【作者】 刘晓华；

【导师】 税青林；

【作者基本信息】 泸州医学院 ， 病理学与病理生理学， 2011， 硕士

【摘要】 目的：构建人端粒酶逆转录酶(hTERT)基因RNA干扰表达载体,同时构建p53上调凋亡调控因子(PUMA)的促表达载体,探讨hTERT基因及PUMA基因单独和联合作用对MCF-7细胞增殖抑制和凋亡效应,为肿瘤联合基因治疗提供实验依据。方法：设计针对hTERT基因的RNA干扰序列GGAACACCAAGAAGTTCATCT(1524-1544),合成具有该靶序列短发夹结构的寡核苷酸,经退火形成双链DNA,用T4 DNA连接酶连接到线性化pYr-2.1质粒U6启动子下游,构建成重组质粒pYr-2.1-hTERT-shRNA。将重组质粒转化感受态细胞DH5a,经摇菌扩增,抽提纯化质粒,通过酶切及测序鉴定确定重组质粒构建成功。同法构建不针对任何基因的阴性对照质粒。另合成PUMA基因的表达序列克隆至pUC57载体上,构建表达载体pUC57-PUMA并鉴定。把pUC57-PUMA和pYr-adshuttle-1分别用BamHⅠ和EcoRⅠ酶切,T4 DNA连接酶连接,构建重组质粒pYr-adshuttle-1-PUMA,经酶切鉴定确认重组质粒pYr-adshuttle-1-PUMA构建成功。乳腺癌MCF-7细胞常规培养,于转染前一日接种于6孔板,将以上两种载体的荧光对照质粒pYr-2.1-EGFP和pYr-adshuttle-1-RFP分别单独、联合转染入细胞,转染后48h,荧光显微镜下观察绿色及红色荧光细胞所占的比例,以此判断转染效率。实验设置hTERT干扰组、PUMA表达组、hTERT/PUMA联合组、干扰阴性对照组(HK group)、表达阴性对照组(adshuttle-1 group)和空白组(blank group),将以上构建的质粒分别转染各组细胞(空白组加等量PBS)。转染后48h通过western blot检测各组细胞中hTERT和PUMA蛋白的相对表达量；流式细胞仪检测细胞的凋亡率和周期分布；CCK-8法于转染后1～4d天检测细胞的生长抑制情况。结果：1.重组质粒pYr-2.1-hTERT-shRNA和pYr-adshuttle-1-PUMA经酶切及测序鉴定证实目的序列插入正确,重组质粒构建成功。2.转染后48h在荧光显微镜下观察,根据荧光细胞所占比例判断转染效率均达60%左右。3.免疫印迹结果分析显示：hTERT干扰组与空白组相比hTERT蛋白的相对表达量下降了54.4%；hTERT/PUMA联合组与空白组相比hTERT蛋白的相对表达量下降了52.3%; PUMA表达组与空白组相比PUMA蛋白的相对表达量增加了168.4%; hTERT/PUMA联合组与空白组相比PUMA蛋白的相对表达量增加了158.6%。4.CCK-8实验结果显示:hTERT组、PUMA组和hTERT/PUMA联合组在1～4d内细胞生长缓慢、增殖显著抑制；hTERT/PUMA联合组对MCF-7细胞的增殖抑制作用明显强于hTERT组和PUMA组。5.流式细胞仪检测细胞凋亡和细胞周期分布显示：转染后48h,hTERT组、PUMA组和hTERT/PUMA联合组细胞凋亡率分别为9.87%、19.02%、31.46%,hTERT组和hTERT/PUMA联合组的细胞周期阻滞于G0/G1期,PUMA组的细胞周期基本无变化,hTERT/PUMA联合组比hTERT组和PUMA组细胞周期阻滞更明显。结论：1.成功构建针对hTERT基因的shRNA表达载体pYr-2.1-hTERT-shRNA,该载体可以特异性地抑制乳腺癌MCF-7细胞中hTERT基因的表达。2.成功构建了针对PUMA基因的促表达载体pYr-adshuttle-1-PUMA,该载体可以在乳腺癌组织中增加PUMA蛋白量的表达量。3.pYr-2.1-hTERT-shRNA和pYr-adshuttle-1-PUMA单独转染人乳腺癌MCF-7细胞后均能促进细胞凋亡、抑制细胞增殖,联合转染后细胞凋亡增加,细胞增殖抑制更明显,二者具有协同效应,hTERT和PUMA联合是肿瘤基因治疗作用靶点的有益选择。更多还原

