EB病毒潜伏期膜蛋白LMP2A对胃癌细胞增殖和凋亡的影响

发布时间：2018-03-03 17:44

本文选题：EB病毒　切入点：重组腺病毒　出处：《青岛大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的构建EBV潜伏期膜蛋白LMP2A编码基因的腺病毒表达载体，初步探讨LMP2A表达对靶细胞SGC-7901的生物学作用。方法RT-PCR扩增获取目的基因LMP2A，采用AdEasy系统构建携带目的基因的重组腺病毒载体pAd-2A，脂质体法将重组质粒pAd-2A转染HEK293细胞包装重组腺病毒。选择EBV阴性胃癌细胞SGC-7901作为靶细胞，试验分重组腺病毒SGC-2A组，病毒对照SGC-GFP组和细胞对照SGC组3组，采用MTT实验、透射电镜观察、流式细胞分析、激光共聚焦等技术检测目的基因LMP2A表达对靶细胞生物学行为的影响。结果①限制性酶切、PCR及测序鉴定证实，目的基因LMP2A正确插入重组质粒，，HEK293细胞成功包装出具有稳定感染性的重组腺病毒，命名为vAd-2A，经测定病毒滴度为3.16×10~(12)pfu／L。②MTT检测结果显示：与SGC-GFP组和SGC组比较，SGC-2A组细胞从接种后第2天起表现出更强的增殖能力。③透射电镜观察SGC-2A组细胞的超微结构无明显改变，而SGC-GFP组和SGC组则出现一系列变化，并可观察到凋亡小体。④激光共聚焦分析表明，SGC-2A组细胞几乎均可观察到较强的cyclinE荧光信号；而SGC-GFP组仅部分细胞可观察到cyclinE荧光信号，且较弱；未感染病毒的SGC组细胞则仅有少量细胞观察到荧光。⑤流式细胞分析感染vAd-2A后48h和72h的SGC-7901细胞S期的比例明显高于SGC-GFP和SGC两对照组。结论成功构建了EBV潜伏期膜蛋白基因LMP2A的重组腺病毒表达载体，并在HEK293细胞中成功包装出重组腺病毒；同时初步证实LMP2A可促进SGC-7901细胞增殖并抑制其凋亡，该作用可能与LMP2A诱导靶细胞cyclinE的表达有关。
[Abstract]:Objective to construct the adenovirus expression vector of EBV latent membrane protein (LMP2A) encoding gene and to explore the biological effect of LMP2A expression on target cell SGC-7901. Methods the target gene LMP2A was amplified by RT-PCR, and the recombinant adenovirus vector pAd-2A was constructed by AdEasy system. The recombinant plasmid pAd-2A was transfected into HEK293 cells to package the recombinant adenovirus by liposome method. The EBV negative gastric cancer cell SGC-7901 was selected as the target cell. The experiment was divided into three groups: recombinant adenovirus SGC-2A group, virus control SGC-GFP group and cell control SGC group. The effects of LMP2A expression on the biological behavior of target cells were detected by MTT experiment, transmission electron microscopy, flow cytometry and laser confocal focusing. Results 1 restriction endonuclease PCR and sequencing confirmed that the recombinant adenovirus with stable infection was successfully packaged by inserting LMP2A into the recombinant plasmid pHEK293. VAd-2A was named as vAd-2A. The results of MTT assay showed that compared with SGC-GFP group and SGC group, SGC-2A cells showed stronger proliferative ability from the second day after inoculation. 3 transmission electron microscope showed no obvious change in ultrastructure of SGC-2A group. In SGC-GFP group and SGC group, a series of changes were observed, and the apoptosis corpuscles were observed by confocal laser analysis. The results showed that cyclinE fluorescence signal was almost observed in SGC-2A group, while cyclinE fluorescence signal was observed in only a few cells in SGC-GFP group. Only a few of the cells in the SGC group without infection observed that the S phase ratio of SGC-7901 cells at 48h and 72h after vAd-2A infection was significantly higher than that in the SGC-GFP and SGC control groups. Conclusion the recombinant adenovirus expression vector of EBV latent membrane protein gene LMP2A was successfully constructed, and the recombinant adenovirus was successfully packaged in HEK293 cells, and it was preliminarily proved that LMP2A could promote the proliferation and inhibit the apoptosis of SGC-7901 cells. This effect may be related to the expression of cyclinE in target cells induced by LMP2A.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R735.2;R373

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