人胃癌组织中肿瘤相关成纤维细胞与胃癌细胞的分离及其转录组学

发布时间：2018-03-22 18:53

  本文选题：胃癌　切入点：胃癌相关成纤维细胞　出处：《贵州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:分离并鉴定胃癌相关成纤维细胞(gastric cancer-associated fibroblasts,CAFs)、癌旁正常成纤维细胞(normal fibroblasts,NFs)及原代胃癌细胞(primary gastric carcinoma cells),比较CAFs与NFs的增殖差异以及对原代胃癌细胞克隆形成的影响,初步探讨CAFs与NFs转录组学的差异。方法:(1)选取临床胃癌患者手术切除的胃癌组织及癌旁正常组织(距癌周5cm以上、病理检测阴性),剪碎后胶原酶消化接种于预先包被的培养瓶中,常规传代培养。用免疫细胞化学染色检测细胞中平滑肌肌动蛋白α(α-smooth muscle actin,α-SMA)和纤维细胞活化蛋白(fibroblast activation protein,FAP)的表达鉴定CAFs与NFs,实时荧光定量PCR(RT-qPCR)检测细胞中α-SMA基因mRNA的表达水平;用HE染色、软琼脂克隆形成实验及免疫细胞化学检测细胞中癌胚抗原(carcino-embryonic antigen,CEA)和角蛋白18(cytokeratin 18,CK-18)的表达鉴定原代胃癌细胞。CCK8实验检测CAFs与NFs的增殖并绘制生长曲线;将原代胃癌细胞分别与CAFs和NFs混合培养,平板克隆形成实验及结晶紫染色检测原代胃癌细胞的克隆形成能力;幽门螺旋杆菌以感染复数1:100分别感染CAFs及原代胃癌细胞,倒置显微镜下观察细菌对细胞的黏附情况。(2)挑选3对临床表型相似、病理诊断为胃腺癌的病人的CAFs与NFs,配对进行转录组学研究,以CAFs/NFs比值差异两倍以上的基因为有意义的差异表达基因,将3个生物学重要组的差异基因两两交集之后并集,利用omicsbean在线软件进行生物信息学分析。结果:(1)成功从胃癌组织中分离出8株CAFs,5株NFs,3株原代胃癌细胞,且均能良好传代培养。免疫细胞化学染色显示α-SMA、FAP的表达在CAFs中成强阳性,在NFs中弱阳性或阴性,且两者均不表达CK-18;RT-qPCR结果显示CAFs中α-SMA基因的m RNA表达量显著高于NFs,差异有统计学意义(P0.05),提示CAFs与NFs分离成功。生长曲线结果显示培养3天后CAFs的生长速度显著高于NFs,培养六天时差异达到最大(P0.05)。(2)HE染色结果显示3株原代胃癌细胞呈现多形性、核大、畸形、核质比增大、核仁数增多,免疫细胞化学染色显示原代胃癌细胞中CEA、CK-18的表达均为强阳性,软琼脂克隆形成实验结果显示3株原代胃癌细胞均能在体外三维生长形成克隆球,提示分离的原代胃癌细胞具有恶性特征,鉴定成功。当CAFs与原代胃癌细胞共培养后,原代胃癌细胞的克隆数(70.4±8.6),显著高于NFs组(35.2±5.7)与空白对照组(33.1±6.6)(P0.05),而NFs组与空白对照组之间差异无统计学意义(P0.05)。此外,幽门螺旋杆菌不仅能够黏附胃癌细胞,也能黏附CAFs。(3)转录组学结果显示,在3个生物学重复组的CAFs中共获得147个差异基因,其中76个基因上调,71个基因下调。通过聚类分析、GO富集与KEGG富集,共富集到1727个生物学过程、84个细胞组分、153个分子功能和10个通路具有统计学意义。结论:1.成功从临床胃癌组织中分离到原代的CAFs、NFs和胃癌细胞。2.证实CAFs在体外的生长速度快于NFs,促进胃癌细胞克隆性生长的能力更强。3.在CAFs中获得147个差异表达的基因,参与肿瘤相关的重要生物学过程和信号通路,可能在胃癌的发生发展中起重要作用。
[Abstract]:Objective: to isolate and identify gastric cancer associated fibroblasts (gastric cancer-associated, fibroblasts, CAFs), adjacent normal fibroblasts (normal, fibroblasts, NFs) and primary gastric cancer cells (primary gastric carcinoma cells), the difference between the CAFs and the proliferation of NFs and the influence on the formation of cloning of primary gastric cancer cells, to investigate the difference of CAFs with the NFs transcriptome. Methods: (1) selection of gastric carcinoma and the clinical resection operation for gastric cancer in adjacent normal tissues (from above, peritumoral 5cm pathological detection, negative) cut after collagenase digestion inoculated the coated culture flask, were cultured. Cells were examined by smooth muscle alpha actin immunocytochemistry (-smooth muscle alpha actin, alpha -SMA) and fibroblast activation protein (fibroblast activation, protein, FAP) the expression of CAFs and NFs, real-time fluorescence quantitative PCR (RT-qPCR) detection and fine The expression level of -SMA alpha gene mRNA in cells; using HE staining, soft agar clone formation experiment and carcinoembryonic antigen immunocytochemistry cells (carcino-embryonic antigen, CEA) and cytokeratin 18 (cytokeratin 18, CK-18) expression of primary gastric cancer cells.CCK8 test CAFs and NFs proliferation and growth the curve; primary gastric cancer cells respectively with CAFs and NFs mixed culture, colony formation assay and crystal violet staining was used to detect the clone forming ability of primary gastric cancer cells; Helicobacter pylori infection with complex 1:100 CAFs were infected and primary gastric cancer cells, observe the adhesion of bacteria to cells under the inverted microscope (2) selection. 