【Abstract】 Objectives:To construct the RNA interference expression vector for hTERT gene and promoting expression vector for PUMA gene, study on the effects of MCF-7 breast cancer cell proliferation inhibition and apoptosis when hTERT and PUMA gene were transfected separately and cooperately, which could provide experimental basis for multi-gene therapy for tumor. Methods:Designed RNA interference target sequence GGAACACCAAG AAGTTCATCT which aimed at hTERT gene, then synthesize two strands of oligonucleotides including a short hairpin structure, then with T4 DNA ligase the double-strand DNA was inserted downstream from the U6 promoter of the linear pYr-2.1-hU6 vector and the recombination expression plasmid pYr-2.1-hTERT-shRNA was constructed. Nextly the recombinant was transformed into susceptible cell E.coli DH5a, then choosing the positive colony to amplify and extracting the plasmid, and the extracted recombinant was constructed successfully by enzyme digestion and sequence analysis. At the same time, the negative control recombinant pYr-2.1-HK that did not aim at any gene was constructed with the same method. In addition, the expression sequence of PUMA gene was inserted into pUC57 vector and was analyzed. Then, the pUC57-PUMA and pYr-adshuttle-1 were digested with BamHI and EcoRI, combined them with T4 DNA ligase, and pYr-adshuttle-1-PUMA was constructed, and the extracted recombinant was constructed successfully by enzyme digestion. MCF-7 cells were cultured, and those in good condition were plated in 6-well cell culture plates on the day before transfection, then transfected pYr-2.1-EGFP and pYr-adshuttle-1-RFP into MCF-7 separately and cooperately, at 48h post transfection, observed the ratio of green and red fluorescence cells under the fluorescence microscope to judge transfection rate. Set up six experimental groups, they were hTERT group, PUMA group, hTERT/PUMA group, HK group, pYr-adshuttle-1 group and blank control group, then recombinant was transfected into those groups except blank group. At 48h after transfection, the relative expression of hTERT and PUMA protein was detected by western blot, and cell apoptosis and cell cycle distribution was detected by FCM. At 1～4d post transfection, the inhibition effects of the cell growth were detected by CCK-8 assay. Results:1. Recombinant plasmids pYr-2.1-hTERT-shRNA and pYr-adshuttle-1-PUMA were identified by enzyme digestion and sequence analysis confirmed that the purposed sequence had been inserted into proper points, and recombining plasmids were constructed successfully.2. Observe the MCF-7 cells under the fluorescence microscope 48h after transfection and judge the transfection rate that were about 60% basing on the proportion of cells shining fluorescence.3. The results of western blot showed that in hTERT group the relative expression quantities of hTERT protein was about 54.4% lower than blank group, and in hTERT/PUMA group the relative expression quantities of hTERT protein was about 52.3% lower than blank group; in PUMA group the relative expression quantities of PUMA protein was about 168.4% higher than blank group, and in hTERT/PUMA group the relative expression quantities of PUMA protein was about 158.6% higher than blank group.4. The results of analysis of CCK-8 showed that in hTERT group、PUMA group and hTERT/PUMA group MCF-7 cells growth velocity were declined, and the proliferation were significantly inhibited, and for hTERT/PUNA group the rate of MCF-7 cells proliferation inhibition was highter significantly than hTERT group and PUMA group.5. Detection of cell apoptosis and cell cycle distribution by FCM showed that in hTERT group、PUMA group and hTERT/PUMA group MCF-7 cell apoptosis rate were 9.87%,19.02% and 31.46%, in hTERT group and hTERT/PUMA group cell cycle was blocked at G0/G1 phase, and the effect of cell cycle stoppage was more significant in hTERT/PUMA group, but in PUMA group cell cycle had no change. Conclusions:1. The RNA interference expression vector pYr-2.1-hTERT-shRNA aimed at hTERT was successfully constructed.2. The promoting expression vector pYr-adshuttle-1-PUMA targeted at PUMA was successfully constructed.3. The effects of pYr-2.1-hTERT-shRNA and pYr-adshuttle-1-PUMA on MCF-7 cell proliferation inhibition were significantly higher than them separately, so it had showed that the effect on inhibiting on breast cancer MCF-7 cells by combining hTERT with PUMA was more efficacious than that by single gene, and there were synergistic effect between hTERT and PUMA, there is a useful option for cancer gene therapy by combining hTERT and PUMA.更多还原