本文選題：胰腺癌 + 吉西他濱�。� 參考：《安徽醫(yī)科大學(xué)》2017年博士論文

【摘要】：[目的]1、研究吉西他濱在胰腺癌治療中的免疫調(diào)節(jié)作用及其具體分子機(jī)制。2、研究ULBP2在胰腺癌患者血清中的表達(dá),探討ULBP2的表達(dá)與患者臨床病理參數(shù)、預(yù)后間的相關(guān)性,探討血清ULBP2水平對(duì)胰腺癌的診斷價(jià)值。[方法]1、ELISA檢測(cè)胰腺癌化療藥物吉西他濱作用下的胰腺癌細(xì)胞株分泌可溶型ULBP2蛋白的水平,以及流式細(xì)胞術(shù)檢測(cè)胰腺癌細(xì)胞株表達(dá)膜型ULBP2蛋白的水平;2、建立胰腺癌細(xì)胞與NK細(xì)胞共培養(yǎng)殺傷體系后,通過CCK8法檢測(cè)吉西他濱對(duì)胰腺癌免疫殺傷敏感性的影響;3、通過定量PCR技術(shù)、western blot檢測(cè)吉西他濱對(duì)胰腺癌細(xì)胞中ADAM10基因和蛋白表達(dá)的影響;進(jìn)一步通過設(shè)計(jì)合成ADAM10 siRNA并轉(zhuǎn)染胰腺癌細(xì)胞,在驗(yàn)證ADAM10表達(dá)水平下調(diào)后,通過CCK8法檢測(cè)ADAM10siRNA對(duì)胰腺癌細(xì)胞增殖的影響,以及通過ELISA檢測(cè)ADAM10siRNA對(duì)胰腺癌細(xì)胞表達(dá)可溶型ULBP2蛋白的影響;4、通過免疫組織化學(xué)染色法檢測(cè)胰腺癌組織標(biāo)本中ADAM10的表達(dá)水平,以及通過ELISA檢測(cè)胰腺癌患者血清中sULBP2的表達(dá)水平,通過統(tǒng)計(jì)學(xué)分析,評(píng)估在胰腺癌血清中sULBP2的表達(dá)與患者臨床資料和術(shù)后預(yù)后的關(guān)系、及其與組織中ADAM10表達(dá)水平間的相關(guān)性;5、收集吉西他濱處理后的胰腺癌細(xì)胞PANC-1和未經(jīng)處理的對(duì)照組細(xì)胞,通過全轉(zhuǎn)錄組測(cè)序,分析處理前后胰腺癌細(xì)胞中環(huán)狀RNA、lncRNA和mRNA的差異表達(dá)。進(jìn)一步通過生物學(xué)分析,將差異基因按其功能及通路進(jìn)行分類,以便進(jìn)一步尋找差異基因中可能參與免疫調(diào)控的基因及通路蛋白。[結(jié)果]1、通過ELISA檢測(cè)細(xì)胞培養(yǎng)上清液,發(fā)現(xiàn)吉西他濱能夠抑制胰腺癌細(xì)胞PANC-1和MIAPACA-2細(xì)胞分泌可溶型ULBP2;而流式細(xì)胞術(shù)的檢測(cè)結(jié)果表明,吉西他濱可提高胰腺癌細(xì)胞PANC-1和MIAPACA2表面ULBP2膜型蛋白的表達(dá)。2、通過建立人NK細(xì)胞系與人胰腺癌細(xì)胞系的共培養(yǎng)體系,并用CCK-8細(xì)胞毒性實(shí)驗(yàn)觀察吉西他濱對(duì)NK細(xì)胞殺傷胰腺癌細(xì)胞效率的影響。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),在抑制培養(yǎng)上清中可溶型sULBP2的分泌表達(dá)后,吉西他濱組NK細(xì)胞的殺傷效明顯高于DMSO對(duì)照組,在培養(yǎng)體系中加入重組的可溶型sULBP2蛋白能逆轉(zhuǎn)NK92細(xì)胞的殺傷活性。3、實(shí)時(shí)定量PCR和western blot結(jié)果表明,吉西他濱能下調(diào)胰腺癌細(xì)胞株P(guān)ANC-1和MIAPACA-2中ADAM10表達(dá)水平。