本文關(guān)鍵詞：新的胃癌細(xì)胞系的建立和胃癌干細(xì)胞異質(zhì)性研究　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胃癌 異質(zhì)性 干細(xì)胞 RNA-seq

【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,胃印戒細(xì)胞型胃癌,因其生物學(xué)行為復(fù)雜、異質(zhì)性強(qiáng)、治療反應(yīng)率低,成為腫瘤相關(guān)死亡的主要原因之一。腫瘤干細(xì)胞因其多向分化能力和自我更新能力,被認(rèn)為是腫瘤異質(zhì)性、治療反應(yīng)率低和復(fù)發(fā)的根源,而靶向腫瘤干細(xì)胞治療被認(rèn)為能夠從根源上治療腫瘤,防止復(fù)發(fā)。本研究將著重闡明胃癌細(xì)胞系中是否存在明顯差異的干細(xì)胞亞群,并建立一個(gè)理想的富含干細(xì)胞的胃癌細(xì)胞系模型和有明顯異質(zhì)性的干細(xì)胞亞群模型,在此模型基礎(chǔ)上探討個(gè)體化治療并初步探討干細(xì)胞的調(diào)控機(jī)制,篩選重要的調(diào)控因子,達(dá)到靶向干細(xì)胞治療的目的。第一部分：為了深入了解胃印戒細(xì)胞癌的生物學(xué)行為并篩選個(gè)體化治療的藥物,我們建立了病人來源的腫瘤移植模型(patient-derived tumor tissue,PDTT)及同一病人來源的胃印戒細(xì)胞癌的細(xì)胞系,命名為GCSR1。多種生物學(xué)行為特征鑒定結(jié)果顯示GCSR1核分裂多見,核邊聚,呈現(xiàn)印戒細(xì)胞樣形態(tài)。細(xì)胞周期時(shí)相S期細(xì)胞比例、瓶壁和軟瓊脂集落形率分別為25%、24.13%和10%,呈現(xiàn)較強(qiáng)的體外生存能力。GCSR1細(xì)胞系經(jīng)連續(xù)傳至80代左右,細(xì)胞形態(tài)、生長(zhǎng)曲線和倍增時(shí)間、細(xì)胞周期、集落形成能力等性狀仍保持相對(duì)穩(wěn)定。干細(xì)胞表型研究發(fā)現(xiàn),GCSR1高表達(dá)干細(xì)胞表面標(biāo)記蛋白CD44和CD133,并且有很強(qiáng)的體內(nèi)成瘤能力,提示GCSR1富含胃癌干細(xì)胞。綜上所述,我們建立了一株穩(wěn)定的富含腫瘤干細(xì)胞的胃印戒細(xì)胞癌細(xì)胞系GCSR1。第二部分：為了篩選指導(dǎo)臨床治療的有效化療藥物,我們結(jié)合細(xì)胞系GCSR1和PDTT模型對(duì)4種一線化療藥物進(jìn)行了篩選。結(jié)果提示：GCSR1細(xì)胞系多藥耐受,僅對(duì)化療藥物表阿霉素(EPI)敏感。結(jié)合第一二部分結(jié)果：我們發(fā)現(xiàn)GCSR1富含干細(xì)胞且多藥耐受。第三部分：為了明確GCSR1細(xì)胞系中干細(xì)胞和耐藥的關(guān)系,并排除異質(zhì)性對(duì)后續(xù)研究的干擾,我們通過有限稀釋法從GCSR1細(xì)胞系中分離培養(yǎng)9個(gè)細(xì)胞亞群。通過流式細(xì)胞術(shù)和微球形成兩種方法篩選干細(xì)胞特性差異明顯的細(xì)胞亞群,結(jié)果表明其中一個(gè)細(xì)胞亞群GCSR1-6高表達(dá)表面標(biāo)記CD133,微球形成能力明顯,具有明顯的干細(xì)胞表型；同時(shí)還存在一株干細(xì)胞特性較弱的細(xì)胞亞群,被命名為GCSR1-11。同時(shí)還發(fā)現(xiàn)GCSR1-6與GCSR1-11相較,運(yùn)動(dòng)能力更強(qiáng),生長(zhǎng)更緩慢,惡性程度更高。最重要的是GCSR1-6出現(xiàn)明顯的5-FU耐受,而GCSR1-11對(duì)5-FU敏感,結(jié)果提示我們GCSR1-6是GCSR1對(duì)5-FU耐受的主要細(xì)胞群。綜上所述,GCSR1-6是一株惡性程度非常高的具有明顯干細(xì)胞特性的細(xì)胞亞群,是GCSR1細(xì)胞系耐藥的主要細(xì)胞亞群,與GCSR1-11細(xì)胞亞群相比,異質(zhì)性非常明顯。因而靶向治療GCSR1-6細(xì)胞亞群可能可以達(dá)到克服耐藥,提高治療效果的目的。為了達(dá)到這個(gè)目的,我們初步探討了GCSR1-6細(xì)胞亞群調(diào)控的機(jī)制,結(jié)果發(fā)現(xiàn)：轉(zhuǎn)錄因子P-catenin在GCSR1-6細(xì)胞核內(nèi)高表達(dá),下調(diào)β-catenin表達(dá)后,GCSR1-6細(xì)胞形態(tài)發(fā)生改變,凋亡增加,干細(xì)胞標(biāo)記CD44下調(diào)。結(jié)果提示,β-catenin是參與干細(xì)胞調(diào)控的重要蛋白。第四部分：為了進(jìn)一步闡述GCSR1-6和GCSR1-11細(xì)胞亞群的基因表達(dá)異質(zhì)性并深入研究干細(xì)胞調(diào)控的分子機(jī)制,我們進(jìn)行了RNA-seq轉(zhuǎn)錄組測(cè)序研究,初步分析的結(jié)果提示,按照P0.05, log2(Fold change)絕對(duì)值1的標(biāo)準(zhǔn),共有328個(gè)基因在GCSR1-6和GCSR1-11中差異表達(dá)。其中153個(gè)基因在GCSR1-6中高表達(dá),175個(gè)基因在GCSR1-6中低表達(dá)。將差異基因輸入KEGG(Kyoto Encyclopedia of Genes and Genomes)信號(hào)通路研究網(wǎng)站發(fā)現(xiàn),共187條信號(hào)通路參與調(diào)控。這些差異基因和信號(hào)通路,為后續(xù)的基礎(chǔ)臨床驗(yàn)證打下基礎(chǔ)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.2

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