抑癌基因PTEN在不同肿瘤细胞株的表达及其对CIK杀伤肿瘤细胞功能的影响

发布时间：2018-03-31 07:12

本文选题：肿瘤　切入点：PTEN　出处：《广东药科大学》2016年硕士论文

【摘要】：目的通过高分辨率熔解曲线(High Resolution Melt,HRM)法分析不同肿瘤细胞系抑癌基因PTEN的突变状态,探讨PTEN基因在不同肿瘤细胞中的突变频率。运用荧光定量PCR的方法检测PTEN基因在各肿瘤细胞系中表达水平差异,转染改变肿瘤细胞PTEN的相对表达水平探讨其对肿瘤细胞增殖、周期、迁移以及相关免疫分子的表达,并用钙黄绿素法测定健康人CIK细胞对不同肿瘤细胞的杀伤效率,分析不同PTEN相对表达水平对CIK细胞杀伤效率的影响,为今后基于PTEN的肿瘤基因疗法和CIK免疫疗法提供一定的理论依据和参考。方法一、不同肿瘤细胞系抑癌基因PTEN的突变研究按照ATCC中相应细胞的培养方法,培养并收集各肿瘤细胞,基因组试剂盒法提取细胞基因组DNA,设计特异性突变筛选引物,运用HRM分析方法筛选各肿瘤细胞PTEN基因突变的状态,并进一步测序验证HRM筛选结果,分析HRM法筛选肿瘤细胞PTEN基因突变的可行性与准确性,确定各不同肿瘤细胞抑癌基因PTEN的突变频率。二、荧光定量PCR法分析不同肿瘤细胞PTEN表达水平差异的研究培养并收集各肿瘤细胞,提取各肿瘤细胞的总RNA,RT逆转录合成c DNA第一链,采用SYBGreen染料荧光定量PCR法检测各肿瘤细胞PTEN的表达水平,并比较分析不同肿瘤细胞PTEN表达水平的差异。三、PTEN对肿瘤细胞生理特性的影响及其表达水平对免疫相关分子表达的影响通过转染的方式改变肿瘤细胞PTEN的相对表达水平,探讨PTEN表达变化对肿瘤细胞增殖、迁移、周期的影响;运用RT-PCR及琼脂糖电泳半定量的方法分析肿瘤细胞PTEN转染前后不同PTEN表达水平与P38MAPK,协同刺激分子CD80/86,B7-H1,B7-H4,以及趋化因子受体CCR6和CCR7表达水平之间的变化关系,为探讨PTEN表达水平差异对CIK杀伤敏感性的影响奠定基础。四、健康人CIK细胞对不同肿瘤细胞杀伤效率差异及与不同肿瘤细胞不同PTEN相对表达水平关系的研究钙黄绿素法测定健康人CIK细胞作用于各不同肿瘤靶细胞的杀伤效率,比较CIK细胞对不同肿瘤靶细胞杀伤效率的差异,并分析CIK细胞杀伤效率与肿瘤靶细胞PTEN相对表达水平的相关性,真核表达质粒pc DNA3.1-PTEN转染Hep G2和Hela肿瘤细胞,比较转染前后CIK细胞对其杀伤效率变化,探讨肿瘤靶细胞PTEN表达变化对CIK细胞对其杀伤敏感性的影响。结果一、各肿瘤细胞系抑癌基因PTEN序列基本正常,较少发生突变。利用HRM法筛选多种不同来源肿瘤细胞PTEN基因突变频率,并且通过sanger法测序验证HRM筛选结果的准确性,结果发现几乎所有肿瘤细胞PTEN基因均呈野生型表达,基因碱基序列与NCBI公布序列(Gen Bank:AH007803.1)完全一致,HRM筛选只发现Jurkat细胞系熔解曲线与其他细胞熔解曲线存在较明显差异,通过TA克隆测序进一步验证了Jurkat细胞系PTEN基因为部分碱基增加突变,测序结果也证明了HRM筛选肿瘤细胞PTEN基因突变的可行性和准确性。二、各不同肿瘤细胞系PTEN相对表达水平存在差异。提取各肿瘤细胞总RNA,Oligo(d T)逆转录合成c DNA,荧光定量PCR检测各样本PTEN m RNA转录水平,结果发现不同肿瘤细胞PTEN相对表达水平差异大小不一,部分存在较明显差异,且与正常人PBMC相比均表达偏低,不同肿瘤细胞不同PTEN表达水平可以为研究PTEN对肿瘤细胞生理活性及CIK对肿瘤细胞免疫反应影响的不同提供基础依据。三、转染提高肿瘤细胞PTEN相对表达水平可以抑制肿瘤细胞增值,阻滞周期运转,减慢肿瘤细胞迁移修复速度,并且可以改变肿瘤细胞免疫相关分子的表达转染Hep G2和Hela细胞使其过表达PTEN,MTT法测定转染前后细胞活力变化,结果证明过表达PTEN可以降低肿瘤细胞增值活力,抑制肿瘤细胞增殖;PI染色法测定转染前后细胞周期变化,结果说明PTEN可以使肿瘤细胞发生G0/G1到S期细胞周期阻滞;划痕迁移修复法测定过表达PTEN对肿瘤细胞迁移修复能力的变化,结果证明过表达PTEN可以削弱肿瘤细胞迁移修复能力。RT-PCR半定量法比较PTEN转染前后Hep G2和Hela细胞的PTEN相对表达水平与P38、CD80/86、B7H1、B7H4、CCR6、CCR7相对表达水平关系,结果证明PTEN表达水平不同可以影响P38、CD80/86、B7H1、B7H4、CCR6、CCR7的表达变化,PTEN影响肿瘤细胞的生理特性及相关免疫分子表达为进一步研究肿瘤细胞PTEN相对表达对CIK对其杀伤效率的影响提供理论依据。四、健康人CIK对不同肿瘤靶细胞杀伤效率差异不同,并与肿瘤靶细胞自身PTEN相对表达水平呈负相关性诱导成的健康人CIK细胞以不同肿瘤细胞为靶细胞,钙黄绿素法测量杀伤效率差异,结果发现健康人CIK细胞对不同肿瘤靶细胞杀伤效率存在差异,并且相关性分析说明CIK细胞杀伤效率与肿瘤靶细胞PTEN相对表达水平之间呈现出明显负相关(P0.05),进一步研究构建重组表达体系pc DNA3.1-PTEN转染肿瘤细胞改变PTEN相对表达水平,发现改变肿瘤靶细胞PTEN表达水平后CIK对其的杀伤效率出现了下调的趋势。结论成功建立HRM筛选肿瘤细胞PTEN基因突变的方法,鉴定了各肿瘤细胞PTEN基因的突变频率和表达水平差异;证明了PTEN可以抑制肿瘤细胞增殖,阻滞周期运转及削弱肿瘤细胞迁移修复,并进一步证明了PTEN表达水平差异可以影响肿瘤细胞相关免疫分子的表达;证明了CIK细胞对不同肿瘤靶细胞杀伤效率存在差异,发现并证实了CIK细胞的免疫杀伤效率与肿瘤靶细胞PTEN的相对表达水平存在负相关。
[Abstract]:Objective by high resolution melting curve (High Resolution, Melt, HRM) method for the analysis of different tumor cell lines mutation of tumor suppressor gene PTEN, to investigate the mutation frequency of PTEN gene in different tumor cells. The expression level of using the method of fluorescence quantitative PCR detection of PTEN gene in various tumor cell lines, relative expression of transfected tumor cells change the level of PTEN to investigate the cycle of tumor cell proliferation, migration and expression of related immune molecules, and calcein method for the determination of healthy human CIK cells on different tumor cell killing efficiency, analysis the relative expression level of CIK cells influence the killing efficiency of different PTEN, PTEN for tumor gene therapy and immunotherapy CIK based on a theoretical and reference method. A study of different tumor cell lines, mutation of tumor suppressor