本文選題：肝癌 + 細(xì)胞培養(yǎng)　； 參考：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：研究背景:由于肝癌的發(fā)病機(jī)制尚未完全清楚,其具有發(fā)展迅速、發(fā)現(xiàn)困難、手術(shù)切除后復(fù)發(fā)概率高等特點(diǎn),因此造成了肝癌的高死亡率。近年來許多學(xué)者提出腫瘤異質(zhì)性與腫瘤干細(xì)胞的觀點(diǎn),以新的角度重新認(rèn)識(shí)肝癌,同時(shí)指出肝癌干細(xì)胞標(biāo)志物將成為肝癌免疫治療的新靶點(diǎn)。研究目的:1.從肝癌以及癌旁組織中分離腫瘤細(xì)胞,純化得到穩(wěn)定傳代的肝癌細(xì)胞株。2.對(duì)8個(gè)細(xì)胞株進(jìn)行細(xì)胞形態(tài)、生長曲線、遷移與侵襲能力、單細(xì)胞克隆能力、生長周期、細(xì)胞核型、分子標(biāo)志物、致瘤能力等生物學(xué)特性進(jìn)行鑒定。研究方法:1.采用肝裂解液消化結(jié)合剪碎方法分離細(xì)胞,通過反復(fù)差速消化、差速貼壁的方法純化腫瘤細(xì)胞并擴(kuò)大培養(yǎng)。2.利用IncuCyteZOOM長時(shí)間動(dòng)態(tài)細(xì)胞成像及分析系統(tǒng)觀察細(xì)胞形態(tài)、繪制細(xì)胞生長曲線、比較細(xì)胞遷移與侵襲能力以及追蹤單細(xì)胞克隆過程;通過HE染色計(jì)算核質(zhì)比、Giemsa染色分析染色體眾數(shù)和核型;裸鼠體內(nèi)致瘤實(shí)驗(yàn);利用熒光定量PCR檢測細(xì)胞上清液中HBV DNA載量;利用免疫熒光技術(shù)檢測腫瘤細(xì)胞中AFP、GPC3、HepPar-1、CK18、CK19、PCNA、Vimentin蛋白表達(dá);利用流式細(xì)胞細(xì)胞儀檢測細(xì)胞周期及肝癌干細(xì)胞表面標(biāo)志物EpCAM、CD13、CD44、CD90、CD24、CD47、CD133、DLK1表達(dá)情況。采用Plasmocin、BM-Cyclin、MRA等3種藥物對(duì)支原體污染的細(xì)胞進(jìn)行處理,以CLARK一步法試劑、Biotool快速檢測試劑和PCR等3種方法進(jìn)行支原體檢測。研究結(jié)果:在體外分離純化不同來源的肝癌細(xì)胞,建立8株穩(wěn)定傳代的肝癌細(xì)胞株,觀察發(fā)現(xiàn)8株細(xì)胞形態(tài)各異,且細(xì)胞核質(zhì)比異常。對(duì)比MTT法與IncuCyteZOOM提供的相差與熒光5種方法,其中細(xì)胞融合度繪制的生長曲線能準(zhǔn)確反映8株細(xì)胞增殖情況,177T細(xì)胞增殖速度最快,倍增時(shí)間29.70±0.84h,以下依次為216T3、78T、83N、216T2、216N、216T1、92N;比較8株細(xì)胞傷口寬度的變化發(fā)現(xiàn)216T2的遷移與侵襲能力明顯強(qiáng)于其它7株細(xì)胞,216T2傷口愈合需45h±0.82h,而穿透基質(zhì)膠只需要22h±0.45h;利用IncuCyteZOOM全孔成像功能進(jìn)行動(dòng)態(tài)追蹤單細(xì)胞克隆形成過程,表明8株細(xì)胞均能夠在體外形成單細(xì)胞克隆,177T克隆能力最強(qiáng),克隆形成率為38/96;而78T最弱,形成率僅4/96。細(xì)胞周期分析表明92N的S期比例最大,為15.22%±0.60%;染色體眾數(shù)分析顯示216N、216T1為亞四倍體,其余6株細(xì)胞均為亞三倍體。采用熒光定量PCR檢測細(xì)胞上清液中HBV DNA載量,結(jié)果顯示除216N、216T1、216T2外,其他5株細(xì)胞HBV DNA陽性;免疫熒光結(jié)果顯示8株細(xì)胞均不表達(dá)AFP、HepPar-1,表達(dá)GPC3、CK19,且8株細(xì)胞GPC3、CK19表達(dá)強(qiáng)度不一,GPC3在78T等細(xì)胞中表達(dá)強(qiáng)度明顯高,在177T中表達(dá)弱;CK19在78T中表達(dá)最強(qiáng)。與EMT相關(guān)的上皮細(xì)胞標(biāo)志CK18在8株細(xì)胞中均強(qiáng)表達(dá),而間質(zhì)細(xì)胞標(biāo)志物Vimentin卻只在部分細(xì)胞的胞漿中表達(dá),對(duì)比發(fā)現(xiàn)216T2細(xì)胞中表達(dá)Vimentin蛋白的細(xì)胞比例明顯高于其他7株細(xì)胞。PCNA在8株細(xì)胞中表達(dá)差異,從而反映了8株細(xì)胞分化程度不同,其中92N細(xì)胞中PCNA陽性細(xì)胞比例最高,分化程度最低。8種肝癌干細(xì)胞標(biāo)志物分析表明,8株細(xì)胞均高比例表達(dá)CD47,但不表達(dá)DLKI,其余標(biāo)志物陽性細(xì)胞在細(xì)胞株中比例不一,除216T2細(xì)胞外其余細(xì)胞均高表達(dá)EpCAM,EpCAM+細(xì)胞高于90%,而216T2細(xì)胞卻表達(dá)其他細(xì)胞低表達(dá)或不表達(dá)的CD133、CD90、CD24等標(biāo)志物,其中CD90+CD44+細(xì)胞高達(dá)64.20%,CD133+CD44+細(xì)胞比例為17%。單株細(xì)胞內(nèi)的細(xì)胞表達(dá)標(biāo)記物也存在差異,例如78T細(xì)胞中CD47+細(xì)胞比例為88%,而CD47+CD44+細(xì)胞為83.8%,CD47+EpCAM+細(xì)胞約為29%。裸鼠體內(nèi)移植實(shí)驗(yàn)顯示除177、216T3外,其余6株細(xì)胞能使裸鼠致瘤,通過解剖均未發(fā)現(xiàn)體內(nèi)肝、肺轉(zhuǎn)移。8株肝癌干細(xì)胞在培養(yǎng)過程中遭到支原體污染,利用3種方法進(jìn)行清除,3種方法進(jìn)行檢測,結(jié)果顯示:一步法試劑檢測顯示Plasmocin處理14 d后支原體消除,快速檢測試劑檢測顯示BM-Cyclin處理21 d徹底消滅支原體,而MRA經(jīng)PCR檢測發(fā)現(xiàn)徹底消除支原體需要14 d;另外,3種藥物交替使用清除支原體效果更加顯著。研究結(jié)論:經(jīng)過多種細(xì)胞生物學(xué)特性的鑒定獲得8個(gè)肝癌細(xì)胞株,各種標(biāo)志物在8株細(xì)胞系中的差異表達(dá)提示肝癌的起源存在異質(zhì)性,同時(shí)單株細(xì)胞表達(dá)干細(xì)胞標(biāo)志物的差異也反映個(gè)體內(nèi)部存在差異性;通過對(duì)肝癌干細(xì)胞標(biāo)志物的分析為繼續(xù)探索肝癌異質(zhì)性與尋找肝癌細(xì)胞免疫治療新靶點(diǎn)奠定基礎(chǔ)。
