【作者】 蔡艳玲；

【导师】 罗小玲；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2011， 硕士

【摘要】 目的构建含有小发夹RNA (shRNA)的针对hTERT基因的重组真核质粒pGPU6/GFP/Neo-hTERT-shRNA,并探讨RNA干扰人端粒酶逆转录酶(human telomerase reverse transcriptase, hTERT)的表达对大肠癌SW480细胞凋亡的影响。方法设计合成3条针对hTERT基因的siRNA,将筛选出最有效的siRNA合成shRNA寡核苷酸片段插入到真核质粒pGPU6/GFP/Neo中,进行酶切和测序鉴定。将构建好的重组真核质粒pGPU6/GFP/Neo-hTERT-shRNA,采用脂质体法转染人大肠癌SW480细胞。在荧光显微镜下观察细胞转染效率及细胞形态学变化。RT-PCR法检测不同转染时间SW480细胞中hTERTmRNA的表达水平。TRAP-PCR-ELISA法检测转染48小时后SW480细胞的端粒酶活性。免疫组化法检测SW480细胞中hTERT蛋白的表达。流式细胞仪检测转染后细胞周期分布。TUNEL法检测转染后细胞凋亡状况。激光共聚焦显微镜观察细胞线粒体跨膜电位(MMP)的变化。透射电镜观察转染后细胞超微结构。结果将筛选出干扰效率较好的的siRNA序列合成shRNA片段后,将其成功插入质粒pGPU6/GFP/Neo中,构成重组真核质粒pGPU6/GFP/ Neo-hTERT-shRNA。SW480细胞以质粒与脂质体比例为1：2.5、转染48h时的转染效率较高,其转染率达59%。RT-PCR结果显示,hTERT-shRNA组的hTERTmRNA表达水平在转染48h时降低较明显,与空白组、脂质体组、NC-shRNA组比较,抑制率分别是39.20%,33.28%,27.95%。TRAP-PCR-ELISA结果示,hTERT-shRNA组SW480细胞端粒酶活性显著降低18.7%,与其它三组比较,差别均有统计学意义(P<0.05)。免疫组化结果示,hTERT-shRNA组被染色的阳性细胞明显少于其他组,经病理图像分析软件分析灰度值后得出,转染48h后hTERT-shRNA组与空白组比较,差异有明显统计学意义(P<0.05),同时与脂质体组和NC-shRNA组比较,差异均有统计学意义(P<0.01)。流式细胞仪结果示,hTERT-shRNA组G0/G1期细胞显著增加,S期细胞减少,提示hTERT-shRNA质粒转染SW480细胞后,使进入静止状态的细胞增多,细胞增殖指数下降约14.2%,与NC-shRNA组比较,差别有显著统计学意义(P<0.05)。TUNEL结果示,与其他组比较,hTERT-shRNA组凋亡细胞数显著增多,凋亡指数为21.5%,明显比其他组增高,差异有统计学意义(P<0.01)。激光共聚焦显微镜观察结果,hTERT-shRNA组可见多个含有核分裂相的细胞,细胞Rh123荧光强度明显增强,MMP显著下降,与其他组比较,差异有统计学意义(P<0.01)。透射电镜观察结果示,SW480细胞体积明显缩小,表面突起及微绒毛减少,甚至消失,细胞核固缩,染色质不均匀地沿核膜下聚集,空泡增多。结论成功构建了针对hTERT基因的重组真核表达质粒pGPU6/GF P/Neo-hTERT-shRNA。它能有效沉默hTERT基因,因而能有效抑制人大肠癌SW480细胞增殖生长,降低端粒酶活性,最终促使肿瘤细胞凋亡。目的研究靶向hTERT基因的重组质粒pGPU6/GFP/Neo-hTERT-shRNA对人大肠癌SW480细胞裸鼠移植瘤的治疗作用。方法于裸鼠右侧腋下皮下注射人大肠癌SW480细胞建立大肠癌移植瘤动物模型,待肿瘤长到一定大小时,随机分为生理盐水组(NS组)、NC-shRNA组和hTERT-shRNA组。各组连续进行相应治疗6次后,观察肿瘤的生长状况,测量肿瘤体积,绘制肿瘤生长曲线,HE染色观察肿瘤组织细胞形态学变化,免疫组化法检测移植瘤组织中hTERT蛋白的表达,TUNEL法检测肿瘤组织中细胞凋亡情况,RT-PCR法检测瘤组织中hTERTmRNA的表达,PCR-TRAP-ELISA法检测肿瘤组织的端粒酶活性。结果所有裸鼠在接种SW480细胞第14天后,皮下肿瘤结节直径达5-7mm,成功构建了裸鼠移植瘤模型,成瘤率为100%。荷瘤裸鼠开始治疗后,hTERT-shRNA组瘤体积增长速度慢于NS组和NC-ShRNA组,并于第18天开始明显减慢。HE染结果示,hTERT-shRNA组肿瘤组织出现局部坏死区,瘤组织细胞形态发生明显改变。免疫组化法检测结果示,hTERT-shRNA组肿瘤组织中hTERT蛋白表达水平下降,可见少量hTERT蛋白阳性细胞,细胞呈浅棕色。TUNEL法检测结果示,hTERT-shRNA组凋亡细胞数明显增多,细胞分布密集,与NS组和NC-shRNA组比较,凋亡指数分别增加29.4%和31.1%,差异有显著统计学意义(P<0.01)；RT-PCR法检测结果示,hTERT-shRNA组hTERT mRNA表达水平较NS组和NC-shRNA组分别下降52.1%和48.3%,差异具有显著性意义(P<0.01)。PCR-TRAP-ELISA法检测结果示,hTERT-shRNA组与NS组和NC-shRNA组比较,端粒酶活性降低较明显,抑制率分别为48.5%和53.0%,差异有统计学意义(P<0.05)。结论重组质粒pGPU6/GFP/Neo-hTERT-shRNA通过下调hTERTmRNA和hTERT蛋白的表达,促进肿瘤细胞的凋亡,从而抑制大肠癌移植瘤的生长。更多还原

【Abstract】 Part I:Constuction of shRNA recombinant eukaryotic expression vector and the effect on apoptosis of colorectal cancer SW480 cellsObjective To construct the recombinant eukaryotic plasmid containing small hairpin RNA (shRNA) targeting hTERT and investigate the effect of RNA-mediated interference hTERT (human telomerase reverse transcriptase,hTERT) expression on the biological behaviour of colorectal cancer sw480 cells.Methods Three small interfering RNA (siRNA) targeting hTERT gene were synthesized chemically, one pair of siRNA fragments which had the most silencing effect was synthesized into oligodeoxynucleotide fragment,and constructed into eukaryotic vector pGPU6/GFP/Neo.The recombinant eukaryotic vector pGPU6/GFP/Neo was identified