新的胃癌细胞系的建立和胃癌干细胞异质性研究

发布时间：2017-12-26 21:01

本文关键词：新的胃癌细胞系的建立和胃癌干细胞异质性研究　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：胃癌的死亡率一直居高不下,其5年生存率徘徊在20%～30%；其中,胃印戒细胞型胃癌,因其生物学行为复杂、异质性强、治疗反应率低,成为肿瘤相关死亡的主要原因之一。肿瘤干细胞因其多向分化能力和自我更新能力,被认为是肿瘤异质性、治疗反应率低和复发的根源,而靶向肿瘤干细胞治疗被认为能够从根源上治疗肿瘤,防止复发。本研究将着重阐明胃癌细胞系中是否存在明显差异的干细胞亚群,并建立一个理想的富含干细胞的胃癌细胞系模型和有明显异质性的干细胞亚群模型,在此模型基础上探讨个体化治疗并初步探讨干细胞的调控机制,筛选重要的调控因子,达到靶向干细胞治疗的目的。第一部分：为了深入了解胃印戒细胞癌的生物学行为并筛选个体化治疗的药物,我们建立了病人来源的肿瘤移植模型(patient-derived tumor tissue,PDTT)及同一病人来源的胃印戒细胞癌的细胞系,命名为GCSR1。多种生物学行为特征鉴定结果显示GCSR1核分裂多见,核边聚,呈现印戒细胞样形态。细胞周期时相S期细胞比例、瓶壁和软琼脂集落形率分别为25%、24.13%和10%,呈现较强的体外生存能力。GCSR1细胞系经连续传至80代左右,细胞形态、生长曲线和倍增时间、细胞周期、集落形成能力等性状仍保持相对稳定。干细胞表型研究发现,GCSR1高表达干细胞表面标记蛋白CD44和CD133,并且有很强的体内成瘤能力,提示GCSR1富含胃癌干细胞。综上所述,我们建立了一株稳定的富含肿瘤干细胞的胃印戒细胞癌细胞系GCSR1。第二部分：为了筛选指导临床治疗的有效化疗药物,我们结合细胞系GCSR1和PDTT模型对4种一线化疗药物进行了筛选。结果提示：GCSR1细胞系多药耐受,仅对化疗药物表阿霉素(EPI)敏感。结合第一二部分结果：我们发现GCSR1富含干细胞且多药耐受。第三部分：为了明确GCSR1细胞系中干细胞和耐药的关系,并排除异质性对后续研究的干扰,我们通过有限稀释法从GCSR1细胞系中分离培养9个细胞亚群。通过流式细胞术和微球形成两种方法筛选干细胞特性差异明显的细胞亚群,结果表明其中一个细胞亚群GCSR1-6高表达表面标记CD133,微球形成能力明显,具有明显的干细胞表型；同时还存在一株干细胞特性较弱的细胞亚群,被命名为GCSR1-11。同时还发现GCSR1-6与GCSR1-11相较,运动能力更强,生长更缓慢,恶性程度更高。最重要的是GCSR1-6出现明显的5-FU耐受,而GCSR1-11对5-FU敏感,结果提示我们GCSR1-6是GCSR1对5-FU耐受的主要细胞群。综上所述,GCSR1-6是一株恶性程度非常高的具有明显干细胞特性的细胞亚群,是GCSR1细胞系耐药的主要细胞亚群,与GCSR1-11细胞亚群相比,异质性非常明显。因而靶向治疗GCSR1-6细胞亚群可能可以达到克服耐药,提高治疗效果的目的。为了达到这个目的,我们初步探讨了GCSR1-6细胞亚群调控的机制,结果发现：转录因子P-catenin在GCSR1-6细胞核内高表达,下调β-catenin表达后,GCSR1-6细胞形态发生改变,凋亡增加,干细胞标记CD44下调。结果提示,β-catenin是参与干细胞调控的重要蛋白。第四部分：为了进一步阐述GCSR1-6和GCSR1-11细胞亚群的基因表达异质性并深入研究干细胞调控的分子机制,我们进行了RNA-seq转录组测序研究,初步分析的结果提示,按照P0.05, log2(Fold change)绝对值1的标准,共有328个基因在GCSR1-6和GCSR1-11中差异表达。其中153个基因在GCSR1-6中高表达,175个基因在GCSR1-6中低表达。将差异基因输入KEGG(Kyoto Encyclopedia of Genes and Genomes)信号通路研究网站发现,共187条信号通路参与调控。这些差异基因和信号通路,为后续的基础临床验证打下基础。
[Abstract]:The mortality rate of gastric cancer has been high. Its 5 year survival rate is 20% to 30%. Among them, gastric signet ring cell type gastric cancer is one of the main causes of tumor related death because of its complex biological behavior, heterogeneity and low response rate. Cancer stem cells are considered to be the source of tumor heterogeneity, low response rate and relapse due to their multidirectional differentiation and self-renewal ability. Targeted cancer stem cell therapy is considered to be able to treat tumors and prevent relapse from the source. This study focuses on stem cell subsets have obvious difference in gastric cancer cell lines, and to establish an ideal stem cells and gastric cancer cell line model with stem cell subsets significantly heterogeneous model, based on the model of individualized treatment and to investigate the regulatory mechanism of stem cell regulation, screening an important factor, to achieve targeted stem cell therapy for the purpose of. The first part: in order to understand the drug biological behavior of gastric signet ring cell carcinoma screening and individualized treatment, we established a patient derived tumor model (patient-derived tumor tissue, PDTT) of gastric signet ring cell carcinoma cell lines and the same patient source, named GCSR1. The results of identification of various biological behavior characteristics showed that the GCSR1 nuclear division was more common and the nuclear side was clustered, showing the form of signet ring cell like. The cell cycle phase S phase cell ratio, the bottle wall and the soft agar colony formation rate were 25%, 24.13% and 10%, respectively, and showed strong ability to survive in vitro. The GCSR1 cell line has been continuously