吉西他滨对胰腺癌细胞的免疫调节作用及其机制研究

发布时间：2018-05-01 23:40

本文选题：胰腺癌 + 吉西他滨　；参考：《安徽医科大学》2017年博士论文

【摘要】：[目的]1、研究吉西他滨在胰腺癌治疗中的免疫调节作用及其具体分子机制。2、研究ULBP2在胰腺癌患者血清中的表达,探讨ULBP2的表达与患者临床病理参数、预后间的相关性,探讨血清ULBP2水平对胰腺癌的诊断价值。[方法]1、ELISA检测胰腺癌化疗药物吉西他滨作用下的胰腺癌细胞株分泌可溶型ULBP2蛋白的水平,以及流式细胞术检测胰腺癌细胞株表达膜型ULBP2蛋白的水平;2、建立胰腺癌细胞与NK细胞共培养杀伤体系后,通过CCK8法检测吉西他滨对胰腺癌免疫杀伤敏感性的影响;3、通过定量PCR技术、western blot检测吉西他滨对胰腺癌细胞中ADAM10基因和蛋白表达的影响;进一步通过设计合成ADAM10 siRNA并转染胰腺癌细胞,在验证ADAM10表达水平下调后,通过CCK8法检测ADAM10siRNA对胰腺癌细胞增殖的影响,以及通过ELISA检测ADAM10siRNA对胰腺癌细胞表达可溶型ULBP2蛋白的影响;4、通过免疫组织化学染色法检测胰腺癌组织标本中ADAM10的表达水平,以及通过ELISA检测胰腺癌患者血清中sULBP2的表达水平,通过统计学分析,评估在胰腺癌血清中sULBP2的表达与患者临床资料和术后预后的关系、及其与组织中ADAM10表达水平间的相关性;5、收集吉西他滨处理后的胰腺癌细胞PANC-1和未经处理的对照组细胞,通过全转录组测序,分析处理前后胰腺癌细胞中环状RNA、lncRNA和mRNA的差异表达。进一步通过生物学分析,将差异基因按其功能及通路进行分类,以便进一步寻找差异基因中可能参与免疫调控的基因及通路蛋白。[结果]1、通过ELISA检测细胞培养上清液,发现吉西他滨能够抑制胰腺癌细胞PANC-1和MIAPACA-2细胞分泌可溶型ULBP2;而流式细胞术的检测结果表明,吉西他滨可提高胰腺癌细胞PANC-1和MIAPACA2表面ULBP2膜型蛋白的表达。2、通过建立人NK细胞系与人胰腺癌细胞系的共培养体系,并用CCK-8细胞毒性实验观察吉西他滨对NK细胞杀伤胰腺癌细胞效率的影响。实验结果发现,在抑制培养上清中可溶型sULBP2的分泌表达后,吉西他滨组NK细胞的杀伤效明显高于DMSO对照组,在培养体系中加入重组的可溶型sULBP2蛋白能逆转NK92细胞的杀伤活性。3、实时定量PCR和western blot结果表明,吉西他滨能下调胰腺癌细胞株PANC-1和MIAPACA-2中ADAM10表达水平。ADAM10 siRNA转染胰腺癌细胞PANC-1和MIAPACA-2,有效地沉默PANC-1和MIAPACA-2中ADAM10蛋白表达水平后同样有效地下调了 PANC-1和MIAPACA-2细胞培养上清中分泌可溶型ULBP2的水平。4、ELISA检测胰腺癌患者血清中ULBP2的表达水平,发现胰腺癌患者的血清中sULBP2浓度显著高于正常健康对照人群,同时胰腺癌患者肿瘤组织的免疫组化结果显示ADAM10主要分布在肿瘤细胞的胞浆内,不同患者的ADAM10染色程度不同。结合患者的临床和病理参数统计发现血清sULBP2浓度与组织中ADAM10表达呈正相关。血清sULBP2浓度与CA199(p=0.013),淋巴结转移(p=0.009)以及患者总生存期(p=0.045)密切相关。5、全转录组测序分析处理前后胰腺癌细胞中环状RNA、lncRNA和mRNA的差异表达,发现72个环状RNA上调,56个环状RNA下调;以及1025个mRNA表达上调,656个mRNA表达下调。通过生物学分析,将差异基因按其功能及通路进行分类,并通过流行的miRNA靶基因预测软件进行差异环状RNA-miRNA相互作用预测,以推断可以作为"miRNA海绵"的环状RNA。[结论]1、吉西他滨抑制胰腺癌细胞ULBP2的蛋白胞外脱落,通过ULBP2可溶型蛋白与膜蛋白竞争结合NK细胞活化受体NKG2D,从而促进NK细胞杀伤胰腺癌细胞。2、吉西他滨下调胰腺癌表达ADAM10蛋白酶,从而抑制其对ULBP2蛋白的蛋白切割。ADAM10siRNA在转染下调胰腺癌细胞中ADAM10蛋白表达后,能够下调胰腺癌细胞培养上清中分泌可溶型ULBP2蛋白。3、胰腺癌患者血清中可溶型ULBP2的表达水平明显高于正常健康志愿者,且与胰腺癌淋巴结转移和患者总生存其有关。血清中ULBP2表达水平与患者肿瘤组织中ADAM10表达呈正相关。4、通过全转录组测序,分析了吉西他滨处理前后胰腺癌细胞中环状RNA、lncRNA和mRNA的差异表达,发现72个环状RNA上调,56个环状RNA下调;以及1025个mRNA表达上调,656个mRNA表达下调,这其中有一些与免疫耐受诱导以及免疫细胞分化调节相关的基因均发生变化,这为下一步研究吉西他滨在胰腺癌免疫调控中的具体信号通路提供基础。
[Abstract]:[Objective]1 to study the immunomodulatory role of gemcitabine in the treatment of pancreatic cancer and its specific molecular mechanism.2, to study the expression of ULBP2 in the serum of patients with pancreatic cancer, to explore the correlation between the expression of ULBP2 and the clinicopathological parameters and prognosis of the patients, and to explore the diagnostic value of serum ULBP2 level for pancreatic adenocarcinoma. [methods]1, ELISA detection of pancreatic carcinogenesis. The level of soluble ULBP2 protein secreted by gemcitabine under the action of gemcitabine and the level of flow cytometry to detect the expression of membrane type ULBP2 protein in pancreatic cancer cell lines. 