本文選題：腫瘤　切入點(diǎn)：PTEN　出處：《廣東藥科大學(xué)》2016年碩士論文

【摘要】：目的通過高分辨率熔解曲線(High Resolution Melt,HRM)法分析不同腫瘤細(xì)胞系抑癌基因PTEN的突變狀態(tài),探討PTEN基因在不同腫瘤細(xì)胞中的突變頻率。運(yùn)用熒光定量PCR的方法檢測PTEN基因在各腫瘤細(xì)胞系中表達(dá)水平差異,轉(zhuǎn)染改變腫瘤細(xì)胞PTEN的相對表達(dá)水平探討其對腫瘤細(xì)胞增殖、周期、遷移以及相關(guān)免疫分子的表達(dá),并用鈣黃綠素法測定健康人CIK細(xì)胞對不同腫瘤細(xì)胞的殺傷效率,分析不同PTEN相對表達(dá)水平對CIK細(xì)胞殺傷效率的影響,為今后基于PTEN的腫瘤基因療法和CIK免疫療法提供一定的理論依據(jù)和參考。方法一、不同腫瘤細(xì)胞系抑癌基因PTEN的突變研究按照ATCC中相應(yīng)細(xì)胞的培養(yǎng)方法,培養(yǎng)并收集各腫瘤細(xì)胞,基因組試劑盒法提取細(xì)胞基因組DNA,設(shè)計(jì)特異性突變篩選引物,運(yùn)用HRM分析方法篩選各腫瘤細(xì)胞PTEN基因突變的狀態(tài),并進(jìn)一步測序驗(yàn)證HRM篩選結(jié)果,分析HRM法篩選腫瘤細(xì)胞PTEN基因突變的可行性與準(zhǔn)確性,確定各不同腫瘤細(xì)胞抑癌基因PTEN的突變頻率。二、熒光定量PCR法分析不同腫瘤細(xì)胞PTEN表達(dá)水平差異的研究培養(yǎng)并收集各腫瘤細(xì)胞,提取各腫瘤細(xì)胞的總RNA,RT逆轉(zhuǎn)錄合成c DNA第一鏈,采用SYBGreen染料熒光定量PCR法檢測各腫瘤細(xì)胞PTEN的表達(dá)水平,并比較分析不同腫瘤細(xì)胞PTEN表達(dá)水平的差異。三、PTEN對腫瘤細(xì)胞生理特性的影響及其表達(dá)水平對免疫相關(guān)分子表達(dá)的影響通過轉(zhuǎn)染的方式改變腫瘤細(xì)胞PTEN的相對表達(dá)水平,探討PTEN表達(dá)變化對腫瘤細(xì)胞增殖、遷移、周期的影響;運(yùn)用RT-PCR及瓊脂糖電泳半定量的方法分析腫瘤細(xì)胞PTEN轉(zhuǎn)染前后不同PTEN表達(dá)水平與P38MAPK,協(xié)同刺激分子CD80/86,B7-H1,B7-H4,以及趨化因子受體CCR6和CCR7表達(dá)水平之間的變化關(guān)系,為探討PTEN表達(dá)水平差異對CIK殺傷敏感性的影響奠定基礎(chǔ)。四、健康人CIK細(xì)胞對不同腫瘤細(xì)胞殺傷效率差異及與不同腫瘤細(xì)胞不同PTEN相對表達(dá)水平關(guān)系的研究鈣黃綠素法測定健康人CIK細(xì)胞作用于各不同腫瘤靶細(xì)胞的殺傷效率,比較CIK細(xì)胞對不同腫瘤靶細(xì)胞殺傷效率的差異,并分析CIK細(xì)胞殺傷效率與腫瘤靶細(xì)胞PTEN相對表達(dá)水平的相關(guān)性,真核表達(dá)質(zhì)粒pc DNA3.1-PTEN轉(zhuǎn)染Hep G2和Hela腫瘤細(xì)胞,比較轉(zhuǎn)染前后CIK細(xì)胞對其殺傷效率變化,探討腫瘤靶細(xì)胞PTEN表達(dá)變化對CIK細(xì)胞對其殺傷敏感性的影響。結(jié)果一、各腫瘤細(xì)胞系抑癌基因PTEN序列基本正常,較少發(fā)生突變。利用HRM法篩選多種不同來源腫瘤細(xì)胞PTEN基因突變頻率,并且通過sanger法測序驗(yàn)證HRM篩選結(jié)果的準(zhǔn)確性,結(jié)果發(fā)現(xiàn)幾乎所有腫瘤細(xì)胞PTEN基因均呈野生型表達(dá),基因堿基序列與NCBI公布序列(Gen Bank:AH007803.1)完全一致,HRM篩選只發(fā)現(xiàn)Jurkat細(xì)胞系熔解曲線與其他細(xì)胞熔解曲線存在較明顯差異,通過TA克隆測序進(jìn)一步驗(yàn)證了Jurkat細(xì)胞系PTEN基因?yàn)椴糠謮A基增加突變,測序結(jié)果也證明了HRM篩選腫瘤細(xì)胞PTEN基因突變的可行性和準(zhǔn)確性。