ADAM10 siRNA轉(zhuǎn)染胰腺癌細(xì)胞PANC-1和MIAPACA-2,有效地沉默PANC-1和MIAPACA-2中ADAM10蛋白表達(dá)水平后同樣有效地下調(diào)了 PANC-1和MIAPACA-2細(xì)胞培養(yǎng)上清中分泌可溶型ULBP2的水平。4、ELISA檢測(cè)胰腺癌患者血清中ULBP2的表達(dá)水平,發(fā)現(xiàn)胰腺癌患者的血清中sULBP2濃度顯著高于正常健康對(duì)照人群,同時(shí)胰腺癌患者腫瘤組織的免疫組化結(jié)果顯示ADAM10主要分布在腫瘤細(xì)胞的胞漿內(nèi),不同患者的ADAM10染色程度不同。結(jié)合患者的臨床和病理參數(shù)統(tǒng)計(jì)發(fā)現(xiàn)血清sULBP2濃度與組織中ADAM10表達(dá)呈正相關(guān)。血清sULBP2濃度與CA199(p=0.013),淋巴結(jié)轉(zhuǎn)移(p=0.009)以及患者總生存期(p=0.045)密切相關(guān)。5、全轉(zhuǎn)錄組測(cè)序分析處理前后胰腺癌細(xì)胞中環(huán)狀RNA、lncRNA和mRNA的差異表達(dá),發(fā)現(xiàn)72個(gè)環(huán)狀RNA上調(diào),56個(gè)環(huán)狀RNA下調(diào);以及1025個(gè)mRNA表達(dá)上調(diào),656個(gè)mRNA表達(dá)下調(diào)。通過生物學(xué)分析,將差異基因按其功能及通路進(jìn)行分類,并通過流行的miRNA靶基因預(yù)測(cè)軟件進(jìn)行差異環(huán)狀RNA-miRNA相互作用預(yù)測(cè),以推斷可以作為"miRNA海綿"的環(huán)狀RNA。[結(jié)論]1、吉西他濱抑制胰腺癌細(xì)胞ULBP2的蛋白胞外脫落,通過ULBP2可溶型蛋白與膜蛋白競(jìng)爭(zhēng)結(jié)合NK細(xì)胞活化受體NKG2D,從而促進(jìn)NK細(xì)胞殺傷胰腺癌細(xì)胞。2、吉西他濱下調(diào)胰腺癌表達(dá)ADAM10蛋白酶,從而抑制其對(duì)ULBP2蛋白的蛋白切割。ADAM10siRNA在轉(zhuǎn)染下調(diào)胰腺癌細(xì)胞中ADAM10蛋白表達(dá)后,能夠下調(diào)胰腺癌細(xì)胞培養(yǎng)上清中分泌可溶型ULBP2蛋白。3、胰腺癌患者血清中可溶型ULBP2的表達(dá)水平明顯高于正常健康志愿者,且與胰腺癌淋巴結(jié)轉(zhuǎn)移和患者總生存其有關(guān)。血清中ULBP2表達(dá)水平與患者腫瘤組織中ADAM10表達(dá)呈正相關(guān)。4、通過全轉(zhuǎn)錄組測(cè)序,分析了吉西他濱處理前后胰腺癌細(xì)胞中環(huán)狀RNA、lncRNA和mRNA的差異表達(dá),發(fā)現(xiàn)72個(gè)環(huán)狀RNA上調(diào),56個(gè)環(huán)狀RNA下調(diào);以及1025個(gè)mRNA表達(dá)上調(diào),656個(gè)mRNA表達(dá)下調(diào),這其中有一些與免疫耐受誘導(dǎo)以及免疫細(xì)胞分化調(diào)節(jié)相關(guān)的基因均發(fā)生變化,這為下一步研究吉西他濱在胰腺癌免疫調(diào)控中的具體信號(hào)通路提供基礎(chǔ)。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.9

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