發(fā)布時(shí)間：2018-06-16 22:19

  本文選題：胰腺癌 + 白細(xì)胞介素35�。� 參考：《天津醫(yī)科大學(xué)》2015年博士論文

【摘要】：背景白細(xì)胞介素35(Interleukin 35,IL-35)是最新發(fā)現(xiàn)的白細(xì)胞介素12(IL-12)細(xì)胞因子家族成員。IL35可能在自身免疫性疾病,炎性疾病,感染性疾病以及某些腫瘤中具有重要的作用,可能成為治療這些疾病的新靶點(diǎn)。在我們的前期研究中發(fā)現(xiàn),與正常組織及細(xì)胞系比較,胰腺癌組織及細(xì)胞高表達(dá)IL-35蛋白。我們?cè)O(shè)計(jì)本實(shí)驗(yàn),進(jìn)一步明確IL-35在胰腺癌組織及細(xì)胞中的表達(dá)方式;從臨床水平、細(xì)胞水平及動(dòng)物實(shí)驗(yàn)水平探索胰腺癌腫瘤源性IL-35的功能及其調(diào)控機(jī)制。方法1.應(yīng)用胰腺癌組織石蠟標(biāo)本,進(jìn)行IL-35兩個(gè)亞基p35和EBI3及IL-35受體兩個(gè)亞基IL12rβ2和gp130的連續(xù)切片免疫組化染色,分析IL-35及其受體的表達(dá)水平及相關(guān)性;搜集病例資料進(jìn)行預(yù)后隨訪,分析IL-35及其受體的表達(dá)水平與臨床病理參數(shù)之間的關(guān)系。2.應(yīng)用Western blot、RT-PCR、ELISA等實(shí)驗(yàn)技術(shù)檢測(cè)胰腺癌5個(gè)細(xì)胞系、Treg細(xì)胞(作為陽性對(duì)照)以及正常胰腺導(dǎo)管細(xì)胞系的IL-35及其受體的表達(dá)水平;應(yīng)用免疫熒光技術(shù)檢測(cè)IL-35及其受體共4個(gè)亞基在胰腺癌細(xì)胞中的表達(dá)細(xì)胞定位;應(yīng)用免疫共沉淀技術(shù)(Co-Immunoprecipitation,COIP)研究胰腺癌細(xì)胞表達(dá)IL-35兩個(gè)亞基的相關(guān)性。3.應(yīng)用分子克隆方法構(gòu)建IL-35過表達(dá)質(zhì)粒及IL-35 shRNA降表達(dá)質(zhì)粒,構(gòu)建慢病毒穩(wěn)定轉(zhuǎn)染系統(tǒng),建立穩(wěn)定轉(zhuǎn)染細(xì)胞系;用Western blot、RT-PCR及ELISA檢測(cè)所構(gòu)建穩(wěn)系表達(dá)IL-35水平;應(yīng)用EDU增殖實(shí)驗(yàn)檢測(cè)所構(gòu)建細(xì)胞表達(dá)的IL-35的生物學(xué)功能。4.用人臍靜脈內(nèi)皮細(xì)胞(Human umbilical vein endothelial cells,HUVEC)粘附實(shí)驗(yàn)及跨內(nèi)皮遷移(Transendothelial migration,TEM)實(shí)驗(yàn)研究IL-35在體外促腫瘤細(xì)胞出血管過程。應(yīng)用轉(zhuǎn)錄組測(cè)序的方法篩選IL-35促進(jìn)腫瘤轉(zhuǎn)移的靶標(biāo);應(yīng)用Western blot及RT-PCR實(shí)驗(yàn)驗(yàn)證測(cè)序結(jié)果;通過阻斷試驗(yàn)進(jìn)一步確定IL-35促腫瘤出血管過程的靶標(biāo)。5.應(yīng)用Western blot、RT-PCR、CHIP及免疫熒光實(shí)驗(yàn)檢測(cè)IL-35調(diào)控靶標(biāo)的分子機(jī)制。6.構(gòu)建nod-sci小鼠腫瘤細(xì)胞出血管過程模型;構(gòu)建nod-scid小鼠胰腺癌原位成瘤模型;體內(nèi)驗(yàn)證il-35促胰腺癌細(xì)胞出血管及促進(jìn)胰腺癌轉(zhuǎn)移作用。結(jié)果1.il-35及其受體在胰腺癌組織中的表達(dá)及其意義。通過對(duì)123例胰腺癌組織標(biāo)本進(jìn)行免疫組化染色發(fā)現(xiàn),il-35兩個(gè)亞基p35及ebi3的表達(dá)在多種胰腺癌病理類型表達(dá)陽性,而對(duì)應(yīng)的正常胰腺組織不表達(dá)或弱表達(dá);分析p35及eib3表達(dá)的相關(guān)性,發(fā)現(xiàn)兩者的分布呈明顯相關(guān)性(r=0.950;p0.0001);將p35及ebi3都高表達(dá)的定義為il-35高表達(dá),其他的為il-35低表達(dá),我們發(fā)現(xiàn)il-35高表達(dá)的占48.8%(60例);我們進(jìn)一步探索il-35表達(dá)水平與臨床病理參數(shù)的關(guān)系,發(fā)現(xiàn)高il-35水平與較晚tnm分期、淋巴結(jié)受累以及血管侵犯相關(guān)(p值分別為0.01,0.027及0.007)。我們發(fā)現(xiàn)較高的il-35表達(dá)水平患者具有較差的總生存期(中位數(shù):13.70±0.722vs18.5±1.50月)及較差的無復(fù)發(fā)生存期(中位數(shù):11.39±0.54vs16.87±2.00);cox風(fēng)險(xiǎn)模型經(jīng)過單因素、多因素分析后得出il-35表達(dá)水平是總生存期及無復(fù)發(fā)生存期的獨(dú)立影響因素。il-35受體兩個(gè)亞基gp130及il12rβ2在胰腺癌組織中均有陽性表達(dá)。進(jìn)一步研究了il-35配體及受體表達(dá)的相關(guān)性,發(fā)現(xiàn)兩者明顯正相關(guān)(r=0.384,p0.0001)。我們將il-35配體及受體綜合考慮分組后發(fā)現(xiàn),il-35高表達(dá)同時(shí)受體表達(dá)陽性的患者的總生存期及無復(fù)發(fā)生存期較其他組的差;這些結(jié)果提示il-35在體內(nèi)通過自分泌或旁分泌作用直接作用于胰腺癌細(xì)胞而發(fā)揮生物學(xué)功能,引起腫瘤進(jìn)展。2.il-35及其受體在胰腺癌細(xì)胞系中的表達(dá)。rt-pcr、westernblot、免疫熒光實(shí)驗(yàn)及elisa實(shí)驗(yàn)發(fā)現(xiàn)5個(gè)胰腺癌細(xì)胞系(panc-1、miapaca-2、cfpac-1、aspc-1及bxpc-3)均表達(dá)il-35及其受體;在miapaca-2及cfpac-1細(xì)胞中用coip實(shí)驗(yàn)證實(shí)了il-35兩個(gè)亞基的結(jié)合。3.成功構(gòu)建了2個(gè)過表達(dá)il-35的胰腺癌細(xì)胞系(panc-1及bxpc-3)和降表達(dá)il-35的兩個(gè)細(xì)胞系(miapaca-2和cfpac-1);經(jīng)cd4+t細(xì)胞體外edu增殖實(shí)驗(yàn)證實(shí),構(gòu)建的il-35穩(wěn)系具備有文獻(xiàn)報(bào)道的抑制cd4+t細(xì)胞增殖的生物學(xué)功能。