【摘要】：第一章LKB1基因慢病毒表達(dá)載體的構(gòu)建與鑒定 【摘要】目的:探討慢病毒系統(tǒng)介導(dǎo)的LKB1基因在HEC-1A細(xì)胞中的過(guò)表達(dá)，為進(jìn)一步研究LKB1基因在子宮內(nèi)膜癌的作用機(jī)制奠定基礎(chǔ)。方法:以PCR擴(kuò)增LKB1克隆質(zhì)粒獲得全長(zhǎng)cDNA，，將LKB1cDNA鏈接到慢病毒載體pWPI，構(gòu)建慢病毒表達(dá)質(zhì)粒LKB1/pWPI。通過(guò)與包裝質(zhì)粒pCMV-Dr8.74和pMD2.G共轉(zhuǎn)染293T細(xì)胞進(jìn)行病毒包裝，用包裝成功后的病毒液侵染子宮內(nèi)膜癌HEC-1A細(xì)胞，熒光定量PCR法檢測(cè)HEC-1A細(xì)胞中LKB1的相對(duì)表達(dá)量。結(jié)果：成功擴(kuò)增LKB1全長(zhǎng)cDNA，和構(gòu)建LKB1重組慢病毒表達(dá)載體LKB1/pWPI。轉(zhuǎn)染包裝293T細(xì)胞后能產(chǎn)生慢病毒顆粒并能有效感染靶細(xì)胞HEC-1A。轉(zhuǎn)染后HEC-1A-LKB1-PWPI細(xì)胞中LKB1的表達(dá)率明顯高于親本細(xì)胞和空白對(duì)照細(xì)胞（p＜0.01）。結(jié)論：成功構(gòu)建攜帶LKB1基因的慢病毒表達(dá)載體，包裝病毒后能有效地感染子宮內(nèi)膜癌HEC-1A細(xì)胞，為進(jìn)一步探討LKB1基因在子宮內(nèi)膜癌中的生物學(xué)效應(yīng)奠定基礎(chǔ)。 第二章LKB1基因?qū)ψ訉m內(nèi)膜癌HEC-1A細(xì)胞生物學(xué)影響的體外實(shí)驗(yàn)研究 目的：探索子宮內(nèi)膜癌HEC-1A細(xì)胞中LKB1基因表達(dá)上調(diào)后對(duì)其生物學(xué)功能的影響。方法：平板克隆實(shí)驗(yàn)檢測(cè)三組細(xì)胞（LKB1-pWPI-HEC-1A、pWPI-HEC-1A、HEC-1A）的單克隆能力，MTT測(cè)定三組細(xì)胞的生長(zhǎng)增殖情況，流式細(xì)胞術(shù)檢測(cè)三組細(xì)胞的生長(zhǎng)周期。結(jié)果：實(shí)驗(yàn)組細(xì)胞（LKB1-pWPI-HEC-1A）與兩對(duì)照組（pWPI-HEC-1A、HEC-1A）比較：①實(shí)驗(yàn)組細(xì)胞單克隆能力較兩個(gè)對(duì)照組減弱（P＜0.05）。②實(shí)驗(yàn)組細(xì)胞生長(zhǎng)速度較兩個(gè)對(duì)照組減慢（P＜0.05）。③實(shí)驗(yàn)組細(xì)胞G0/G1期細(xì)胞的比例明顯高于兩個(gè)對(duì)照組（P＜0.05）。結(jié)論：LKB1基因抑制了HEC-1A細(xì)胞的單克隆能力，同時(shí)其對(duì)于子宮內(nèi)膜癌HEC-1A細(xì)胞的生長(zhǎng)起到抑制作用，將細(xì)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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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