[Abstract]:Background: because the pathogenesis of liver cancer is not completely clear, it has the characteristics of rapid development, difficult detection and high recurrence probability after resection. Therefore, the high mortality of liver cancer is caused. In recent years, many scholars have proposed the viewpoint of tumor heterogeneity and cancer stem cells to re recognize liver cancer with a new angle, and point out the dry fine of liver cancer. Cellular markers will be a new target for immunotherapy of liver cancer. 1. the tumor cells were isolated from liver cancer and para cancer tissue, and the cell morphology, growth curve, migration and invasion ability, single cell Clonization, growth cycle, cell karyotype, molecular marker, and molecular marker, were purified from the liver cancer cell line.2.. Identification of biological characteristics such as tumor ability. Study methods: 1. the cells were separated by the digestion of liver lysate and the method of shearing and shredding. The tumor cells were purified by repeated differential digestion and differential adherence, and the cell morphology was observed by IncuCyteZOOM long time dynamic cell imaging and analysis system, and the cell growth curve was drawn, and the cell growth curve was plotted. 1. Compared with cell migration and invasion ability and tracking single cell cloning process, HE staining was used to calculate the karyoplasm ratio, Giemsa staining analysis of chromosomal numbers and karyotypes, nude mice in vivo tumorigenesis experiment, HBV DNA load in cell supernatant by fluorescence quantitative PCR, and AFP, GPC3, HepPar-1, CK18, CK19, PCNA in tumor cells by immunofluorescence technique. Vimentin protein expression, using flow cytometry to detect cell cycle and the surface markers of liver cancer stem cells EpCAM, CD13, CD44, CD90, CD24, CD47, CD133, DLK1 expression. Use Plasmocin, BM-Cyclin, MRA and other 3 kinds of drugs to treat Mycoplasma contaminated cells, with one step reagent, rapid detection reagent and 3 species. Methods the results of mycoplasma detection were carried out. The results were as follows: 8 hepatocellular carcinoma cells were isolated and purified from different sources in vitro, and 8 cells with stable passages were established. The results showed that 8 cells were different in morphology and abnormal cell nuclear ratio. The difference and fluorescence provided by the MTT method and IncuCyteZOOM were compared and the growth curves of cell fusion degree were obtained. Accurately reflecting the proliferation of 8 cells, the proliferation rate of 177T cells was the fastest, the doubling time was 29.70 + 0.84h, and the following were 216T3,78T, 83N, 216T2216N, 216T1,92N. The changes of the wound width of the 8 cells showed that the migration and invasion ability of 216T2 was stronger than the other 7 cells. The wound healing of 216T2 needed 45h + 0.82h, and the penetration of matrix gel only needed 2. 2H + 0.45h; using the function of IncuCyteZOOM full hole imaging to dynamically trace single cell clone formation process, which showed that 8 cells were able to form single cell clone in vitro, the ability of 177T cloning was the strongest, the clone formation rate was 38/96, and 78T was the weakest, and the formation rate of 4/96. cell cycle analysis showed that the proportion of S phase of 92N was the largest, and the chromosome was 15.22% 0.60%. The analysis showed that 216N, 216T1 were subtetraploid, and the other 6 cells were subtriploid. Fluorescence quantitative PCR was used to detect HBV DNA load in cell supernatant. The results showed that except 216N and 216T1216T2, the other 5 cells were positive for HBV DNA, and the immunofluorescence results showed that 8 cells did not express AFP, HepPar-1, expressed GPC3, and 8 cells 9 expression