LKB1慢病毒表達載體的構建及其在子宮內膜癌HEC-1A細胞中的表達
發(fā)布時間：2018-08-30 10:27
【摘要】：第一章LKB1基因慢病毒表達載體的構建與鑒定 【摘要】目的:探討慢病毒系統(tǒng)介導的LKB1基因在HEC-1A細胞中的過表達，為進一步研究LKB1基因在子宮內膜癌的作用機制奠定基礎。方法:以PCR擴增LKB1克隆質粒獲得全長cDNA，，將LKB1cDNA鏈接到慢病毒載體pWPI，構建慢病毒表達質粒LKB1/pWPI。通過與包裝質粒pCMV-Dr8.74和pMD2.G共轉染293T細胞進行病毒包裝，用包裝成功后的病毒液侵染子宮內膜癌HEC-1A細胞，熒光定量PCR法檢測HEC-1A細胞中LKB1的相對表達量。結果：成功擴增LKB1全長cDNA，和構建LKB1重組慢病毒表達載體LKB1/pWPI。轉染包裝293T細胞后能產生慢病毒顆粒并能有效感染靶細胞HEC-1A。轉染后HEC-1A-LKB1-PWPI細胞中LKB1的表達率明顯高于親本細胞和空白對照細胞（p＜0.01）。結論：成功構建攜帶LKB1基因的慢病毒表達載體，包裝病毒后能有效地感染子宮內膜癌HEC-1A細胞，為進一步探討LKB1基因在子宮內膜癌中的生物學效應奠定基礎。 第二章LKB1基因對子宮內膜癌HEC-1A細胞生物學影響的體外實驗研究 目的：探索子宮內膜癌HEC-1A細胞中LKB1基因表達上調后對其生物學功能的影響。方法：平板克隆實驗檢測三組細胞（LKB1-pWPI-HEC-1A、pWPI-HEC-1A、HEC-1A）的單克隆能力，MTT測定三組細胞的生長增殖情況，流式細胞術檢測三組細胞的生長周期。結果：實驗組細胞（LKB1-pWPI-HEC-1A）與兩對照組（pWPI-HEC-1A、HEC-1A）比較：①實驗組細胞單克隆能力較兩個對照組減弱（P＜0.05）。②實驗組細胞生長速度較兩個對照組減慢（P＜0.05）。③實驗組細胞G0/G1期細胞的比例明顯高于兩個對照組（P＜0.05）。結論：LKB1基因抑制了HEC-1A細胞的單克隆能力，同時其對于子宮內膜癌HEC-1A細胞的生長起到抑制作用，將細胞周期阻滯于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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
本文編號：2212796
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
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