本文選題：胃癌　切入點：胃癌相關(guān)成纖維細胞　出處：《貴州醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:分離并鑒定胃癌相關(guān)成纖維細胞(gastric cancer-associated fibroblasts,CAFs)、癌旁正常成纖維細胞(normal fibroblasts,NFs)及原代胃癌細胞(primary gastric carcinoma cells),比較CAFs與NFs的增殖差異以及對原代胃癌細胞克隆形成的影響,初步探討CAFs與NFs轉(zhuǎn)錄組學的差異。方法:(1)選取臨床胃癌患者手術(shù)切除的胃癌組織及癌旁正常組織(距癌周5cm以上、病理檢測陰性),剪碎后膠原酶消化接種于預(yù)先包被的培養(yǎng)瓶中,常規(guī)傳代培養(yǎng)。用免疫細胞化學染色檢測細胞中平滑肌肌動蛋白α(α-smooth muscle actin,α-SMA)和纖維細胞活化蛋白(fibroblast activation protein,FAP)的表達鑒定CAFs與NFs,實時熒光定量PCR(RT-qPCR)檢測細胞中α-SMA基因mRNA的表達水平;用HE染色、軟瓊脂克隆形成實驗及免疫細胞化學檢測細胞中癌胚抗原(carcino-embryonic antigen,CEA)和角蛋白18(cytokeratin 18,CK-18)的表達鑒定原代胃癌細胞。CCK8實驗檢測CAFs與NFs的增殖并繪制生長曲線;將原代胃癌細胞分別與CAFs和NFs混合培養(yǎng),平板克隆形成實驗及結(jié)晶紫染色檢測原代胃癌細胞的克隆形成能力;幽門螺旋桿菌以感染復(fù)數(shù)1:100分別感染CAFs及原代胃癌細胞,倒置顯微鏡下觀察細菌對細胞的黏附情況。(2)挑選3對臨床表型相似、病理診斷為胃腺癌的病人的CAFs與NFs,配對進行轉(zhuǎn)錄組學研究,以CAFs/NFs比值差異兩倍以上的基因為有意義的差異表達基因,將3個生物學重要組的差異基因兩兩交集之后并集,利用omicsbean在線軟件進行生物信息學分析。結(jié)果:(1)成功從胃癌組織中分離出8株CAFs,5株NFs,3株原代胃癌細胞,且均能良好傳代培養(yǎng)。免疫細胞化學染色顯示α-SMA、FAP的表達在CAFs中成強陽性,在NFs中弱陽性或陰性,且兩者均不表達CK-18;RT-qPCR結(jié)果顯示CAFs中α-SMA基因的m RNA表達量顯著高于NFs,差異有統(tǒng)計學意義(P0.05),提示CAFs與NFs分離成功。生長曲線結(jié)果顯示培養(yǎng)3天后CAFs的生長速度顯著高于NFs,培養(yǎng)六天時差異達到最大(P0.05)。(2)HE染色結(jié)果顯示3株原代胃癌細胞呈現(xiàn)多形性、核大、畸形、核質(zhì)比增大、核仁數(shù)增多,免疫細胞化學染色顯示原代胃癌細胞中CEA、CK-18的表達均為強陽性,軟瓊脂克隆形成實驗結(jié)果顯示3株原代胃癌細胞均能在體外三維生長形成克隆球,提示分離的原代胃癌細胞具有惡性特征,鑒定成功。當CAFs與原代胃癌細胞共培養(yǎng)后,原代胃癌細胞的克隆數(shù)(70.4±8.6),顯著高于NFs組(35.2±5.7)與空白對照組(33.1±6.6)(P0.05),而NFs組與空白對照組之間差異無統(tǒng)計學意義(P0.05)。此外,幽門螺旋桿菌不僅能夠黏附胃癌細胞,也能黏附CAFs。(3)轉(zhuǎn)錄組學結(jié)果顯示,在3個生物學重復(fù)組的CAFs中共獲得147個差異基因,其中76個基因上調(diào),71個基因下調(diào)。通過聚類分析、GO富集與KEGG富集,共富集到1727個生物學過程、84個細胞組分、153個分子功能和10個通路具有統(tǒng)計學意義。結(jié)論:1.成功從臨床胃癌組織中分離到原代的CAFs、NFs和胃癌細胞。2.證實CAFs在體外的生長速度快于NFs,促進胃癌細胞克隆性生長的能力更強。3.在CAFs中獲得147個差異表達的基因,參與腫瘤相關(guān)的重要生物學過程和信號通路,可能在胃癌的發(fā)生發(fā)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.

【學位授予單位】：貴州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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