intensity was different, GPC3 expression in 78T and other cells was high, and expressed weakly in 177T. CK19 was the strongest in 78T. EMT related epithelial marker CK18 was strongly expressed in 8 cells, but interstitial cell marker Vimentin was expressed only in the cytoplasm of some cells, and the expression of Vimentin protein in 216T2 cells was found. The proportion of cells was significantly higher than that of the other 7 cell.PCNA cells in 8 cells, which reflected the difference in the differentiation of 8 cells, among which the proportion of PCNA positive cells in 92N cells was the highest. The lowest differentiation degree of.8 stem cell markers showed that the 8 cells expressed CD47 at a high proportion, but did not express DLKI, and the other markers were positive and thin. The proportion of cell in cell line is different, except for 216T2 cells, the other cells all express EpCAM, EpCAM+ cells are higher than 90%, while 216T2 cells express CD133, CD90, CD24 and other markers of low expression or non expression of other cells, including CD90+CD44+ cells as high as 64.20%, CD133+CD44+ cell ratio is also stored in the cell expression markers of 17%. single cell. In the difference, for example, the proportion of CD47+ cells in 78T cells was 88%, and that of CD47+CD44+ cells was 83.8%, and that of CD47+EpCAM+ cells in 29%. nude mice showed that except 177216T3, the other 6 cells could cause nude mice to be tumor, and none of them were found in the body, and the.8 strain of the lung cancer stem cells were contaminated by Mycoplasma during the culture process, and 3 were used in the culture. 3 methods were detected. The results showed that one step reagent detection showed that the Mycoplasma was eliminated after Plasmocin treatment 14 d, and the rapid detection reagent showed that BM-Cyclin treatment 21 d completely eliminated mycoplasma, and MRA after PCR detected the total elimination of Mycoplasma for 14 d; in addition, the 3 drugs used alternately to clear Mycoplasma effect. The results were more significant. Conclusion: 8 hepatoma cell lines were obtained through the identification of various cell biological characteristics. The differential expression of various markers in 8 cell lines suggests the heterogeneity of the origin of liver cancer, and the difference in the expression of stem cell markers in single cell expression also reflects the differences within the individual; by the standard of liver cancer stem cells. The analysis of chronicles provides the basis for further exploration of heterogeneity of hepatocellular carcinoma and finding new targets for immunotherapy of hepatocellular carcinoma cells.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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3 宋關(guān)斌;肝癌細(xì)胞力學(xué)特性及其與細(xì)胞周期的關(guān)系研究[D];重慶大學(xué);2002年

4 翟文龍;內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)肝癌細(xì)胞對(duì)阿霉素耐藥的機(jī)制[D];鄭州大學(xué);2011年

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6 周珍珍;MTDH/AEG-1在肝癌靶向性肺轉(zhuǎn)移中的作用和分子機(jī)制研究[D];華中科技大學(xué);2011年

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9 于洋;肝癌中醫(yī)臨床信息數(shù)據(jù)庫系統(tǒng)的構(gòu)建及應(yīng)用[D];第二軍醫(yī)大學(xué);2009年

10 孫穎;PBCA納米粒載體介導(dǎo)的肝癌HSV-TK/GCV基因治療的體外實(shí)驗(yàn)研究[D];中南大學(xué);2004年

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10 周慧江;復(fù)發(fā)性肝癌的早期診斷和治療分析[D];浙江大學(xué);2004年

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