2, after establishing a co culture killing system of pancreatic cancer cells and NK cells, the effect of zilitabine on the immuno killing sensitivity of pancreatic cancer was detected by CCK8 method; 3 The effects of gemcitabine on the expression of ADAM10 and protein in pancreatic cancer cells were detected by quantitative PCR technique and Western blot, and ADAM10 siRNA was designed and transfected into pancreatic cancer cells. The effect of ADAM10siRNA on the proliferation of pancreatic cancer cells was detected by CCK8 method and the ELISA detection was detected by ELISA. The effect of ADAM10siRNA on the expression of soluble ULBP2 protein in pancreatic cancer cells. 4, the expression level of ADAM10 in pancreatic cancer tissues was detected by immunohistochemical staining, and the expression level of sULBP2 in the serum of pancreatic cancer patients was detected by ELISA. The expression of sULBP2 in the serum of pancreatic cancer and the patient's presence were evaluated by statistical analysis. The correlation between bed data and postoperative prognosis and the correlation with the level of ADAM10 expression in the tissue; 5. Collect the PANC-1 and untreated control groups of the pancreatic cancer cells treated with gemcitabine, and analyze the differential expression of cyclic RNA, lncRNA and mRNA in pancreatic cancer cells before and after the whole transcriptional sequence. Further through biology The differential gene was classified according to its function and pathway in order to further find the genes and pathway proteins that may participate in the immunoregulation of the differential genes. [result]1, by ELISA detection of cell culture supernatant, it was found that gemcitabine could inhibit the secretion of soluble ULBP2 in pancreatic cancer cells PANC-1 and MIAPACA-2 cells; and flow cytometry The results showed that gemcitabine could improve the expression of ULBP2 membrane protein.2 on the surface of pancreatic cancer cells PANC-1 and MIAPACA2. By establishing co culture system of human NK cell line and human pancreatic cancer cell line, the effect of gemcitabine on the cytotoxicity of pancreatic cancer cells by NK cells was observed by CCK-8 cytotoxicity test. After the secretory expression of soluble sULBP2 in the culture supernatant, the killing effect of NK cells in the gemcitabine group was significantly higher than that of the DMSO control group. The recombinant soluble sULBP2 protein added to the culture system could reverse the killing activity of NK92 cells, and the real-time quantitative PCR and Western blot results showed that gemcitabine could downregulate the PANC-1 and MIAP of pancreatic cancer cell lines. ADAM10 expression level.ADAM10 siRNA transfected to pancreatic cancer cells PANC-1 and MIAPACA-2, effectively silencing the expression level of ADAM10 protein in PANC-1 and MIAPACA-2 effectively also effectively regulated the level of soluble ULBP2 in the culture supernatant of PANC-1 and MIAPACA-2 cells, and detected the expression level of the serum in the patients with pancreatic adenocarcinoma. The serum concentration of sULBP2 in the patients with pancreatic cancer was significantly