本文關(guān)鍵詞： 肝細胞癌 程序性死亡因子配體1 HBx蛋白 免疫治療 乙型肝炎病毒　出處：《青島大學》2017年碩士論文　論文類型：學位論文

【摘要】：背景原發(fā)性肝癌是最常見的惡性腫瘤之一,其腫瘤相關(guān)死亡率在我國惡性腫瘤中排名居第二位。手術(shù)切除、肝臟移植、放化療、姑息治療雖然能讓患者得到康復,但療效有限。最近幾年以來,免疫療法研究進展迅速,已成為繼上述方法后最具希望的腫瘤治療方式之一。是以,對肝癌免疫治療進行深入探究具有深遠的意義。程序性死亡因子配體1(PD-L1)作為免疫監(jiān)測點因子,在多種腫瘤細胞中異常上調(diào),從而抑制T淋巴細胞的免疫活性,造成腫瘤的免疫逃逸。PD-L1的表達受多種機制調(diào)控,部分研究發(fā)現(xiàn)其表達與病毒感染有關(guān),但具體的機制仍然不是很清楚。乙型肝炎病毒(hepatitis B virus,HBV)感染是肝癌最常見的病因之一,其中的X基因表達產(chǎn)物(HBx)在肝癌發(fā)生與發(fā)展過程中起著至關(guān)重要的作用。目的闡明HBx和PD-L1在肝細胞癌中的作用,探索HBx蛋白對肝細胞癌PD-L1表達的影響,并深入研究其中的機制。研究HBx蛋白在肝癌患者中的臨床意義。方法免疫熒光染色法檢測SMMC7721(乙肝陰性的肝癌細胞)和PLC/PRF/5(乙肝陽性的肝癌細胞)中HBx和PD-L1的含量及定位。實時熒光定量法檢測SMMC7721和PLC/PRF/5細胞中HBx和PD-L1在m RNA上的表達水平。Western Blot法檢測SMMC7721和PLC/PRF/5細胞中HBx和PD-L1在蛋白上的表達水平。相關(guān)性曲線表明HBx和PD-L1在基因和蛋白表達水平上的相關(guān)性。質(zhì)粒轉(zhuǎn)染技術(shù)將HBx質(zhì)粒和空載質(zhì)粒轉(zhuǎn)染到SMMC7721細胞,并用實時熒光定量法和Western Blot法檢測HBx和PD-L1在基因和蛋白水平上的表達變化。si RNA沉默技術(shù)應用于PLC/PRF/5細胞后用實時熒光定量法和Western Blot法檢測HBx和PD-L1在基因和蛋白水平上的表達變化。Western Blot法檢測各種細胞中NF-κB,p-NF-κB,JAK2,p-JAK2,STAT3,p-STAT3蛋白表達的變化,探索其中的分子機制。Transwell法測定各種細胞遷移和侵襲能力的大小。免疫組織化學法檢測肝癌患者組織標本中PD-L1的表達。結(jié)果HBx能上調(diào)SMMC7721細胞中PD-L1的表達,并且兩者呈正相關(guān)。抑制HBx后,PLC/PRF/5細胞中PD-L1的表達也隨之下降。HBx轉(zhuǎn)染和沉默后NF-κB,JAK2,STAT3蛋白表達量基本沒變化,而p-NF-κB,p-JAK2,p-STAT3隨著HBx的變化而變化。對細胞遷移和侵襲能力方面的影響,轉(zhuǎn)染HBx后SMMC7721的能力升高,而沉默HBx后PLC/PRF/5細胞的能力下降。感染HBV的肝癌患者組織標本中PD-L1表達的陽性率高于未感染HBV的肝癌患者。結(jié)論HBx能誘導肝細胞癌細胞PD-L1的表達,而si RNA能沉默這個過程,降低PD-L1的表達。HBx可能通過活化NF-κB、JAK/STAT信號通路中的某些核心因子介導肝癌細胞中PD-L1的表達。HBx轉(zhuǎn)染后肝癌細胞的遷移和侵襲能力都有所增強,暗示預防乙肝感染或許能降低肝細胞癌的遷移和侵襲能力。預防HBV的感染和切斷PD-1/PD-L1分子路徑的傳遞能降低PD-L1的表達,降低癌細胞的遷移和侵襲能力,這可能為乙肝相關(guān)性肝細胞癌的治療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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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