雷公藤甲素对原发性渗出性淋巴瘤细胞的影响及分子机制研究

发布时间：2018-06-21 05:13

本文选题：雷公藤甲素 + 卡波氏肉瘤相关疱疹病毒　；参考：《武汉大学》2016年博士论文

【摘要】：目的：卡波氏肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus, KSHV)又被称为人疱疹病毒8型,属于疱疹病毒γ亚科的一种双链DNA病毒。人类是KSHV的天然宿主,KSHV感染后可在人体内长期潜伏。KSHV是一种肿瘤病毒,与多种人类恶性肿瘤密切相关,主要包括卡波氏肉瘤(Kaposi's sarcoma, KS),原发性渗出性淋巴瘤(primary effusion lymphoma, PEL)和多中心卡斯特莱曼疾病(multicentric Castleman's disease, MCD)。其中PEL属于一种罕见的非霍奇金淋巴瘤,好发生在KSHV感染的免疫缺陷或器官移植的患者,常规抗肿瘤治疗效果欠佳,预后极差。目前临床上使用的抗病毒药物主要是阿昔洛韦,其不但作用于病毒DNA复制,同时也影响人细胞的DNA复制。因此,探索发现新型的毒副作用小的靶向性小分子抑制剂并探究其分子机制对于研发有效的治疗方法至关重要。雷公藤甲素(Triprolide,TP)是从中草药雷公藤中分离得到的一种具有生物学活性的小分子化合物,其生物学活性主要包括抗炎、抗肿瘤、免疫抑制及抗生育等。本研究旨在观察TP对于KSHV阳性的PEL细胞的影响并初步探索其分子机制。方法：通过CCK-8 (Cell Counting Kit-8)试剂盒检测PEL细胞(BCBL-1、BC-3和JSC-1)经不同浓度TP处理不同时间后细胞活力的变化。通过流式细胞仪检测TP诱导PEL细胞周期阻滞及细胞凋亡的情况。通过Western blotting检测PEL细胞经不同方法处理后,目的蛋白表达水平的变化。通过荧光实时定量技术(quantitative real-time polymerase chain reaction, qPCR)检测PEL细胞经TP处理后目的蛋白mRNA水平的变化以及TP对PEL细胞中KSHV病毒粒子产量的影响。通过酶联免疫吸附试验(Enzyme-linked immunosorbent assay, ELISA)检测TP对PEL细胞分泌IL-6水平的影响。通过双荧光素酶报告基因检测技术(Dual luciferase reporter assay)检测TP对人端粒酶反转录酶(Human telomerase reverse transcriptase, hTERT)不同区段启动子活性的影响。NOD/SCID (Non-obese diabetic/severe combined immunodeficient)小鼠经BCBL-1细胞致瘤后,观察TP对小鼠体重、腹水量、腹水细胞中KSHV潜伏相关核抗原1(Latency-associatednuclear antigen 1, LANA1)表达及对脾脏的影响。结果：TP能明显降低KSHV阳性PEL细胞的活力,而对KSHV阴性正常人外周血单个核细胞(Peripheral blood mononuclear cells, PBMCs)及KSHV阴性EBV阳性P3HR-1细胞增殖抑制影响不明显；TP能诱导PEL细胞的凋亡,导致BC-3和JSC-1细胞阻滞在G0/G1期,BCBL-1细胞阻滞在G2/M期；TP能明显降低PEL细胞中LANA1的表达水平,而对其mRNA水平影响不大；TP通过蛋白酶降解途径诱导LANA1水平的下调,并可影响其半衰期；TP能降低诱导KSHV进入裂解期后的BCBL-1细胞内、外病毒粒子的产量；TP通过降低PEL细胞中hTERT不同区段启动子的活性,下调其mRNA水平,进而导致hTERT蛋白水平的下降；TP能降低PEL细胞中转录因子Spl (specificity protein 1)的表达；在PEL细胞中knockdown转录因子Spl (specificity protein 1)能降低PEL细胞的活力及hTERT的表达,诱导细胞凋亡。在PEL细胞中knockdown转录因子Sp1能增加PEL细胞对TP诱导的细胞增殖抑制及细胞凋亡的敏感性;TP能明显抑制NOD/SCID小鼠腹水的增长及BCBL-1细胞对脾脏的浸润。结论：在体内和体外,TP均能降低PEL细胞中LANA1的表达水平,抑制KSHV阳性PEL细胞的增殖,诱导细胞凋亡；TP可通过降低PEL细胞Sp1的表达,抑制hTERT的转录,进而抑制PEL细胞的增殖及永生化。通过对TP在KSHV阳性PEL细胞中抗病毒及抗肿瘤作用的研究,为后续TP及其衍生物用于治疗KSHV相关恶性肿瘤的基础理论研究奠定了一定的基础。
[Abstract]:Objective: Kaposi's sarcoma associated herpesvirus (Kaposi's sarcoma-associated, herpesvirus, KSHV) is also called human herpesvirus 8, a double stranded DNA virus belonging to the subfamily of herpes simplex virus gamma. Humans are the natural host of KSHV, after KSHV infection in the human body can long-term latent.KSHV is a tumor virus, and a variety of human malignant tumor. Cut, including Kaposi's sarcoma (Kaposi's sarcoma, KS), primary effusion lymphoma (primary effusion, lymphoma, PEL) and multi center (multicentric Castleman's disease, Castleman's disease MCD). The PEL is a rare type of non Hodgkin's lymphoma, occurs in immunodeficiency or organ transplantation KSHV infection. Patients with poor general anti-tumor treatment effect the prognosis. The current clinical use of antiviral drugs is mainly A Silowe, which not only effects on viral DNA replication, but also affect the replication of human cell DNA. Therefore, the discovery of new low toxicity targeting small molecule inhibitors and explore the molecular mechanism for the development of there Is effective. Triptolide (Triprolide, TP) is a small molecule bioactive compounds have been isolated from a Chinese herb Tripterygium wilfordii, its biological activities including anti-inflammatory, antitumor, immune suppression and anti fertility. The purpose of this study is to observe the effect of TP