of MCF-7 cells after infection were observed by fluorescence microscope. RT-PCR and Western-Blot were used to detect the transcription and expression of EM-4 gene in MCF-7 cells. The half inhibitory concentration (IC50) of ADM5-Fu-CTX on MCF-7 breast cancer cells before and after infection was determined by CCK method. The apoptosis of MCF-7 cells was detected by flow cytometry, and the effect of the expression of Ring-4 gene on the expression of apoptotic genes in MCF-7 cells was detected by semi-quantitative RT-PCR. Results: the recombinant adenovirus vector Ad-ING4 was successfully infected with human MCF-7 breast cancer cells. With the extension of infection time, MCF-7 breast cancer cells induced by Ad-ING4 could be seen to become round, shedding and shrinking under fluorescence microscope. The aggregation became more and more obvious, and the number of cell adhesion was decreased, and the expression of the recombinant adenovirus vector in MCF-7 cells was detected by RT-PCR after transfection of the recombinant adenovirus vector into MCF-7 cells. Western blot analysis showed that there was an expression of EM-4 gene related protein in infected MCF-7 cells. CCK-8 test showed that chemosensitivity of MCF-7 cells to chemotherapeutic drugs was enhanced with the presence of EM-4 gene expression, and the combination of Ad-ING4 and chemotherapeutic drugs was also observed. RT-PCR analysis showed that the expression of Bax and survivin in MCF-7 cells increased after transfection of Ad-ING4, and the expression of Bcl-2 and survivin decreased in MCF-7 cells. The mechanism may be related to the decrease of Bcl-2 / Bax ratio and the inhibition of survivin expression by ING4. Conclusion after transfection of ING4 gene, the proliferation of MCF-7 cells was significantly inhibited, and its sensitivity to chemotherapeutic drugs was higher and apoptosis was more likely. This phenomenon may be achieved by changing the expression levels of BaxanBcl-2 and survivin.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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