肿瘤源性IL-35促胰腺癌转移及机制研究

发布时间：2018-06-16 22:19

本文选题：胰腺癌 + 白细胞介素35　；参考：《天津医科大学》2015年博士论文

【摘要】：背景白细胞介素35(Interleukin 35,IL-35)是最新发现的白细胞介素12(IL-12)细胞因子家族成员。IL35可能在自身免疫性疾病,炎性疾病,感染性疾病以及某些肿瘤中具有重要的作用,可能成为治疗这些疾病的新靶点。在我们的前期研究中发现,与正常组织及细胞系比较,胰腺癌组织及细胞高表达IL-35蛋白。我们设计本实验,进一步明确IL-35在胰腺癌组织及细胞中的表达方式;从临床水平、细胞水平及动物实验水平探索胰腺癌肿瘤源性IL-35的功能及其调控机制。方法1.应用胰腺癌组织石蜡标本,进行IL-35两个亚基p35和EBI3及IL-35受体两个亚基IL12rβ2和gp130的连续切片免疫组化染色,分析IL-35及其受体的表达水平及相关性;搜集病例资料进行预后随访,分析IL-35及其受体的表达水平与临床病理参数之间的关系。2.应用Western blot、RT-PCR、ELISA等实验技术检测胰腺癌5个细胞系、Treg细胞(作为阳性对照)以及正常胰腺导管细胞系的IL-35及其受体的表达水平;应用免疫荧光技术检测IL-35及其受体共4个亚基在胰腺癌细胞中的表达细胞定位;应用免疫共沉淀技术(Co-Immunoprecipitation,COIP)研究胰腺癌细胞表达IL-35两个亚基的相关性。3.应用分子克隆方法构建IL-35过表达质粒及IL-35 shRNA降表达质粒,构建慢病毒稳定转染系统,建立稳定转染细胞系;用Western blot、RT-PCR及ELISA检测所构建稳系表达IL-35水平;应用EDU增殖实验检测所构建细胞表达的IL-35的生物学功能。4.用人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVEC)粘附实验及跨内皮迁移(Transendothelial migration,TEM)实验研究IL-35在体外促肿瘤细胞出血管过程。应用转录组测序的方法筛选IL-35促进肿瘤转移的靶标;应用Western blot及RT-PCR实验验证测序结果;通过阻断试验进一步确定IL-35促肿瘤出血管过程的靶标。5.应用Western blot、RT-PCR、CHIP及免疫荧光实验检测IL-35调控靶标的分子机制。6.构建nod-sci小鼠肿瘤细胞出血管过程模型;构建nod-scid小鼠胰腺癌原位成瘤模型;体内验证il-35促胰腺癌细胞出血管及促进胰腺癌转移作用。结果1.il-35及其受体在胰腺癌组织中的表达及其意义。通过对123例胰腺癌组织标本进行免疫组化染色发现,il-35两个亚基p35及ebi3的表达在多种胰腺癌病理类型表达阳性,而对应的正常胰腺组织不表达或弱表达;分析p35及eib3表达的相关性,发现两者的分布呈明显相关性(r=0.950;p0.0001);将p35及ebi3都高表达的定义为il-35高表达,其他的为il-35低表达,我们发现il-35高表达的占48.8%(60例);我们进一步探索il-35表达水平与临床病理参数的关系,发现高il-35水平与较晚tnm分期、淋巴结受累以及血管侵犯相关(p值分别为0.01,0.027及0.007)。我们发现较高的il-35表达水平患者具有较差的总生存期(中位数:13.70±0.722vs18.5±1.50月)及较差的无复发生存期(中位数:11.39±0.54vs16.87±2.00);cox风险模型经过单因素、多因素分析后得出il-35表达水平是总生存期及无复发生存期的独立影响因素。il-35受体两个亚基gp130及il12rβ2在胰腺癌组织中均有阳性表达。进一步研究了il-35配体及受体表达的相关性,发现两者明显正相关(r=0.384,p0.0001)。我们将il-35配体及受体综合考虑分组后发现,il-35高表达同时受体表达阳性的患者的总生存期及无复发生存期较其他组的差;这些结果提示il-35在体内通过自分泌或旁分泌作用直接作用于胰腺癌细胞而发挥生物学功能,引起肿瘤进展。2.il-35及其受体在胰腺癌细胞系中的表达。rt-pcr、westernblot、免疫荧光实验及elisa实验发现5个胰腺癌细胞系(panc-1、miapaca-2、cfpac-1、aspc-1及bxpc-3)均表达il-35及其受体;在miapaca-2及cfpac-1细胞中用coip实验证实了il-35两个亚基的结合。3.成功构建了2个过表达il-35的胰腺癌细胞系(panc-1及bxpc-3)和降表达il-35的两个细胞系(miapaca-2和cfpac-1);经cd4+t细胞体外edu增殖实验证实,构建的il-35稳系具备有文献报道的抑制cd4+t细胞增殖的生物学功能。4.体外功能实验证实肿瘤细胞过表达il-35后,huvec粘附及tem能力增强;而肿瘤细胞降表达il-35后huvec粘附及tem能力下降。转录组测序发现IL-35过表达后细胞间粘附分子1(intercellular adhesion molecule 1,ICAM1)表达水平明显上调,Western blot实验验证了ICAM1表达水平受IL-35的调控;瓶颈试验发现,当将过表达IL-35细胞的ICAM1分子下调后,该细胞系的HUVEC粘附及TEM能力不再有明显的增强。ICAM1抗体能部分阻断发现IL-35过表达引起的HUVEC粘附能力增强。5.肿瘤细胞受到IL-35蛋白刺激后JAK-STAT信号通路被激活,磷酸化STAT1(p-STAT1)及磷酸化STAT4(p-STAT4)表达水平明显上升,并且在细胞核内可见p-STAT1及p-STAT4聚集。IL-35是通过gp130:gp130同源受体,激活P-STAT1:P-STAT1同源二聚体入核后激活人PDAC细胞ICAM1表达的。6.体内出血管实验发现IL-35通过调控ICAM1的表达,促进了Pan02细胞出血管过程;体内胰腺原位成瘤实验发现IL-35通过调控ICAM1的表达,促进了肿瘤的转移。7.IL-35能诱导人胰腺癌细胞的EBI3及p35基因的自激活,促进IL-35表达水平增加,提高IL-35阳性肿瘤细胞的比率。提高了胰腺癌细胞中IL-35的表达强度及表达广度,诱导了肿瘤转移潜能的播散。结论1.IL-35蛋白及其受体在胰腺癌组织及细胞系中异常过表达;胰腺癌组织IL-35表达水平较高的患者预后较差。2.IL-35过表达后将激活JAK-STAT信号通路,促进p-STAT1:p-STAT1同源二聚体的生成,入核后通过上调ICAM1的表达,从而增强胰腺癌细胞的HUVEC粘附能力及TEM能力。通过促进肿瘤细胞血管粘附及出血管过程进而增强肿瘤的转移能力。3.IL-35能促进人胰腺癌细胞IL-35基因的自激活,进一步促进了肿瘤的转移能力。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.9

【共引文献】