in KSHV positive cells and PEL preliminary exploration Its molecular mechanism. Methods: the CCK-8 (Cell Counting Kit-8) PEL cell detection kit (BCBL-1, BC-3 and JSC-1) the changes of cell viability in different time after treatment with different concentrations of TP. Cell cycle arrest and induced apoptosis of PEL cells by flow cytometry in TP conditions. PEL cell line was detected by Western blotting with different methods After the treatment, the expression of the target protein. By real-time quantitative technique (quantitative real-time polymerase chain reaction, qPCR) detection of PEL cell effect on KSHV virus particles in PEL cells yield via changes in protein mRNA levels after treatment with TP and TP by enzyme linked immunosorbent assay (Enzyme-linked immunosor Bent assay, ELISA) to study the effect of TP on the level of IL-6 secretion of PEL cells. Using the dual luciferase reporter assay technique (Dual luciferase reporter assay TP) detection of human telomerase reverse transcriptase (Human telomerase reverse transcriptase, hTERT) of different segments of promoter activity of.NOD/SCID (Non-obese diabetic/severe com Bined immunodeficient) mice by BCBL-1 cells induced by the tumor, the effect of TP on mice body weight, volume of ascites, ascites cells KSHV latency associated nuclear antigen 1 (Latency-associatednuclear antigen 1, LANA1) expression and effect on spleen. Results: TP can significantly reduce the KSHV positive PEL cell viability of KSHV negative normal peripheral blood mononuclear Cells (Peripheral blood mononuclear cells, PBMCs) and KSHV negative EBV positive P3HR-1 cell proliferation inhibition effect is not obvious; TP can induce apoptosis of PEL cells, resulting in BC-3 and JSC-1 cells were blocked in G0/G1 phase, BCBL-1 cells were arrested in G2/M phase; TP can significantly reduce the expression level of LANA1 in PEL cells, but have little effect on the the level of mRNA LANA1; TP induced down-regulation level through the proteasomal degradation pathway, and may influence its half-life; TP can reduce KSHV induced during lytic after BCBL-1 cells, outer virus particle production; by reducing TP hTERT PEL cells in different sections of promoter activity, reduce the mRNA level, which leads to a decrease in hTERT protein levels TP; Reduce the transcription factor Spl in PEL cells (specificity protein 1) expression in PEL cells; knockdown transcription factor Spl (specificity protein 1) expression can reduce the activity and hTERT of PEL cells, induce apoptosis in PEL cells. Knockdown transcription factor Sp1 can inhibit cell proliferation and increase of apoptosis induced by TP PEL cell The sensitivity; infiltration growth of TP can significantly inhibit NOD/SCID of mouse ascites and BCBL-1 cells of the spleen. Conclusion: in vivo and in vitro, TP could reduce the expression level of LANA1 in PEL cells, inhibit KSHV positive PEL cell proliferation, induce cell apoptosis; TP can inhibit the expression of PEL Sp1 cells, transcription inhibition hTERT, inhibit PEL cell proliferation and immortalization. Through the study of TP in KSHV positive PEL cells in antiviral and antitumor effects, lays a foundation for theoretical research for the treatment of malignant KSHV tumors and its derivatives used for subsequent TP.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.1

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