原发性肝癌细胞系的建立与生物学鉴定

发布时间：2018-07-06 18:37

本文选题：肝癌 + 细胞培养　；参考：《天津医科大学》2016年硕士论文

【摘要】：研究背景:由于肝癌的发病机制尚未完全清楚,其具有发展迅速、发现困难、手术切除后复发概率高等特点,因此造成了肝癌的高死亡率。近年来许多学者提出肿瘤异质性与肿瘤干细胞的观点,以新的角度重新认识肝癌,同时指出肝癌干细胞标志物将成为肝癌免疫治疗的新靶点。研究目的:1.从肝癌以及癌旁组织中分离肿瘤细胞,纯化得到稳定传代的肝癌细胞株。2.对8个细胞株进行细胞形态、生长曲线、迁移与侵袭能力、单细胞克隆能力、生长周期、细胞核型、分子标志物、致瘤能力等生物学特性进行鉴定。研究方法:1.采用肝裂解液消化结合剪碎方法分离细胞,通过反复差速消化、差速贴壁的方法纯化肿瘤细胞并扩大培养。2.利用IncuCyteZOOM长时间动态细胞成像及分析系统观察细胞形态、绘制细胞生长曲线、比较细胞迁移与侵袭能力以及追踪单细胞克隆过程;通过HE染色计算核质比、Giemsa染色分析染色体众数和核型;裸鼠体内致瘤实验;利用荧光定量PCR检测细胞上清液中HBV DNA载量;利用免疫荧光技术检测肿瘤细胞中AFP、GPC3、HepPar-1、CK18、CK19、PCNA、Vimentin蛋白表达;利用流式细胞细胞仪检测细胞周期及肝癌干细胞表面标志物EpCAM、CD13、CD44、CD90、CD24、CD47、CD133、DLK1表达情况。采用Plasmocin、BM-Cyclin、MRA等3种药物对支原体污染的细胞进行处理,以CLARK一步法试剂、Biotool快速检测试剂和PCR等3种方法进行支原体检测。研究结果:在体外分离纯化不同来源的肝癌细胞,建立8株稳定传代的肝癌细胞株,观察发现8株细胞形态各异,且细胞核质比异常。对比MTT法与IncuCyteZOOM提供的相差与荧光5种方法,其中细胞融合度绘制的生长曲线能准确反映8株细胞增殖情况,177T细胞增殖速度最快,倍增时间29.70±0.84h,以下依次为216T3、78T、83N、216T2、216N、216T1、92N;比较8株细胞伤口宽度的变化发现216T2的迁移与侵袭能力明显强于其它7株细胞,216T2伤口愈合需45h±0.82h,而穿透基质胶只需要22h±0.45h;利用IncuCyteZOOM全孔成像功能进行动态追踪单细胞克隆形成过程,表明8株细胞均能够在体外形成单细胞克隆,177T克隆能力最强,克隆形成率为38/96;而78T最弱,形成率仅4/96。细胞周期分析表明92N的S期比例最大,为15.22%±0.60%;染色体众数分析显示216N、216T1为亚四倍体,其余6株细胞均为亚三倍体。采用荧光定量PCR检测细胞上清液中HBV DNA载量,结果显示除216N、216T1、216T2外,其他5株细胞HBV DNA阳性;免疫荧光结果显示8株细胞均不表达AFP、HepPar-1,表达GPC3、CK19,且8株细胞GPC3、CK19表达强度不一,GPC3在78T等细胞中表达强度明显高,在177T中表达弱;CK19在78T中表达最强。与EMT相关的上皮细胞标志CK18在8株细胞中均强表达,而间质细胞标志物Vimentin却只在部分细胞的胞浆中表达,对比发现216T2细胞中表达Vimentin蛋白的细胞比例明显高于其他7株细胞。PCNA在8株细胞中表达差异,从而反映了8株细胞分化程度不同,其中92N细胞中PCNA阳性细胞比例最高,分化程度最低。8种肝癌干细胞标志物分析表明,8株细胞均高比例表达CD47,但不表达DLKI,其余标志物阳性细胞在细胞株中比例不一,除216T2细胞外其余细胞均高表达EpCAM,EpCAM+细胞高于90%,而216T2细胞却表达其他细胞低表达或不表达的CD133、CD90、CD24等标志物,其中CD90+CD44+细胞高达64.20%,CD133+CD44+细胞比例为17%。单株细胞内的细胞表达标记物也存在差异,例如78T细胞中CD47+细胞比例为88%,而CD47+CD44+细胞为83.8%,CD47+EpCAM+细胞约为29%。裸鼠体内移植实验显示除177、216T3外,其余6株细胞能使裸鼠致瘤,通过解剖均未发现体内肝、肺转移。8株肝癌干细胞在培养过程中遭到支原体污染,利用3种方法进行清除,3种方法进行检测,结果显示:一步法试剂检测显示Plasmocin处理14 d后支原体消除,快速检测试剂检测显示BM-Cyclin处理21 d彻底消灭支原体,而MRA经PCR检测发现彻底消除支原体需要14 d;另外,3种药物交替使用清除支原体效果更加显著。研究结论:经过多种细胞生物学特性的鉴定获得8个肝癌细胞株,各种标志物在8株细胞系中的差异表达提示肝癌的起源存在异质性,同时单株细胞表达干细胞标志物的差异也反映个体内部存在差异性;通过对肝癌干细胞标志物的分析为继续探索肝癌异质性与寻找肝癌细胞免疫治疗新靶点奠定基础。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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