HBx蛋白在肝细胞癌中上调PD-L1表达的作用及机制

发布时间：2018-02-01 12:54

本文关键词： 肝细胞癌程序性死亡因子配体1 HBx蛋白免疫治疗乙型肝炎病毒　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景原发性肝癌是最常见的恶性肿瘤之一,其肿瘤相关死亡率在我国恶性肿瘤中排名居第二位。手术切除、肝脏移植、放化疗、姑息治疗虽然能让患者得到康复,但疗效有限。最近几年以来,免疫疗法研究进展迅速,已成为继上述方法后最具希望的肿瘤治疗方式之一。是以,对肝癌免疫治疗进行深入探究具有深远的意义。程序性死亡因子配体1(PD-L1)作为免疫监测点因子,在多种肿瘤细胞中异常上调,从而抑制T淋巴细胞的免疫活性,造成肿瘤的免疫逃逸。PD-L1的表达受多种机制调控,部分研究发现其表达与病毒感染有关,但具体的机制仍然不是很清楚。乙型肝炎病毒(hepatitis B virus,HBV)感染是肝癌最常见的病因之一,其中的X基因表达产物(HBx)在肝癌发生与发展过程中起着至关重要的作用。目的阐明HBx和PD-L1在肝细胞癌中的作用,探索HBx蛋白对肝细胞癌PD-L1表达的影响,并深入研究其中的机制。研究HBx蛋白在肝癌患者中的临床意义。方法免疫荧光染色法检测SMMC7721(乙肝阴性的肝癌细胞)和PLC/PRF/5(乙肝阳性的肝癌细胞)中HBx和PD-L1的含量及定位。实时荧光定量法检测SMMC7721和PLC/PRF/5细胞中HBx和PD-L1在m RNA上的表达水平。Western Blot法检测SMMC7721和PLC/PRF/5细胞中HBx和PD-L1在蛋白上的表达水平。相关性曲线表明HBx和PD-L1在基因和蛋白表达水平上的相关性。质粒转染技术将HBx质粒和空载质粒转染到SMMC7721细胞,并用实时荧光定量法和Western Blot法检测HBx和PD-L1在基因和蛋白水平上的表达变化。si RNA沉默技术应用于PLC/PRF/5细胞后用实时荧光定量法和Western Blot法检测HBx和PD-L1在基因和蛋白水平上的表达变化。Western Blot法检测各种细胞中NF-κB,p-NF-κB,JAK2,p-JAK2,STAT3,p-STAT3蛋白表达的变化,探索其中的分子机制。Transwell法测定各种细胞迁移和侵袭能力的大小。免疫组织化学法检测肝癌患者组织标本中PD-L1的表达。结果HBx能上调SMMC7721细胞中PD-L1的表达,并且两者呈正相关。抑制HBx后,PLC/PRF/5细胞中PD-L1的表达也随之下降。HBx转染和沉默后NF-κB,JAK2,STAT3蛋白表达量基本没变化,而p-NF-κB,p-JAK2,p-STAT3随着HBx的变化而变化。对细胞迁移和侵袭能力方面的影响,转染HBx后SMMC7721的能力升高,而沉默HBx后PLC/PRF/5细胞的能力下降。感染HBV的肝癌患者组织标本中PD-L1表达的阳性率高于未感染HBV的肝癌患者。结论HBx能诱导肝细胞癌细胞PD-L1的表达,而si RNA能沉默这个过程,降低PD-L1的表达。HBx可能通过活化NF-κB、JAK/STAT信号通路中的某些核心因子介导肝癌细胞中PD-L1的表达。HBx转染后肝癌细胞的迁移和侵袭能力都有所增强,暗示预防乙肝感染或许能降低肝细胞癌的迁移和侵袭能力。预防HBV的感染和切断PD-1/PD-L1分子路径的传递能降低PD-L1的表达,降低癌细胞的迁移和侵袭能力,这可能为乙肝相关性肝细胞癌的治疗挖掘新的思路和方向。
[Abstract]:Background Primary liver cancer is one of the most common malignant tumors, its tumor-related mortality ranks second among malignant tumors in China. Although palliative therapy can make patients recover, the curative effect is limited. In recent years, immunotherapy research has made rapid progress, and has become one of the most promising cancer treatment methods after the above methods. It is of great significance to explore the immunotherapy of liver cancer. As an immunological monitoring point factor, the programmed death factor ligand 1pd L1 is upregulated in various tumor cells. Thus, the immune activity of T lymphocytes was inhibited and the expression of PD-L1 was regulated by many mechanisms. Some studies have found that the expression of PD-L1 is related to virus infection. However, the specific mechanism is still not clear. Hepatitis B virus HBV) infection is one of the most common causes of liver cancer. The X gene expression product (HBX) plays an important role in the carcinogenesis and development of HCC. Objective to elucidate the role of HBx and PD-L1 in hepatocellular carcinoma. To explore the effect of HBx protein on the expression of PD-L1 in hepatocellular carcinoma. To study the clinical significance of HBx protein in patients with liver cancer. Methods Immunofluorescence staining was used to