4.體外功能實(shí)驗(yàn)證實(shí)腫瘤細(xì)胞過表達(dá)il-35后,huvec粘附及tem能力增強(qiáng);而腫瘤細(xì)胞降表達(dá)il-35后huvec粘附及tem能力下降。轉(zhuǎn)錄組測(cè)序發(fā)現(xiàn)IL-35過表達(dá)后細(xì)胞間粘附分子1(intercellular adhesion molecule 1,ICAM1)表達(dá)水平明顯上調(diào),Western blot實(shí)驗(yàn)驗(yàn)證了ICAM1表達(dá)水平受IL-35的調(diào)控;瓶頸試驗(yàn)發(fā)現(xiàn),當(dāng)將過表達(dá)IL-35細(xì)胞的ICAM1分子下調(diào)后,該細(xì)胞系的HUVEC粘附及TEM能力不再有明顯的增強(qiáng)。ICAM1抗體能部分阻斷發(fā)現(xiàn)IL-35過表達(dá)引起的HUVEC粘附能力增強(qiáng)。5.腫瘤細(xì)胞受到IL-35蛋白刺激后JAK-STAT信號(hào)通路被激活,磷酸化STAT1(p-STAT1)及磷酸化STAT4(p-STAT4)表達(dá)水平明顯上升,并且在細(xì)胞核內(nèi)可見p-STAT1及p-STAT4聚集。IL-35是通過gp130:gp130同源受體,激活P-STAT1:P-STAT1同源二聚體入核后激活人PDAC細(xì)胞ICAM1表達(dá)的。6.體內(nèi)出血管實(shí)驗(yàn)發(fā)現(xiàn)IL-35通過調(diào)控ICAM1的表達(dá),促進(jìn)了Pan02細(xì)胞出血管過程;體內(nèi)胰腺原位成瘤實(shí)驗(yàn)發(fā)現(xiàn)IL-35通過調(diào)控ICAM1的表達(dá),促進(jìn)了腫瘤的轉(zhuǎn)移。7.IL-35能誘導(dǎo)人胰腺癌細(xì)胞的EBI3及p35基因的自激活,促進(jìn)IL-35表達(dá)水平增加,提高IL-35陽性腫瘤細(xì)胞的比率。提高了胰腺癌細(xì)胞中IL-35的表達(dá)強(qiáng)度及表達(dá)廣度,誘導(dǎo)了腫瘤轉(zhuǎn)移潛能的播散。結(jié)論1.IL-35蛋白及其受體在胰腺癌組織及細(xì)胞系中異常過表達(dá);胰腺癌組織IL-35表達(dá)水平較高的患者預(yù)后較差。2.IL-35過表達(dá)后將激活JAK-STAT信號(hào)通路,促進(jìn)p-STAT1:p-STAT1同源二聚體的生成,入核后通過上調(diào)ICAM1的表達(dá),從而增強(qiáng)胰腺癌細(xì)胞的HUVEC粘附能力及TEM能力。通過促進(jìn)腫瘤細(xì)胞血管粘附及出血管過程進(jìn)而增強(qiáng)腫瘤的轉(zhuǎn)移能力。3.IL-35能促進(jìn)人胰腺癌細(xì)胞IL-35基因的自激活,進(jìn)一步促進(jìn)了腫瘤的轉(zhuǎn)移能力。
[Abstract]:Background interleukin 35 (Interleukin 35, IL-35) is the most newly discovered member of the interleukin 12 (IL-12) cytokine family,.IL35, which may play an important role in autoimmune diseases, inflammatory diseases, infectious diseases and some tumors. It may be a new target for the treatment of these diseases. Compared with normal tissue and cell lines, pancreatic cancer tissues and cells express IL-35 protein. We designed this experiment to further clarify the expression of IL-35 in pancreatic cancer tissues and cells, and explore the function and regulation mechanism of pancreatic cancer derived IL-35 from clinical level, cell level and animal experimental level. Method 1. the pancreatic cancer group was used in the pancreatic cancer group. IL-35 two subunits p35 and EBI3 and IL-35 receptor two subunits IL12r beta 2 and gp130 were immunohistochemical staining to analyze the expression level and correlation of IL-35 and its receptor, and to collect case data for prognosis, and to analyze the relationship between the expression level of IL-35 and its receptor and the clinicopathological parameters in.2. application Wes. Tern blot, RT-PCR, ELISA and other experimental techniques were used to detect 5 cell lines of pancreatic cancer, Treg cells (as positive controls) and the expression level of IL-35 and their receptors in normal pancreatic ductal cell lines. Immunofluorescence technique was used to detect the expression of 4 subunits of IL-35 and its receptor in pancreatic cancer cells, and immunoprecipitation (Co-) technique (Co-). Immunoprecipitation, COIP) study the correlation of pancreatic cancer cells to express IL-35 two subunits,.3. application molecular cloning