LKB1慢病毒表达载体的构建及其在子宫内膜癌HEC-1A细胞中的表达

发布时间：2018-08-30 10:27

【摘要】：第一章LKB1基因慢病毒表达载体的构建与鉴定【摘要】目的:探讨慢病毒系统介导的LKB1基因在HEC-1A细胞中的过表达，为进一步研究LKB1基因在子宫内膜癌的作用机制奠定基础。方法:以PCR扩增LKB1克隆质粒获得全长cDNA，，将LKB1cDNA链接到慢病毒载体pWPI，构建慢病毒表达质粒LKB1/pWPI。通过与包装质粒pCMV-Dr8.74和pMD2.G共转染293T细胞进行病毒包装，用包装成功后的病毒液侵染子宫内膜癌HEC-1A细胞，荧光定量PCR法检测HEC-1A细胞中LKB1的相对表达量。结果：成功扩增LKB1全长cDNA，和构建LKB1重组慢病毒表达载体LKB1/pWPI。转染包装293T细胞后能产生慢病毒颗粒并能有效感染靶细胞HEC-1A。转染后HEC-1A-LKB1-PWPI细胞中LKB1的表达率明显高于亲本细胞和空白对照细胞（p＜0.01）。结论：成功构建携带LKB1基因的慢病毒表达载体，包装病毒后能有效地感染子宫内膜癌HEC-1A细胞，为进一步探讨LKB1基因在子宫内膜癌中的生物学效应奠定基础。第二章LKB1基因对子宫内膜癌HEC-1A细胞生物学影响的体外实验研究目的：探索子宫内膜癌HEC-1A细胞中LKB1基因表达上调后对其生物学功能的影响。方法：平板克隆实验检测三组细胞（LKB1-pWPI-HEC-1A、pWPI-HEC-1A、HEC-1A）的单克隆能力，MTT测定三组细胞的生长增殖情况，流式细胞术检测三组细胞的生长周期。结果：实验组细胞（LKB1-pWPI-HEC-1A）与两对照组（pWPI-HEC-1A、HEC-1A）比较：①实验组细胞单克隆能力较两个对照组减弱（P＜0.05）。②实验组细胞生长速度较两个对照组减慢（P＜0.05）。③实验组细胞G0/G1期细胞的比例明显高于两个对照组（P＜0.05）。结论：LKB1基因抑制了HEC-1A细胞的单克隆能力，同时其对于子宫内膜癌HEC-1A细胞的生长起到抑制作用，将细胞周期阻滞于G0/G1期。
[Abstract]:Chapter 1 Construction and identification of lentivirus expression vector of LKB1 gene [abstract] AIM: to investigate the overexpression of LKB1 gene mediated by lentivirus system in HEC-1A cells. To further study the role of LKB1 gene in endometrial carcinoma mechanism. Methods: the LKB1 clone plasmid was amplified by PCR and the full-length cDNA, was obtained. The LKB1cDNA was linked to the lentivirus vector pWPI, to construct the lentivirus expression plasmid LKB1/pWPI.. The viral packaging was carried out by co-transfecting 293T cells with packaging plasmids pCMV-Dr8.74 and pMD2.G. The viral solution was used to infect endometrial carcinoma HEC-1A cells. The relative expression of LKB1 in HEC-1A cells was detected by fluorescence quantitative PCR. Results: the full-length cDNA, of LKB1 was amplified successfully and the recombinant lentivirus expression vector LKB1/pWPI. was constructed. After transfection of packaging 293T cells, lentivirus particles can be produced and HEC-1A. of target cells can be infected effectively. The expression rate of LKB1 in HEC-1A-LKB1-PWPI cells after transfection was significantly higher than that in parent cells and blank control cells (p < 0. 01). Conclusion: the lentivirus expression vector carrying LKB1 gene can be successfully constructed to infect endometrial carcinoma HEC-1A cells effectively after packaging virus, which lays a foundation for further study of the biological effect of LKB1 gene in endometrial carcinoma. Chapter II effects of LKB1 gene on the biology of endometrial carcinoma HEC-1A cells in vitro objective: to explore the effect of up-regulation of LKB1 gene expression on the biological function of endometrial carcinoma HEC-1A cells. Methods: the monoclonal ability of three groups of cells (LKB1-pWPI-HEC-1A,HEC-1A) was detected by plate cloning assay. The growth and proliferation of three groups of cells were measured by flow cytometry. The growth cycle of three groups of cells was detected by flow cytometry. Results: compared with the two control groups (pWPI-HEC-1A,HEC-1A), the Monoclonal ability of the LKB1-pWPI-HEC-1A cells in the experimental group was lower than that in the two control groups (P < 0. 05). 2. The growth rate of the cells in the experimental group was slower than that in the two control groups (P < 0. 05). The proportion of cells was significantly higher than that of two control groups (P < 0.05). Conclusion the cell cycle of HEC-1A cells was blocked by the cell cycle arrest of the cell cycle of HEC-1A cell line, which inhibited the growth of HEC-1A cells and inhibited the Monoclonal ability of HEC-1A cells by the gene of 1: LKB1.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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