TAMs中RIP140对细胞极化和肝癌细胞侵袭、增殖的影响及其分子机制研究

发布时间：2018-04-22 00:02

本文选题：肝癌 + 肿瘤相关巨噬细胞　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:探讨肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)中RIP140的表达对巨噬细胞极化和对肝癌细胞侵袭、增殖的影响及其分子机制。方法:(1)慢病毒介导小鼠腹腔巨噬细胞(peritoneal macrophages.PMs)RIP140的过表达,Western blot和Real-time PCR(q RT-PCR)分别检测PMs中RIP140蛋白以及核酸表达水平,流式细胞仪分析慢病毒转染率;(2)Western blot、细胞免疫荧光和q RT-PCR检测肝癌条件培养基(hepatocellular carcinoma conditioned medium,HCM)刺激PMs24 h后TAMs中RIP140的表达变化;(3)HCM刺激PMs以及HCM刺激过表达RIP140的PMs,q RT-PCR检测TAMs极化指标以及NF-κB和IL-6的表达,Transwell实验和细胞流式凋亡实验检测肝癌细胞的侵袭和凋亡;(4)肝癌细胞和PMs以4∶1比例注射于BALB/c裸鼠皮下,建立裸鼠皮下肝癌模型,成瘤癌组织HE染色和免疫组化评定肝癌组织大体生长情况和肝癌细胞增殖能力。结果:(1)慢病毒介导PMs RIP140的过表达,病毒转染率较高(约60%),RIP140过表达明显;HCM刺激PMs后,TAMs中RIP140表达降低;(2)HCM诱导TAMs呈M2型极化,并且与肿瘤生长密切相关的NF-κB/IL-6通路处于激活状态;TAMs可促进肝癌细胞侵袭和增殖,抑制肝癌细胞凋亡。(3)TAMs过表达RIP140可抑制HCM介导的TAMs M2型极化并抑制NF-κB/IL-6通路的激活,减少IL-6的释放;此外,TAMs过表达RIP140可抑制肝癌细胞的侵袭和增殖[PCNA+细胞数:对照组(117.3±13.1)vs过表达组(56.9±7.4),P0.05],并促进肝癌细胞的凋亡[凋亡率:对照组(28.7±3.6%)vs过表达组(43.1±2.9%),P0.05]。结论:过表达RIP140的TAMs抑制肝癌细胞的侵袭和增殖。其机制与TAMs过表达RIP140后抑制TAMs M2型极化有关。
[Abstract]:Aim: to investigate the effect of RIP140 expression in tumor-associated macrophages (tumor-associated macrophages) on macrophage polarization, invasion and proliferation of hepatoma cells and its molecular mechanism. Methods the overexpression of macrophages.PMs)RIP140 in murine peritoneal macrophages mediated by lentivirus was detected by Western blot and Real-time PCR(q RT-PCR respectively. Flow cytometry analysis of lentivirus transfection rate: Western blot.The cell immunofluorescence and Q RT-PCR were used to detect the expression of RIP140 in TAMs after PMs24 h stimulated by hepatocarcinoma conditioned medium (carcinoma conditioned medium). The RIP140 expression in TAMs was detected by PMs stimulated by PMs and PMs by PMs stimulated by HCM and the TAMs electrode by PMsTQ RT-PCR stimulated by HCM. The expression of NF- 魏 B and IL-6 was detected by Transwell assay and flow cytometry. The invasion and apoptosis of hepatoma cells were detected by transwell assay and flow cytometry. (4) hepatoma cells and PMs were injected subcutaneously into BALB/c nude mice at 4:1. The subcutaneous liver cancer model of nude mice was established, and HE staining and immunohistochemistry were used to evaluate the gross growth and proliferation of hepatoma cells. Results PMs RIP140 overexpression mediated by lentivirus had a high transfection rate (about 60% RIP140 overexpression). The expression of RIP140 in PMs induced by lentivirus-induced TAMs showed M2 polarization. Furthermore, the activation of NF- 魏 B/IL-6 pathway, which is closely related to tumor growth, can promote the invasion and proliferation of hepatocellular carcinoma cells, and inhibit the overexpression of TAMs M2 type polarization mediated by HCM, inhibit the activation of NF- 魏 B/IL-6 pathway and reduce the release of IL-6. In addition, RIP140 overexpression of tams inhibited the invasion and proliferation of HCC cells [PCNA cell number: 56.9 卤7.4 PCNA cells in the control group: 117.3 卤13.1)vs overexpression group, P0.05], and promoted apoptosis of HCC cells [apoptosis rate: 43.1 卤2.9 in the control group, 28.7 卤3.6%)vs overexpression group, P0.05]. Conclusion: TAMs overexpression of RIP140 inhibits the invasion and proliferation of hepatoma cells. The mechanism is related to the inhibition of M 2 polarization of TAMs after TAMs overexpression of RIP140.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

【参考文献】