二、各不同腫瘤細(xì)胞系PTEN相對表達(dá)水平存在差異。提取各腫瘤細(xì)胞總RNA,Oligo(d T)逆轉(zhuǎn)錄合成c DNA,熒光定量PCR檢測各樣本PTEN m RNA轉(zhuǎn)錄水平,結(jié)果發(fā)現(xiàn)不同腫瘤細(xì)胞PTEN相對表達(dá)水平差異大小不一,部分存在較明顯差異,且與正常人PBMC相比均表達(dá)偏低,不同腫瘤細(xì)胞不同PTEN表達(dá)水平可以為研究PTEN對腫瘤細(xì)胞生理活性及CIK對腫瘤細(xì)胞免疫反應(yīng)影響的不同提供基礎(chǔ)依據(jù)。三、轉(zhuǎn)染提高腫瘤細(xì)胞PTEN相對表達(dá)水平可以抑制腫瘤細(xì)胞增值,阻滯周期運(yùn)轉(zhuǎn),減慢腫瘤細(xì)胞遷移修復(fù)速度,并且可以改變腫瘤細(xì)胞免疫相關(guān)分子的表達(dá)轉(zhuǎn)染Hep G2和Hela細(xì)胞使其過表達(dá)PTEN,MTT法測定轉(zhuǎn)染前后細(xì)胞活力變化,結(jié)果證明過表達(dá)PTEN可以降低腫瘤細(xì)胞增值活力,抑制腫瘤細(xì)胞增殖;PI染色法測定轉(zhuǎn)染前后細(xì)胞周期變化,結(jié)果說明PTEN可以使腫瘤細(xì)胞發(fā)生G0/G1到S期細(xì)胞周期阻滯;劃痕遷移修復(fù)法測定過表達(dá)PTEN對腫瘤細(xì)胞遷移修復(fù)能力的變化,結(jié)果證明過表達(dá)PTEN可以削弱腫瘤細(xì)胞遷移修復(fù)能力。RT-PCR半定量法比較PTEN轉(zhuǎn)染前后Hep G2和Hela細(xì)胞的PTEN相對表達(dá)水平與P38、CD80/86、B7H1、B7H4、CCR6、CCR7相對表達(dá)水平關(guān)系,結(jié)果證明PTEN表達(dá)水平不同可以影響P38、CD80/86、B7H1、B7H4、CCR6、CCR7的表達(dá)變化,PTEN影響腫瘤細(xì)胞的生理特性及相關(guān)免疫分子表達(dá)為進(jìn)一步研究腫瘤細(xì)胞PTEN相對表達(dá)對CIK對其殺傷效率的影響提供理論依據(jù)。四、健康人CIK對不同腫瘤靶細(xì)胞殺傷效率差異不同,并與腫瘤靶細(xì)胞自身PTEN相對表達(dá)水平呈負(fù)相關(guān)性誘導(dǎo)成的健康人CIK細(xì)胞以不同腫瘤細(xì)胞為靶細(xì)胞,鈣黃綠素法測量殺傷效率差異,結(jié)果發(fā)現(xiàn)健康人CIK細(xì)胞對不同腫瘤靶細(xì)胞殺傷效率存在差異,并且相關(guān)性分析說明CIK細(xì)胞殺傷效率與腫瘤靶細(xì)胞PTEN相對表達(dá)水平之間呈現(xiàn)出明顯負(fù)相關(guān)(P0.05),進(jìn)一步研究構(gòu)建重組表達(dá)體系pc DNA3.1-PTEN轉(zhuǎn)染腫瘤細(xì)胞改變PTEN相對表達(dá)水平,發(fā)現(xiàn)改變腫瘤靶細(xì)胞PTEN表達(dá)水平后CIK對其的殺傷效率出現(xiàn)了下調(diào)的趨勢。結(jié)論成功建立HRM篩選腫瘤細(xì)胞PTEN基因突變的方法,鑒定了各腫瘤細(xì)胞PTEN基因的突變頻率和表達(dá)水平差異;證明了PTEN可以抑制腫瘤細(xì)胞增殖,阻滯周期運(yùn)轉(zhuǎn)及削弱腫瘤細(xì)胞遷移修復(fù),并進(jìn)一步證明了PTEN表達(dá)水平差異可以影響腫瘤細(xì)胞相關(guān)免疫分子的表達(dá);證明了CIK細(xì)胞對不同腫瘤靶細(xì)胞殺傷效率存在差異,發(fā)現(xiàn)并證實(shí)了CIK細(xì)胞的免疫殺傷效率與腫瘤靶細(xì)胞PTEN的相對表達(dá)水平存在負(fù)相關(guān)。
[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.

【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R730.5

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