gene PTEN in cultured ATCC cells in corresponding culture method. Raise and collect the tumor cell, extracting genomic DNA genome kit method, design specific mutation screening primers, using the HRM analysis method of screening the tumor cell PTEN gene mutation status, and further sequencing HRM screening results, analysis of the accuracy and feasibility of screening of tumor cell PTEN gene mutation by HRM, to determine the mutation frequency of each different tumor cells of tumor suppressor gene PTEN. Two, analysis of the differences in the expression levels of the culture and collection of the tumor cells in different tumor cell PTEN fluorescence quantitative PCR method, total RNA was extracted from the tumor cell, RT reverse transcription of C first strand of DNA expression level by SYBGreen dye fluorescence quantitative PCR method for detection of PTEN tumor cells comparative analysis of different tumor cells, and PTEN expression. Three, the expression and expression of immune related molecular effects of PTEN on physiological characteristics of tumor cells The relative expression changes of tumor cells by transfection of PTEN the level of expression of PTEN on the migration of tumor cell proliferation, cycle effect; level and P38MAPK expression in different tumor cells before and after PTEN PTEN transfection analysis by semi quantitative RT-PCR and agarose gel electrophoresis, costimulatory molecules CD80/86, B7-H1, B7-H4, and the change trend chemokine receptors CCR6 and CCR7 expression levels, lay the foundation for the study of PTEN expression level difference sensitivity effect on CIK. Four healthy people, CIK cell killing efficiency on different tumor cells and tumor cells with different PTEN expression determination in different tumor target cell killing efficiency function of CIK cells in healthy individuals study on the relationship between the level of calcein biotin method, differences of the efficiency of killing CIK cells on different tumor cells, and analyze the CIK cell killing efficiency and swelling The relative expression level of correlation between PTEN tumor target cells, eukaryotic expression plasmid PC DNA3.1-PTEN was transfected into Hep G2 cells and Hela cells, comparing the transfection efficiency of CIK cells before and after the change of the killing of tumor cells, the expression of PTEN on sensitivity of the effect on cytotoxicity of CIK cells. As a result, the tumor suppressor gene PTEN sequence normal tumor cell lines, fewer mutations. Using HRM method for screening different tumor cell derived PTEN gene mutation frequency and accuracy by the method of Sanger sequencing of HRM screening results, we found that almost all tumor cells showed a wild-type PTEN gene expression, gene sequence with the published NCBI sequence (Gen Bank:AH007803.1) exactly the same, only HRM screening found that there were obvious differences between Jurkat cells and other cells melting melting curve, through TA sequencing to verify the Jurkat cell line PTEN Gene mutation increases part of the base, the sequencing results also demonstrated that HRM gene mutation screening of tumor cell PTEN. The feasibility and accuracy of two, the relative expression level differences in different tumor cell lines. The tumor cell PTEN extraction of total RNA, Oligo (D T) C was synthesized with DNA, fluorescence quantitative PCR detection of each sample PTEN m RNA the