method to construct IL-35 overexpression plasmid and IL-35 shRNA descending expression plasmid, construct the stable transfection system of lentivirus, establish stable transfection cell line, and use Western blot, RT-PCR and ELISA detection to construct the stable expression IL-35 level; should The EDU proliferation test was used to detect the biological function of IL-35 expressed by the constructed cells.4. using human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVEC) adhesion experiment and trans endothelial migration (Transendothelial migration, TEM) experiment. Select the target of IL-35 to promote tumor metastasis; use the Western blot and RT-PCR test to verify the sequencing results; the target.5. for IL-35 promoting the tumor angiogenesis process by blocking test, Western blot, RT-PCR, CHIP, and immunofluorescence test to detect the molecular mechanism of IL-35 regulation target. Construction of NOD-SCID mouse pancreatic cancer in situ tumor model; in vivo verifying that IL-35 promotes pancreatic cancer cells to produce blood vessels and promotes pancreatic cancer metastasis. Results the expression and significance of 1.il-35 and its receptor in pancreatic cancer tissue. By immunohistochemical staining of 123 pancreatic cancer tissue specimens, IL-35 two subunits p35 and ebi3 The expression was expressed in the pathological types of pancreatic cancer, and the corresponding normal pancreatic tissue was not expressed or weak. The correlation of p35 and eib3 expression was analyzed (r=0.950; P0.0001). The high expression of p35 and ebi3 was defined as the high expression of IL-35, and the others were low expression of IL-35. We found the high expression of IL-35. 48.8% (60 cases); we further explored the relationship between IL-35 expression and clinicopathological parameters, and found that high IL-35 levels were associated with late TNM staging, lymph node involvement, and vascular invasion (P values were 0.01,0.027 and 0.007 respectively). We found that higher IL-35 expression levels had a poor overall survival (median: 13.70 + 0.722vs18.). 5 + 1.50 months) and poor recurrence free survival (median: 11.39 + 0.54vs16.87 + 2); Cox risk model was analyzed by single factor and multiple factor analysis. The expression level of IL-35 was the independent influence factor of the total and non recurrent survival time. The.Il-35 receptor two subunits gp130 and il12r beta 2 were positive in the pancreatic cancer tissues. The correlation of IL-35 ligand and receptor expression was found to be significantly positive correlation (r=0.384, P0.0001). We found that IL-35 ligands and receptors were grouped together and found that the total and non recurrent survival periods of the patients with IL-35 high expression and receptor positive were worse than those in the other groups; these results suggest that IL-35 is autocrine in the body or in the body. Paracrine action directly acts on pancreatic cancer cells and plays biological functions, causing tumor progression.2.il-35 and its receptor expression in pancreatic cancer cell