TNF-α-VEGF-D轴与胆囊癌微淋巴管生成的关系及其分子机制

发布时间：2018-05-11 06:09

本文选题：胆囊癌 + TNF-α　；参考：《福建医科大学》2016年博士论文

【摘要】：【研究背景】本实验室前期研究发现,炎症因子TNF-α可促进胆囊癌的微淋巴管生成;且通过免疫组化检测分析发现VEGF-D(血管内皮生长因子-D)的表达与胆囊癌的淋巴管生成及淋巴结转移正相关。而VEGF-D是否与TNF-α介导的胆囊癌微淋巴管生成有关,尚未见研究报道。因此,本课题拟围绕两部分内容开展研究:1、TNF-α-VEGF-D轴对裸鼠胆囊癌原位移植瘤微淋巴管生成的影响;2、TNF-α促胆囊癌细胞VEGF-D表达的分子机制。旨在通过动物实验、启动子活性分析及信号通路研究等阐明TNF-α-VEGF-D轴与胆囊癌微淋巴管生成的关系及分子机制。【方法】1、用本实验室已构建的胆囊癌细胞株NOZ/sh VEGF-D(RNAi沉默VEGF-D)、NOZ/sh CTRL(转染阴性对照序列)及空白对照NOZ细胞,建立裸鼠胆囊癌原位移植瘤模型。建模后2周开始,实验组腹腔注射TNF-α(2μg/kg),对照组注射等体积生理盐水,每3天注射一次,连续注射3周后处死并解剖裸鼠,取原位癌组织及转移淋巴结标本制作石蜡切片,利用免疫组化及HE染色检测微淋巴管密度(LVD)及转移淋巴结,分析TNF-α-VEGF-D轴对裸鼠胆囊癌原位移植瘤微淋巴管生成的影响。2、通过DNA重组技术构建一系列含VEGF-D启动子截短片段的萤火虫荧光素酶报告质粒,用双荧光素酶检测系统分析VEGF-D启动子活性。3、应用软件TFbind、Promoter Scan对VEGF-D启动子上潜在的AP-1和/或NF-κB结合位点进行预测。4、通过碱基定点突变、电泳迁移率变动分析(EMSA)和染色质免疫沉淀(Ch IP)确定VEGF-D基因启动子上的AP-1和/或NF-κB结合位点及TNF-α对启动子活性的影响。5、应用RNAi沉默技术、蛋白特异阻断剂抑制实验检测TNF-α-VEGF-D轴上的信号通路分子。6、统计学分析方法:总体均数的比较采用t检验,显著性水平α=0.05,P0.05为有统计学差异。【结果】1、TNF-α-VEGF-D轴对裸鼠胆囊癌微淋巴管生成及淋巴转移的影响:1)淋巴管密度计数结果显示,TNF-α干预NOZ组、NOZ/sh CTRL组、NOZ/sh VEGF-D组小鼠后,三组小鼠的淋巴管密度分别为23.73±2.17、23.20±2.18、10.07±1.83,与无TNF-α干预组比较(三组分别为11.73±2.28、14.27±1.36、5.67±1.25),差异均有显著性(P0.05)。但TNF-α对NOZ/sh VEGF-D组的促淋巴管生成作用显著低于NOZ组和NOZ/sh CTRL组(P0.05)。表明TNF-α可通过VEGF-D促进裸鼠胆囊癌原位移植瘤组织的微淋巴管生成。2)TNF-α干预NOZ组、NOZ/sh CTRL组、NOZ/sh VEGF-D组小鼠后,三组小鼠的淋巴结转移发生率均比无TNF-α干预组增高,但TNF-α对NOZ/sh VEGF-D组的淋巴结转移发生率低于NOZ组和NOZ/sh CTRL组,提示TNF-α可能通过VEGF-D促进裸鼠胆囊癌淋巴转移。2、TNF-α促胆囊癌细胞VEGF-D表达的分子机制1)成功构建一系列含VEGF-D启动子截短片段的重组质粒并经测序鉴定序列正确;双荧光素酶系统检测结果显示重组质粒PGL4-988较PGL4-717、PGL4-444较PGL4-325、PGL4-154较PGL4-57,均呈现出更强的荧光活性,差异有统计学意义(均P0.05)。提示VEGF-D启动子转录起始位点ATG上游-988至-717nt、-444至-325nt、-154至-57nt三个区间可能存在调控VEGF-D转录活性的调控位点。2)TFbind、Promoter Scan软件预测结果显示,VEGF-D启动子转录起始位点ATG上游-444至-325nt区间存在两个潜在的AP-1结合位点:GTGTGTCAT(-401至-393nt)和CTGAGATAC(-345至-337nt),而-988至-717nt、-154至-57nt区间未检测到AP-1位点;三个区间均未检测到NF-κB结合位点。3)定点诱变、电泳迁移率变动分析(EMSA)和染色质免疫沉淀(Ch IP)实验证实转录因子AP-1可直接与VEGF-D启动子-444至-325nt区间的两个AP-1位点结合,且TNF-α可以增强AP-1与这两个位点的结合。4)AP-1位点突变对TNF-α诱导VEGF-D启动子活性的影响:“PGL4-AP1mut1+TNF-α”组较“PGL4-444+TNF-α”组(2.77±0.23 vs 3.98±0.15)、“PGL4-AP1 mut2+TNF-α”组较“PGL4-444+TNF-α”组(3.14±0.33 vs 3.98±0.15),荧光活性明显减弱,差异均有统计学意义(P0.05)。表明TNF-α可通过两个AP-1结合位点调控VEGF-D的启动子活性。5)沉默AP-1对TNF-α诱导VEGF-D启动子活性及蛋白表达的影响:TNF-α诱导si AP-1 NOZ细胞的PGL4-444活性较NOZ细胞的弱(2.69±0.34 vs 5.88±0.15),差异有统计学意义(P0.05);TNF-α诱导si AP-1 NOZ细胞的VEGF-D蛋白表达较NOZ细胞的弱(0.59±0.13 vs 1.39±0.10),差异有统计学意义(P0.05)。表明TNF-α通过AP-1增强VEGF-D启动子活性并上调VEGF-D蛋白表达。6)MAPKs抑制剂对TNF-α诱导VEGF-D启动子活性的影响:“PD98059+TNF-α”组NOZ细胞的PGL4-444活性(1.74±0.23)较TNF-α组(5.88±0.15)明显减弱(P0.05),“SP600125+TNF-α”组的PGL4-444活性(5.80±0.31)和“SB203580+TNF-α”组的PGL4-444活性(6.49±0.54)与TNF-α组比较差异均无显著性(P0.05)。表明TNF-α通过ERK1/2通路增强VEGF-D启动子活性。7)MAPKs抑制剂对TNF-α诱导VEGF-D蛋白表达的影响:“PD98059+TNF-α”组NOZ细胞的VEGF-D蛋白表达(0.14±0.03)较TNF-α组(0.33±0.02)明显减少(P0.05),“SP600125+TNF-α”组的VEGF-D蛋白表达(0.34±0.05)和“SB203580+TNF-α”组的VEGF-D蛋白表达(0.32±0.03)与TNF-α组比较差异均无显著性(P0.05)。表明TNF-α通过ERK1/2通路上调VEGF-D蛋白表达。【结论】1、TNF-α可通过VEGF-D促进裸鼠胆囊癌原位移植瘤的微淋巴管生成。2、VEGF-D启动子-444至-325nt区间存在两个有活性的AP-1结合位点,且TNF-α可增强AP-1与这两个位点的结合;未检测到NF-κB结合位点。3、TNF-α通过“ERK1/2-AP-1”通路增强VEGF-D启动子活性并上调VEGF-D蛋白表达。