transmitted to the 80 generation. Cell morphology, growth curve and multiplication time, cell cycle and colony forming ability remain relatively stable. Stem cell phenotype studies showed that GCSR1 overexpressed the surface marker proteins CD44 and CD133 of stem cells, and had strong tumorigenicity in vivo, suggesting that GCSR1 was rich in gastric cancer stem cells. To sum up, we have established a stable gastric signet ring cell line, GCSR1, which is rich in tumor stem cells. The second part: in order to screen effective chemotherapeutic drugs to guide clinical treatment, we screened 4 front-line chemotherapeutic drugs by combining the cell line GCSR1 and the PDTT model. The results suggest that GCSR1 cell line multidrug resistance to chemotherapeutic drugs, only epirubicin (EPI) sensitive. Combined with the results of the first two parts, we found that GCSR1 is rich in stem cells and multidrug tolerance. The third part: in order to clarify the relationship between stem cell and drug resistance in GCSR1 cell line, and exclude the interference of heterogeneity on subsequent research, we isolated 9 cell subsets from GCSR1 cell line by limited dilution method. Two methods of screening stem cell characteristics significantly different cell subsets by flow cytometry and the results show that the GCSR1-6 microspheres, a subpopulation of high expression of surface markers of CD133 microsphere formation significantly, with apparent stem cell phenotype; there is also a stem cell characteristics of weak cell subsets was named GCSR1-11. At the same time, GCSR1-6 was found to be more athletic, slower and more malignant than GCSR1-11. The most important thing is that GCSR1-6 has obvious 5-FU tolerance, and GCSR1-11 is sensitive to 5-FU. The results suggest that GCSR1-6 is the main cell group of GCSR1 for 5-FU tolerance. In conclusion, GCSR1-6 is a highly malignant cell subset with obvious stem cell characteristics. It is a major cell subset of GCSR1 cell line. The heterogeneity is very obvious compared with GCSR1-11 cell subsets. Therefore, the target therapy of GCSR1-6 cell subgroup may be able to overcome the resistance and improve the therapeutic effect. To achieve this goal, we preliminarily discussed the mechanism of GCSR1-6 cell subset regulation. It was found that transcription factor P-catenin was highly expressed in GCSR1-6 cell nucleus, and the expression of -catenin was down regulated. GCSR1-6 cells morphologically changed and apoptosis increased. Stem cell marker CD44 was down regulated. The results suggest that beta -catenin is an important protein involved in the regulation of stem cells. The fourth part: in order to further elaborate the GCSR1-6 and GCSR1-11 cell subsets of gene expression and molecular mechanism of heterogeneity and in-depth study of stem cell regulation, we sequenced the transcriptome studies RNA-seq, preliminary analysis results suggest that, according to P0.05, log2 (Fold change) absolute value standard of 1, a total of 328 genes in GCSR1-6 and GCSR1- 11 difference. 153 of these genes were highly expressed in GCSR1-6, and 175 genes were low in GCSR1-6. The difference gene input KEGG (Kyoto Encyclopedia of Genes and Genomes) signal pathway research site found that a total of 187 signal pathways involved in the regulation. These differential genes and signaling pathways provide a basis for subsequent basic clinical validation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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