by restriction enzyme digestion and sequencing.The constructed recombinant eukaryotic vector pGPU6/GFP/Neo-hTERT-shRNA was transfected into human colorectal cancer SW480 cells by lipofectamine.Cells carrying efficiency and morphology changes was observed by fluorescence microscope.The expression level of hTERT mRNA in SW480 cells in different times was detected by RT-PCR analysis. The telomerase activity of SW480 cells after transfected 48h was examined by TRAP-PCR-ELISA analysis.The hTERT protein expression of SW480 cells was detected by immunohistochemical method.The distribution of cell cycle was analyzed by FCCS.The apoptosis changes of SW480 cells were monitored by TUNEL assay.The changes of mitochondrial membrane potential (MMP) were detected by laser confocal microscope. The ultrastructure changes of SW480 cells after transfected was examined by TEM (The transmission electron microscope).Results The shRNA fragment was successfully inserted into the eukaryotic plasmid pGPU6/GFP/Neo to constitute recombinant eukaryotic plasmid pGPU6/GFP/Neo-hTERT-shRNA.The transfection efficiency of SW480 cells was higher when the ratio of plasmid and Lipofectamine at 1:2.5 and transfected 48h, and the transfected percentage of cell is 59%. RT-PCR assay showed that the hTERTmRNA expression level of hTERT-shRNA group reduced remarkably, compared with the blank group, liposome group, NC-shRNA group, its inhibition rate was respectively 39.2%,33.28%, 27.95%. TRAP-PCR-ELISA showed that the telomerase activity in the hTERT-shRNA group after transfection decreased for 18.7% significantly,compared with another three groups,the difference was statistic significance(P<0.05).Immunohistochemistry assay showed that the number of stained positive SW480 cell in hTERT-shRNA group was less markedly than that of in other three groups. Through pathological image software assay, the difference between hTERT-shRNA group and blank group had clear statistic significance (P<0.05), as well as there were statistic significance compared with Lipofectamine group and NC-shRNA group (P<0.01).FACS method showed that the SW480 cells number at phase G0/G1 increased obviously in hTERT-shRNA group. This pointed out that the silence S W480 cells increased after transfection plasmid hTERT-shRNA, compared with NC-shRNA group, the proliferation index(PI) went down about 14.2%,there was obvious statistics significance (P<0.05). TUNEL assay showed that the number of apoptotic cells for hTERT-shRNA group increased remarkably and the apoptosis index(AI) was evidently higher than that of other groups, it had obvious statistics significance (P<0.01). Transmission Electron Microscope assay showed that nucleolus fissions seen in some cells, MMP went down markedly and fluorescent intensity of Rh 123 enhanced for hTERT-shRNA group, compared with other groups, the difference had statistic significance(P<0.01). Transmission Electron Microscope:the volume of SW480 cells got smaller significantly, the minovillus and protrusions reduce, some disappeared even, chromatin gathered unevenly along the nuclear membrane. Lunularosome could be seen and more