[Abstract]:[background] a preliminary study in our laboratory found that inflammatory factor TNF- alpha could promote the formation of microlymphatic vessels in gallbladder carcinoma, and the expression of VEGF-D (vascular endothelial growth factor -D) is positively related to lymphatic formation and lymph node metastasis of gallbladder cancer by immunohistochemical detection. And whether VEGF-D is associated with TNF- alpha in gallbladder cancer Guan Shengcheng has not yet seen the research report. Therefore, this subject is intended to carry out a study around two parts: 1, the effect of TNF- alpha -VEGF-D axis on the formation of microlymphatic vessels in the orthotopic xenografts in nude mice; 2, the molecular mechanism of TNF- alpha promoting the expression of VEGF-D in gallbladder cancer cells. The relationship between TNF- alpha -VEGF-D axis and the formation of microlymphatic duct in gallbladder carcinoma. [Methods] 1. The model of the orthotopic xenografts in nude mice was established by using the gallbladder cancer cell line NOZ/sh VEGF-D (RNAi silent VEGF-D), NOZ/sh CTRL (transfected negative control sequence) and the blank control NOZ cells. After 2 weeks, the experimental group was established. Intraperitoneal injection of TNF- alpha (2 u g/kg), injection of equal volume of normal saline in the control group, injected once every 3 days, and after 3 weeks of continuous injection, were killed and dissected in nude mice. The paraffin sections were made in situ and metastatic lymph nodes. The microlymphatic vessel density (LVD) and metastatic lymph nodes were detected by immunohistochemistry and HE staining, and the TNF- alpha -VEGF-D axis on the nude mice was analyzed. The effect of.2 on the formation of microlymphangioma in the tumor in situ transplantation tumor, a series of firefly luciferase reporter plasmids containing VEGF-D promoter were constructed by DNA recombination technology, and the VEGF-D promoter activity.3 was analyzed with the dual luciferase detection system, the application software TFbind, Promoter Scan on the potential AP-1 and / or NF- kappa B binding on the VEGF-D promoter. The loci predict.4, through base directed mutagenesis, electrophoresis mobility change analysis (EMSA) and chromatin immunoprecipitation (Ch IP) determination of AP-1 and / or NF- kappa B binding sites on VEGF-D gene promoter and the effect of TNF- alpha on promoter activity,.5, RNAi silencing technique, and egg white specific blocker inhibition test on TNF- alpha axis .6, statistical analysis: t test, significant level of alpha =0.05, and P0.05 were statistically different. [results] 1, the effect of TNF- alpha -VEGF-D axis on the formation and lymphatic metastasis of gallbladder carcinoma in nude mice: 1) the lymphatic density count results showed that TNF- alpha intervention in NOZ group, NOZ/sh CTRL group, NOZ/sh VEGF-D After group mice, the lymphatic density of the three groups were 23.73 + 2.17,23.20 + 2.18,10.07 + 1.83 respectively, compared with the non TNF- alpha intervention group (three groups were 11.73 + 2.28,14.27 + 1.36,5.67 1.25), and the difference was significant (P0.05). But TNF- alpha was significantly lower than the NOZ group and NOZ/sh CTRL group. TNF- alpha could promote the microlymphatic.2 in the orthotopic xenografts in nude mice by VEGF-D. TNF- alpha intervention in the NOZ group, NOZ/sh CTRL group and NOZ/sh VEGF-D group mice, the incidence of lymph node metastasis in the three groups was higher than that in the non TNF- alpha intervention group, but the incidence of lymph node metastasis in the NOZ/sh group was lower than that of the TNF- alpha intervention group. RL group, suggesting that TNF- alpha may promote the lymphatic metastasis of gallbladder cancer in nude mice by VEGF-D and the molecular mechanism of VEGF-D expression of TNF- a gallbladder cancer cells 1.) a series of recombinant plasmids containing VEGF-D promoter truncated fragments were successfully constructed and the sequencing identification sequence was correct. The results of the double luciferase system test showed that the recombinant plasmid PGL4-988 was more than PGL4-717, PGL4-4. 