lines.Rt-pcr. Westernblot, immunofluorescence test and ELISA experiment found that 5 pancreatic cancer cell lines (PANC-1, miapaca-2, CFPAC-1, AsPC-1 and BXPC-3) all express IL-35 and its receptor; in miapac In A-2 and CFPAC-1 cells, the combination of CoIP experiments confirmed that the binding.3. of two subunits of IL-35 successfully constructed 2 pancreatic cancer cell lines (PANC-1 and BXPC-3) and two cell lines (miapaca-2 and CFPAC-1) to express IL-35 over the expression of IL-35. The biological function of cell proliferation.4. in vitro proved that after the tumor cells overexpressed IL-35, the adhesion of HUVEC and the ability of TEM increased, while the HUVEC adhesion and TEM ability decreased after the tumor cells expressed IL-35. The transcriptional sequence found that the expression of intercellular adhesion molecule 1 (intercellular adhesion molecule 1, ICAM1) was obvious after IL-35 overexpression. Up regulation, the Western blot experiment confirmed that the expression level of ICAM1 was regulated by IL-35, and the bottleneck test found that when the ICAM1 molecule of the overexpressed IL-35 cells was downregulated, the HUVEC adhesion and TEM ability of the cell line no longer significantly enhanced the.ICAM1 antibody partially blocking the HUVEC adhesion caused by the IL-35 overexpression enhanced.5. tumor cells. When the IL-35 protein was stimulated, the JAK-STAT signaling pathway was activated, the expression level of phosphorylated STAT1 (p-STAT1) and phosphorylated STAT4 (p-STAT4) was obviously increased, and the aggregation.IL-35 of p-STAT1 and p-STAT4 was found in the nucleus through gp130:gp130 homologous receptor, activating the P-STAT1:P-STAT1 identical source two polymer to activate the expression of the human PDAC cells In vivo blood vessel experiment found that IL-35 promoted the blood vessel process of Pan02 cells by regulating the expression of ICAM1, and found that IL-35 could induce the self activation of EBI3 and p35 gene in human pancreatic cancer cells by regulating the expression of ICAM1 in vivo, and promoted the increase of IL-35 expression level and IL-35 Yang. The ratio of sexual tumor cells increased the intensity and breadth of expression of IL-35 in pancreatic cancer cells and induced the spread of tumor metastasis potential. Conclusion 1.IL-35 protein and its receptor are abnormal overexpression in pancreatic cancer tissue and cell lines, and the poor prognosis of patients with higher IL-35 expression in pancreatic cancer tissues will activate JAK-ST after.2.IL-35 overexpression. AT signaling pathway promotes the formation of p-STAT1:p-STAT1 homologous two polymer and enhances the HUVEC adhesion and TEM ability of pancreatic cancer cells by up regulation of ICAM1 expression..3.IL-35 can promote the self excitation of the IL-35 gene in human pancreatic cancer cells by promoting the adhesion of the tumor cells and the vascular process and then enhancing the metastasis ability of the tumor. To live, to further promote the metastasis of tumor.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.9