detect SMMC7721 (Hepatitis B negative hepatoma cells). And PLC / PRF / 5 (Hepatitis B positive hepatoma cells). The content and localization of HBx and PD-L1 in SMMC7721 and PLC/PRF/5 cells. Real-time fluorescence quantitative detection of HBx and PD-L1 in SMMC7721 and PLC/PRF/5 cells. Expression level of RNA. Blot assay was used to detect the expression of HBx and PD-L1 in SMMC7721 and PLC/PRF/5 cells. The correlation curve showed that HBx and PD-L1 were on gene and protein surface. HBx plasmid and empty plasmid were transfected into SMMC7721 cells by plasmid transfection technique. Expression changes of HBx and PD-L1 at gene and protein level were detected by real-time fluorescence quantitative assay and Western Blot assay. Si. Application of RNA silencing technique to PLC/PRF/5 cells by real-time fluorescence quantitative assay and Western. Blot assay was used to detect the expression of HBx and PD-L1 at the level of gene and protein. Western Blot assay was used to detect NF- 魏 B in all kinds of cells. The expression of p-NF- 魏 B, JAK2, p-JAK2, STAT3and p-STAT3. The molecular mechanism was explored. Transwell method was used to determine the migration and invasion ability of various cells. Immunohistochemical method was used to detect the expression of PD-L1 in the tissues of patients with liver cancer. Results HBx could be used to detect the expression of PD-L1. The expression of PD-L1 was up-regulated in SMMC7721 cells. Inhibition of PD-L1 expression in PLC / PRF / 5 cells after HBx also decreased. HBX transfection and silencing NF- 魏 B ~ (2) JAK2. The expression of STAT3 protein was almost unchanged, while p-NF- 魏 Bnp-JAK2pSTAT3 changed with the change of HBx, and the effect on cell migration and invasion ability. The ability of SMMC7721 was increased after transfection of HBx. The positive rate of PD-L1 expression in HCC samples infected with HBV was higher than that in HCC patients without HBV. Conclusion HBx can inhibit the ability of PLC/PRF/5 cells. To induce the expression of PD-L1 in hepatocellular carcinoma cells. Si RNA silenced this process and reduced the expression of PD-L1. HBX may activate NF- 魏 B. Some core factors in the JAK/STAT signaling pathway mediated the expression of PD-L1 in hepatoma cells. HBX transfection enhanced the migration and invasion of HCC cells. It suggests that the prevention of hepatitis B infection may reduce the ability of migration and invasion of hepatocellular carcinoma. Preventing HBV infection and cutting off the transmission of PD-1/PD-L1 molecule pathway can reduce the expression of PD-L1. Reducing the ability of migration and invasion of cancer cells may open up new ideas and directions for the treatment of Hepatitis B associated hepatocellular carcinoma.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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