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

发布时间：2018-03-25 06:15

本文选题：膀胱癌细胞　切入点：IL-2基因　出处：《兰州大学》2014年硕士论文

【摘要】：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-α、 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-α 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westenblot检测五种共表达载体IL-2、TNF-α、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(P0.01)；MTT结果亦显示转染共表达载体后,细胞增殖受到明显抑制(P0.01),加入25ug/ml GCV后细胞抑制率明显增高(P0.01)。结论：包含UPⅡ启动子驱动的HSV-TK基因、并分别搭配IL-2、TNF-α、TRAIL、 IL-2-TNF-α (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。
[Abstract]:Objective: To evaluate the construction of five successful co expression gene combined with cytokines gene Dutch act in urinary tissue specific eukaryotic expression vector pBud-UPII-TK/CMV-IL-2, pBud-UPII-TK/CMV-TNF- alpha, pBud-UPII-TK/CMV-TRAIL, pBud-UPII-TK/CMV-IT, pBud-UPII-TK/CMV-ITR on the inhibition of human bladder cancer cell 5637.
Methods: constructed parallel sequencing, dual promoter vector pBud-UPII-TK/CMV-IL-2 PCR and restriction endonuclease proof, pBud-UPII-TK/CMV-TNF- alpha pBud-UPII-TK/CMV-TRAIL, pBud-UPII-TK/CMV-IT, pBud-UPII-TK/CMV-ITR were transfected into bladder cancer cell 5637, the resistance signs were resistant to zeocin cloning, application of westenblot detection of five kinds of co expression vector IL-2, TNF- alpha, TRAIL expression the protein, compared with non transfected bladder cancer cells transfected with vector pBud4.1 and 5637 target cells; PCR analysis before and after transfection and transfection of different carriers after IL-2, TRAIL, TNF-a expression of mRNA, a non transfected bladder cancer cells 5637 as control, the expression of mRNA of MTT; difference detection method five kinds of target cells and non transfected 5637 cells transfected with vector and the inhibition rate of pBud4.1, and to observe the 25ug/ml GCV after five The effect of carriers on the inhibition rate of bladder cancer cell 5637 was analyzed. The apoptosis rate of five target cells and 5637 cells without transfected bladder cancer cells was detected by flow cytometry, and the results were statistically analyzed.
Results: westen blot showed that transfection of five co expression vector after cytokine protein expression was significantly higher than that of the corresponding target cells without transfection and 5637 vector transfected IL-2 cells; RT-PCR confirmed, TRAIL 5637 cells, TNF-a mRNA expression was significantly increased; flow cytometry showed that transfection of five co expression after the carrier, the apoptosis rate was significantly higher than that of empty vector transfection group and non transfection group (P0.01); MTT results also showed that co expression vector after transfection, cell proliferation was significantly inhibited (P0.01), GCV after adding 25ug/ml cell inhibition rate was significantly higher (P0.01).
Conclusion: II contains the UP promoter and HSV-TK gene driven, and collocation of TNF- alpha, IL-2, TRAIL, IL-2-TNF- alpha (IT), IL-2-TRAIL (ITR) five kinds of dual promoter recombinant eukaryotic expression plasmid, can effectively accelerate the apoptosis of bladder cancer cell 5637, under the action of GCV cells of bladder cancer 5637 the cell inhibition rate increased significantly, which laid the foundation for the further study of eukaryotic expression of specific cytokines and cytokine Dutch act gene combined with urinary bladder cancer tissue treatment applied to the carrier.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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