【作者】 陈绍坤；

【导师】 税青林；

【作者基本信息】 泸州医学院 ， 中西医结合基础， 2008， 硕士

【摘要】 目的:构建腺病毒介导的人端粒酶逆转录酶(hTERT)基因和端粒重复序列结合因子2(TRF2)基因的RNA干扰(RNAi)表达载体,探讨hTERT和TRF2蛋白在MCF-7细胞中表达的相关性,以及hTERT和TRF2双基因干扰对乳腺癌治疗的协同性,为联合干扰hTERT和TRF2基因作为肿瘤基因治疗新策略的应用提供实验依据。方法:设计针对hTERT和TRF2基因的RNA干扰靶序列,合成具有该靶序列短发夹结构的寡核苷酸,退火形成双链DNA,通过T4 DNA连接酶连接到线性化pGenesil-1质粒U6启动子下游,构建重组表达质粒pGenesil-shRNA-hTERT和pGenesil-shRNA -TRF2。随后将U6-hTERT-shRNA和U6-TRF2-shRNA表达框克隆到入门载体pENTRTM1A,再通过LR体外同源重组将表达框(U6-shRNA-hTERT、U6-shRNA-TRF2)转移至pAd/PL-DEST腺病毒表达质粒上,并在HEK293细胞内包装为具有感染能力的病毒颗粒rAd-hTERT和rAd-TRF2。同法构建不针对任何基因的shRNA阴性对照rAd-HK,空病毒对照rAd-blank和表达绿色荧光蛋白(EGFP)的腺病毒rAd -EGFP。培养MCF-7细胞,接种指数生长期细胞于6孔板,转染rAd–EGFP以测定最佳感染强度和最佳感染时间。然后设置空白组、空病毒组、阴性对照组、rAd-hTERT组、rAd-TRF2组、rAd-hTERT/rAd-TRF2联合组,分别以50MOI转染人乳腺癌MCF-7细胞。于转染后48h裂解各组细胞提取总蛋白,通过western blot检测各组hTERT和TRF2蛋白的相对表达量。同时于转染后1~6d用MTT法检测细胞增殖情况;PI染色后流式细胞仪检测转染48h后细胞周期分布情况。结果:①重组质粒pGenesil-shRNA-hTERT和pGenesil-shRNA-TRF2经Xbal酶切鉴定和测序鉴定证实目的序列已准确插入到预计位点,重组质粒构建成功。②重组腺病毒rAd-hTERT和rAd-TRF2经酶切鉴定和PCR鉴定证实包装成功。③以50MOI转染rAd–EGFP于MCF-7细胞后48h荧光显微镜下观察,转染效率达99%左右。④免疫印迹分析显示: rAd-hTERT组和rAd-hTERT/rAd-TRF2联合组hTERT蛋白的相对表达量与其它四组相比均下降82%左右,而rAd-hTERT组与rAd-hTERT/rAd-TRF2联合组之间hTERT蛋白表达差异无显著性( P>0.05 ); rAd-TRF2组和rAd-hTERT/rAd-TRF2联合组TRF2蛋白的相对表达量与其它四组相比均下降83%左右,而rAd-TRF2组与rAd-hTERT/rAd-TRF2联合组之间TRF2蛋白表达差异无显著性(P>0.05)。⑤MTT实验结果显示:rAd-hTERT组、rAd-TRF2组以及rAd-hTERT/rAd-TRF2联合组在1～6d范围内细胞生长速度减慢、增殖显著抑制;但rAd-hTERT/rAd-TRF2联合组对MCF-7细胞增殖的抑制作用明显强于rAd-hTERT组和rAd-TRF2组,且细胞增殖抑制高峰持续时间明显长于rAd-hTERT组和rAd-TRF2组。⑥通过流式细胞仪检测细胞周期分布显示: rAd-hTERT组、rAd-TRF2组以及rAd-hTERT/ rAd-TRF2联合组于转染后48h,MCF-7细胞周期阻滞于G0/G1期、增殖指数(PI)显著下降;其中rAd-hTERT/rAd-TRF2联合组比rAd-hTERT组和rAd-TRF2组细胞周期阻滞更显著、细胞增殖指数(PI)下降更明显。结论:①成功构建了腺病毒介导的靶向hTERT和TRF2基因的RNA干扰真核表达载体rAd-hTERT、rAd-TRF2。②针对hTERT和TRF2基因的RNAi靶序列设计有效,将rAd-hTERT和rAd-TRF2转染人乳腺癌MCF-7细胞48h后可显著抑制hTERT蛋白和TRF2蛋白表达,但hTERT蛋白和TRF2蛋白表达无相关性。③单独转染rAd-hTERT和rAd-TRF2于人乳腺癌MCF-7细胞后细胞周期阻滞于G0/G1期、细胞增殖抑制,但联合转染rAd-hTERT和rAd-TRF2后细胞增殖抑制更明显、持续时间更长,说明联合干扰hTERT和TRF2基因可获得更有效的肿瘤基因治疗效果。更多还原