44 compared with PGL4-325 and PGL4-154, compared with PGL4-57, they all showed stronger fluorescence activity, and the difference was statistically significant (P0.05). It suggested that the transcription start site of VEGF-D promoter was -988 to -717nt, -444 to -325nt, -154 to -57nt three regions may have regulatory sites regulating the transcriptional activity. There are two potential AP-1 binding sites in the upstream -444 to -325nt interval of the promoter of VEGF-D promoter ATG: GTGTGTCAT (-401 to -393nt) and CTGAGATAC (-345 to -337nt). Analysis (EMSA) and chromatin immunoprecipitation (Ch IP) experiments confirmed that transcription factor AP-1 can be directly associated with the two AP-1 loci of the VEGF-D promoter -444 to -325nt interval, and TNF- alpha can enhance the.4 of AP-1 with these two loci. The +TNF- Alpha Group (2.77 + 0.23 vs 3.98 + 0.15), "PGL4-AP1 mut2+TNF- alpha" group was compared to the "PGL4-444+TNF- alpha" group (3.14 + 0.33 vs 3.98 + 0.15), and the fluorescence activity was significantly reduced, the difference was statistically significant (P0.05). It showed that TNF- alpha could regulate the promoter activity of VEGF-D through two AP-1 binding sites. The effect of activity and protein expression: the PGL4-444 activity of TNF- alpha induced Si AP-1 NOZ cells was weaker than that of NOZ cells (2.69 + 0.34 vs 5.88 + 0.15), and the difference was statistically significant (P0.05); TNF- alpha induced Si AP-1 NOZ cells was weaker than that of the cells (0.59 + 0.13 1.39 + 0.10), and the difference was statistically significant. AP-1 enhanced the activity of VEGF-D promoter and up-regulated the expression of VEGF-D protein.6) the effect of MAPKs inhibitor on the activity of VEGF-D promoter induced by TNF- A: PGL4-444 activity of NOZ cells in "PD98059+TNF- a" group (1.74 + 0.23) was significantly lower than that in TNF- Alpha Group (5.88 + 0.15). The activity of PGL4-444 (6.49 + 0.54) in the group of alpha group was not significantly different from that in the TNF- Alpha Group (P0.05). It showed that TNF- alpha enhanced the activity of VEGF-D promoter through the ERK1/2 pathway. The effect of MAPKs inhibitor on the expression of VEGF-D protein induced by TNF- alpha: "PD98059+TNF- alpha" group of NOZ cells (0.14 + 0.03) was significantly lower than that of the group (0.33 + 0.02). Less (P0.05), the expression of the expression of VEGF-D protein (0.34 + 0.05) in the "SP600125+TNF- alpha" group and the expression of VEGF-D protein in the group of "SB203580+TNF- alpha" (0.32 + 0.03) had no significant difference from that of the TNF- Alpha Group (P0.05). It showed that TNF- a increased the expression of VEGF-D protein through the ERK1/2 pathway. [knot] 1, TNF- alpha could promote the orthotopic transplantation of gallbladder carcinoma in nude mice. The microlymphatic vessels of the tumor produce.2, and there are two active AP-1 binding sites in the -444 to -325nt interval of the VEGF-D promoter, and TNF- a enhances the binding of AP-1 to these two loci; NF- kappa B binding site.3 is not detected. TNF- alpha enhances the activity of the promoter through the "ERK1/2-AP-1" pathway and up-regulated the expression of the protein.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.8

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