higher than that in normal healthy controls. At the same time, the immunohistochemical results of the tumor tissues of the patients with pancreatic cancer showed that ADAM10 was mainly distributed in the cytoplasm of the tumor cells, and the degree of ADAM10 staining in different patients was different. The serum sULBP2 concentration and group were found with the clinical and pathological parameters of the patients. The expression of ADAM10 in the fabric was positively correlated. The serum concentration of sULBP2 was closely related to CA199 (p=0.013), lymph node metastasis (p=0.009) and the total survival time (p=0.045). The differential expression of cyclic RNA, lncRNA and mRNA in the pancreatic cancer cells before and after the whole transcriptional analysis, 72 cyclic RNA up-regulated, 56 cyclic RNA down; and 1025 The expression was down regulated and 656 mRNA expressions were downregulated. Through biological analysis, the differential genes were classified according to their functions and pathways, and differential cyclic RNA-miRNA interaction was predicted through the popular miRNA target gene prediction software to deduce the circular RNA.[conclusion that could be used as "miRNA sponge", and gemcitabine inhibited the ULBP2 eggs of pancreatic cancer cells. Out of leukocyte exfoliate, ULBP2 soluble protein and membrane protein are competitive combined with NK cell activation receptor NKG2D, thus promoting NK cells to kill pancreatic cancer cell.2, and gemcitabine downregulation the expression of ADAM10 protease in pancreatic cancer, thus inhibiting the expression of ULBP2 protein protein cutting.ADAM10siRNA in the expression of ADAM10 protein in pancreatic cancer cells. The soluble type of ULBP2 protein.3 was secreted in the supernatant of pancreatic cancer cell culture. The expression level of soluble ULBP2 in serum of pancreatic cancer patients was significantly higher than that of normal healthy volunteers, and it was related to lymph node metastasis of pancreatic cancer and the total survival of the patients. The level of ULBP2 expression in serum was positively correlated with the expression of ADAM10 in the tumor tissues of the patients, and the expression of ADAM10 was positively correlated with the expression of.4 in the tumor tissue. The transcriptional group was sequenced and analyzed the differential expression of cyclic RNA, lncRNA and mRNA in the pancreatic cancer cells before and after gemcitabine treatment, and found that 72 ring-shaped RNA up-regulated, 56 cyclic RNA down-regulation, and 1025 mRNA expressions up and 656 mRNA expressions downregulated, among which some genes associated with immune tolerance induction and immune cell differentiation regulation were found. This provides the basis for further research on specific signaling pathways of gemcitabine in the immunoregulation of pancreatic cancer.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9

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