【Abstract】 Objectives: To construct the adenovirus-mediated RNA interference expression vector aimed at hTERT and TRF2 gene ,explore the relativity of hTERT and TRF2 protein expression in MCF-7 cells,and the therapy cooperativity on breast cancer by combinate hTERT and TRF2 gene ,which could be provide experimental evidence for interference combination hTERT and TRF2 gene as a new effective way of tumor gene therapy .Methods: Designed RNA interference target sequence aimed at hTERT and TRF2 gene, then synthesize two strands of oligonucleotides including a hairpin structure,than the double-strand DNA was inserted downstream from the U6 promoter of the linear pGenesil-1 vector by T4 DNA ligase and the recombination expression plasmid pGenesil-shRNA-hTERT and pGenesil-shRNA-TRF2 was constructed. Then the expression cassette of U6-hTERT-shRNA and U6-TRF2-shRNA were subcloned into entry vector pENTRTM1A. Followed by homologous recombination technique ,the expression frame(U6-shRNA-hTERT、U6-shRNA-TRF2)was integrated into adenovirus expression plasmid pAd/PL-DEST, and the recombinant adenovirus were packaged in HEK293 cells,gained rAd-hTERT and rAd-TRF2 .At the same time, constructed the negative control recombinant adenovirus rAd-HK that did not aim at any gene、blank adenovirus controlr Ad-blank、rAd–EGFP that express green fluorescence protein by the same method. MCF-7 cells that were in good condition were plated in 6-well cell culture plates,and transfected rAd–EGFP into MCF-7 cells to judge optimal MOI and transfection time. Then set up six experimental groups, they were blank control group、rAd-blank group、rAd-HK group、rAd-hTERT group、rAd-TRF2 group and rAd-hTERT/rAd-TRF2 group.They were transfected into MCF-7 cells separately at 50MOI . At 48 hours after transfection, the total protein from each well of cells was isolated for Western blot to detect the relative expression of hTERT and TRF2 protein in every group. At the same time,at 1～6d after transfection, the inhibition effects of the cell growth were detected by MTT assay;and the cell cycle was observed by means of flow cytometry FCM by PI staining after gathered every group cells at 48h after transfection . Results:①The recombinant plasmids pGenesil-shRNA-hTERT and pGenesil-shRNA -TRF2 were digested with xbal to agarose gel electrophoresis and sequence analysis ,showed that the target sequence had inserted into the predicted site precisely and the recombining plasmids were successfully constructed.②Recombinant adenovirus rAd-hTERT and rAd-TRF2 were digested with PI-SceI and I-CeuI to agarose gel electrophoresis and PCR analysis ,showed that recombinant adenovirus rAd-hTERT and rAd-TRF2 were successfully constructed.③Observe the MCF-7 cells under the fluorescence microscope 48h after transfection rAd–EGFP at 50MOI, transfection efficiency was about 99%.④The result of analysis of the western blottong showed that the relative expression quantities of hTERT protein at rAd-hTERT group and rAd-hTERT+ TRF2 group was about 82% lower than the others groups,but the difference between rAd-hTERT group and rAd-hTERT+ TRF2 group had no statistical significance (P>0.05);the relative expression quantities of TRF2 protein at rAd-TRF2 group and rAd-hTERT+ TRF2 group was about 83% lower than the others groups,but the difference between rAd-TRF2 group and rAd-hTERT+ TRF2 group had no statistical significance (P>0.05).⑤The result of analysis of MTT showed that rAd-hTERT group、rAd-TRF2 group and rAd-hTERT/rAd -TRF2 group MCF-7 cells growth velocity were step down, and proliferation were inhibited significantly at 1～6d after transfection, but rAd-hTERT/rAd -TRF2 group MCF-7 cells proliferation inhibition rate were higher than rAd-hTERT group or rAd-TRF2 group,and the persistence time of MCF-7 cells proliferation inhibition peak at rAd-hTERT/rAd-TRF2 group were longer than rAd-hTERT group or rAd-TRF2 group.⑥Detection of cell cycle distribution by FCM at 48h after transfection,showed that MCF-7cell cycle was arrested at G0/G1 phase and cell proliferation was inhibited at rAd-hTERT group、rAd-TRF2 group and rAd-hTERT/rAd-TRF2 group,but the effect of cell cycle stoppage and cell proliferation inhibition were more significant at rAd-hTERT/rAd-TRF2 group .Conclusions:①Adenovirus-mediated RNA interference expression vector aimed at hTERT and TRF2(rAd-hTERT and rAd-TRF2)were successfully constructed.②The target sequence of RNA interference which aimed at hTERT and TRF2 gene were designed efficiently,hTERT and TRF2 protein expression were inhibited significantly at 48h after rAd-hTERT and rAd-TRF2 were transfected into MCF-7 cells,but protein expression of hTERT and TRF2 have no relativity.③MCF-7 cell cycle was arrested at G0/G1 phase、cell proliferation was inhibited when rAd-hTERT or rAd-TRF2 were transfected into MCF-7 cells separately,but MCF-7 cell proliferation inhibition rate are more significant and persistence time are langer when combinate rAd-hTERT and rAd-TRF2,show that it could be possibly a new effective way of tumor gene therapy that interference combinate hTERT and TRF2 gene.更多还原