兰州大学机构库 >学院待认领
转染自杀基因联合多细胞因子的组织特异性真核表达载体在膀胱癌细胞的抑制效应研究
Alternative TitleThe Inhibitory Effect of Suicide Gene Transfection Combined Multi-Tissue-Specific Cytokine Eukaryotic Expression Vector For Bladder Cancer
郝有诚
Thesis Advisor田俊强
2014-05-28
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword膀胱癌细胞 IL-2基因 TRAIL基因 TNF-a基因
Abstract目的:探讨已经构建成功的五种共表达自杀基因联合多细胞因子基因的泌尿组织特异性真核表达载体pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-ɑ、pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR对人膀胱癌细胞5637的抑制作用。 方法:将已经构建并行测序、PCR及限制性核酸内切酶酶切证明的双启动子载体pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-ɑ、pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR分别转染至膀胱癌细胞5637,通过抗性标志筛选出抗zeocin克隆,应用westen blot检测五种共表达载体IL-2、TNF-ɑ、TRAIL蛋白质的表达量,将其与未转染膀胱癌细胞5637以及转染空载体pBud4.1的靶细胞做比较;PCR分析转染前后以及转染不同载体后IL-2、TRAIL、TNF-a mRNA的表达,设未转染膀胱癌细胞5637作为对照,对比其mRNA的表达量;MTT的方法检测五种靶细胞与未转染以及转染空载体pBud4.1的5637细胞抑制率的区别,并观察加入25ug/ml GCV后五种载体对膀胱癌细胞5637细胞抑制率的影响;使用流式细胞仪检测五种靶细胞与未转染膀胱癌细胞5637的凋亡率,将其结果进行统计学分析。 结果:westen blot结果显示,转染五种共表达载体后,靶细胞相应的细胞因子蛋白的表达明显高于未转染以及空载体5637细胞;RT-PCR证实转染后5637细胞中IL-2、TRAIL、TNF-a的mRNA表达量明显增加;流式细胞仪结果提示转染五种共表达载体后,细胞凋亡率明显高于转染空白载体组和未转染组(P<0.01);MTT结果亦显示转染共表达载体后,细胞增殖受到明显抑制(P<0.01) ,加入25ug/ml GCV后细胞抑制率明显增高(P<0.01)。 结论:包含UPII启动子驱动的HSV-TK基因、并分别搭配IL-2、TNF-ɑ、TRAIL、IL-2-TNF-ɑ(IT)、IL-2-TRAIL(ITR)的五种双启动子重组真核表达质粒,可有效加快膀胱癌细胞5637的凋亡,在GCV的作用下,膀胱癌细胞5637的细胞抑制率明显增加,为进一步研究细胞因子联合及自杀基因联合多细胞因子的泌尿组织特异性真核表达载体应用于膀胱癌的治疗奠定了基础。
Other AbstractObjective Explore five kinds of multi-joint suicide gene expression of cytokines urinary tissue-specific gene eukaryotic expression vector pBud-UPII-TK/CMV-IL-2,pBud-UPII-TK/CMV-TNF-ɑ,pBud-UPII-TK/CMV-TRAIL,pBud-UPII-TK/CMV-IT,pBud-UPII-TK/CMV-ITR’s inhibition for human bladder cancer 5637. Methods Transfect the Dual promoter vector pBud-UPII-TK/CMV-IL-2, pBud-UPII-TK/CMV-TNF-ɑ, pBud-UPII-TK/CMV-TRAIL, pBud-UPII-TK/CMV-IT, pBud-UPII-TK/CMV-ITR into bladder cancer cells 5637,which we built and parallel sequencing, PCR and restriction endonuclease digestion proved before, screened Zeocin resistant clones by resistance marker. detection the expression of five vector IL-2, TNF-ɑ, TRAIL protein expression by westen blot, compared it with untransfected bladder cancer cells 5637 transfected with empty vector and target cells pBud4.1; analysis IL-2/TNF-a and TRAIL mRNA expression before and after transfection and transfection of different carriers, Let untransfected bladder cancer cell 5637 as a control, compare the difference expression of its mRNA ; MTT was used to detect the difference between five target cells, non-transfected and transfected with the empty vector pBud4.1 5637 cells’s inhibition rate, And observe the effects of inhibition rate five vector on bladder cancer 5637 cell after adding 25ug/ml GCV; Using flow cytometry detect apoptosis rate’s difference between five target cells and untransfected bladder cancer cell 5637, and its results were Statistical analysised. Results Westen blot results showed that after five co-transfected expression vectors expressing target cells corresponding cytokine protein was significantly higher than non-transfected cells and the empty vector 5637; RT-PCR confirmed in 5637 after transfection cells were IL-2, TRAIL , TNF-a mRNA expression was significantly increased; flow cytometry results after five tips were transfected with an expression vector, the apoptotic rate was significantly higher than the group transfected with empty vector and non-transfected group (P <0.01); MTT the results also show that co-expression vector transfected cell proliferation was significantly inhibited (P <0.01), the inhibition rate was significantly increased after adding 25ug/ml GCV (P <0.01). Conclusion UPII promoter-driven HSV-TK gene, with IL-2, TNF-ɑ, TRAIL, IL-2-TNF-ɑ (IT), IL-2-TRAIL (ITR) these five dual promoters reorganization eukaryotic expression plasmid, which can effectively accelerate apoptosi...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222162
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
郝有诚. 转染自杀基因联合多细胞因子的组织特异性真核表达载体在膀胱癌细胞的抑制效应研究[D]. 兰州. 兰州大学,2014.
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