level of transcription, the results showed that different tumor cells PTEN relative expression level differences in size, there are obvious differences, and compared with normal PBMC were low expression, different tumor cells with different expression levels of PTEN can provide the basis for the research of PTEN has different effects on the physiological activity of tumor cells and CIK on tumor cell immune response. Three, improve the transfection of tumor cell PTEN relative expression level can inhibit tumor cell proliferation, block cycle, slow down the speed of tumor cell migration and repair, can change the tumor Cell immune related molecules G2 and Hep transfected Hela cells which overexpressed PTEN were measured before and after transfection, cell viability by MTT, results show that overexpression of PTEN could reduce tumor cell proliferation, inhibit tumor cell proliferation; cell cycle was measured before and after transfection by PI staining, the results indicated that PTEN can make the tumor cells G0/G1 to S cell cycle arrest; Determination of expression of PTEN on tumor cell migration ability to repair the scratch migration repair method, results showed that overexpression of PTEN can reduce tumor cell migration ability to repair the relative expression level of P38 and.RT-PCR semi quantitative method to compare PTEN Hep and Hela cells before and after transfection of G2 PTEN CD80/86, B7H1, B7H4. CCR6, the relative expression level of CCR7, results show that the expression of PTEN can influence the P38, CD80/86, B7H1, B7H4, CCR6, the expression of CCR7, PTEN influence tumor cell physiology The expression characteristics and related immune molecules for further study of tumor cell PTEN relative expression of CIK and to provide a theoretical basis for the effect of killing efficiency. Four healthy people, CIK killing efficiency on different tumor cells, and tumor cells PTEN and its relative expression in different tumor cells as the target cells was negatively correlated to induction healthy human CIK cells, calcein method to measure the killing efficiency differences, the results showed that health human killer CIK cells on different tumor cells efficiency difference and correlation analysis showed that the efficiency and the relative expression of PTEN tumor cells showed a significant negative correlation between CIK cell (P0.05), further study on the recombinant expression system of PC DNA3.1-PTEN transfection of tumor cells to change the relative expression level of PTEN, found that the change of tumor target cells the expression level of PTEN CIK after the killing efficiency appeared The downward trend. Conclusion the establishment of HRM method for screening tumor cell PTEN gene mutations, the mutation frequency of PTEN gene in tumor cells and the expression level of the identification; proves that PTEN can inhibit tumor cell proliferation and migration of tumor cells to repair operation and weaken the block cycle, and further proves the expression difference of PTEN expression level can affect tumor cells related immune molecules; that CIK cells on different tumor cell killing efficiency differences, found and confirmed the existence of negative correlation with the relative expression level of cytotoxicity of tumor cells in PTEN CIK cells.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.5

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