3 of the clinical phenotype is similar to that of CAFs and NFs for pathological diagnosis of gastric cancer patients, matched for transcriptome analysis, significant differentially expressed genes by genetic differences in CAFs/NFs ratio of more than two times, 3 After a group of biologically important differences in the intersection of 22 genes, bioinformatics analysis using omicsbean software. Results: (1) the success of 8 strains of CAFs were isolated from the gastric cancer tissues, 5 NFs strains, 3 strains of primary gastric cancer cells, and were well subcultured. Immunocytochemical staining showed that the alpha -SMA. The expression of FAP strongly positive in CAFs, in NFs weakly positive or negative, and there was no expression of CK-18; RT-qPCR m RNA CAFs showed that the expression of alpha -SMA gene was significantly higher than that in NFs, the difference was statistically significant (P0.05), suggesting that CAFs and NFs from success. Growth curve showed that the growth of cultures 3 days after CAFs was significantly higher than that in NFs, cultured for six days to reach the maximum difference (P0.05). (2) the results of HE staining showed that 3 strains of primary gastric cancer cells showed pleomorphic nuclei, deformity, nucleocytoplasmic ratio increases, the number of nucleolus, immune staining of primary gastric cancer CEA cells, the expression of CK-18 were strongly positive, soft agar colony formation assay showed that 3 strains of primary gastric cancer cells can form clones in vitro three-dimensional growth of primary gastric cancer cells that isolated with malignant features, identification of success. When CAFs and primary gastric cancer cells were co cultured after primary cloning the number of gastric cancer cells (70.4 + 8.6), significantly higher than that of group NFs (35.2 + 5.7) and control group (33.1 + 6.6) (P0.05), while there was no significant difference between the NFs group and the control group (P0.05). In addition, Helicobacter pylori gastric cancer cell adhesion can not only, also can adhere CAFs. (3) the transcriptome results show that the repeated group in 3 CAFs biology 147 genes, including 76 genes up-regulated and 71 genes were downregulated. Through cluster analysis, GO enrichment and KEGG enrichment, all set to 1727 84 biological processes, cellular component, molecular function and 153 10 A statistically significant pathway. Conclusion: 1. successfully isolated from primary clinical gastric cancer tissues CAFs, NFs and.2. CAFs confirmed gastric cancer cell growth rate in vitro was faster than NFs, stronger ability to promote.3. cell clonality of gastric cancer was 147 CAFs difference in the expression of genes involved in tumor related. The important biological processes and pathways, may play an important role in the development of gastric cancer.

【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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