vacuolizations increased in part of cells.Conclusions Recombinant eukaryotic expression vector pGPU6/GFP/ Neo-hTERT-shRNA targeting hTERT gene was successfully construct.It c ould silence hTERT gene effectively, be able to inhibit the proliferation andgrowth of human colorectal cancer SW480 cells, reduce the telomer ase activity and promote apoptosis of tumor cells finally. Objective To investigate the treatment effect of recombinant plasmid pGPU6/GFP/Neo-hTERT-shRNA targeting hTERT gene on human colorectal cancer SW480 cells in transplanted nude mice.Methods Human colorectal cancer SW480 cells were subcutaneously implanted under the skin of the right armpit to establish nude mice models of colorectal cancer, after the tumors grew to a definite size, the mice were randomly divided into three groups:normal saline (NS group), NC-shRNA group and hTERT-shRNA group. Each group was respectively treated for 6 consecutive times, the growth status of the tumor was observed, tumor volume was measured, tumor growth curve was drawn, tumor tissue morphology was observed with HE staining, the expression of hTERT protein in the tumor was detected by immunohistochemistry, cell apoptosis was inspected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL), the expression of hTERT mRNA was checked by RT-PCR and the telomerase activity was detected by PCR-TRAP-ELISA.Results All nude mouce after implanted SW480 cells subcutaneously at the 14th day had formed tumors and the diameters of tumor nodules were up to 5-7mm.The transplanted nude mice models had been constructed successfully. The rate is of 100% into tumors.After nude mouce starting treatment, the growth of tumor volume in hTERT-shRNA group became slower than the NS group and the NC-ShRNA group, and significantly slower at the beginning of the 18th days. HE assay showed that partial tumor tissue in hTERT-shRNA group presented necrosis and tumor cells morphology changed obviously. Immunohistochemistry detection showed that the expression levels of hTERT protein in tumor tissue of hTERT-shRNA group decreased, and a small amount of hTERT protein positive cells had been seen. TUNEL assay showed that the number of apoptotic cells in hTERT-shRNA group increased significantly and distributed densely. Compared with the NS group and the NC-shRNA group, the apoptosis index in hTERT-shRNA group increased by 29.4% and 31.1% separately,the difference was statistically significant (P<0.01). RT-PCR analysis showed that the expression levels of hTERT mRNA in hTERT-shRNA group compared with NS group and NC-shRNA group decreased by 52.1% and 48.3% respectively,and the differences were significant(P<0.01).PCR-TRAP-ELISA analysis showed that the telomerase activity of hTERT-shRNA group was observably lower than NS group and NC-shRNA group, the inhibition rates were 48.5% and 53.0% respectively, the differences were statistically significant (P<0.05).Conclusions Recombinant plasmid pGPU6/GFP/Neo-hTERT-shRNA inhibits the growth and promotes apoptosis of implanted human colorectal cancer by down-regulating the expression of hTERT mRNA和hTERT protein in tumor tissues.更多还原