【共引文獻(xiàn)】

相關(guān)期刊論文 前9條

1 陳倩倩;黎霏霏;曾今誠;徐軍發(fā);;IL-12家族抗腫瘤作用的研究進(jìn)展[J];國際檢驗(yàn)醫(yī)學(xué)雜志;2014年11期

2 林曉悅;梁艷芳;康東平;陳倩倩;黎菲菲;曾今誠;;p35和EBI3mRNA在結(jié)腸癌組織中的表達(dá)及意義[J];中國醫(yī)藥科學(xué);2014年13期

3 王鎮(zhèn)南;黃顥;林瓊燕;鄒琳;唐志;李淑慧;;IL-35在宮頸癌根治術(shù)后復(fù)發(fā)和轉(zhuǎn)移中的預(yù)測(cè)價(jià)值[J];廣州醫(yī)科大學(xué)學(xué)報(bào);2015年01期

4 楊景英;陳學(xué)利;吳文欽;曾今誠;徐軍發(fā);;白細(xì)胞介素-12家族與感染性疾病相關(guān)研究進(jìn)展[J];臨床檢驗(yàn)雜志;2014年06期

5 陳爽;鞠曉紅;鄭文_g;趙良中;;IL-35與人類疾病關(guān)系的研究進(jìn)展[J];免疫學(xué)雜志;2014年07期

6 劉艷;曾今誠;梁小泉;;鼻咽癌組織EBI3和p35蛋白的表達(dá)及臨床意義[J];海南醫(yī)學(xué);2014年18期

7 閆永嘉;何向輝;;IL-35結(jié)構(gòu)功能及其調(diào)控Treg細(xì)胞免疫抑制功能的研究進(jìn)展[J];天津醫(yī)藥;2014年12期

8 鄭相慧;武維恒;;抑制性細(xì)胞因子白介素35及其與疾病關(guān)系的研究進(jìn)展[J];中華全科醫(yī)學(xué);2015年02期

9 王志會(huì);;IL-35在免疫相關(guān)疾病中的研究進(jìn)展[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2015年02期

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1 閆永嘉;IL-35基因修飾間充質(zhì)干細(xì)胞治療小鼠潰瘍性結(jié)腸炎的實(shí)驗(yàn)研究[D];天津醫(yī)科大學(xué);2014年

2 陶千山;IL-35誘導(dǎo)急性髓細(xì)胞白血病細(xì)胞免疫逃逸的作用及機(jī)制[D];安徽醫(yī)科大學(xué);2014年

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1 李百慧;自噬在ALD-DNA誘導(dǎo)SLE發(fā)病中的作用：調(diào)節(jié)巨噬細(xì)胞BAFF的表達(dá)[D];蘇州大學(xué);2013年

2 臺(tái)適;冠心病患者血漿IL-35水平及臨床意義的探討[D];中南大學(xué);2013年

3 金鵬;IL-35在胰腺導(dǎo)管腺癌患者外周血中的表達(dá)[D];天津醫(yī)科大學(xué);2014年

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