- 學位論文
探討細胞激素TIMP-1於EB病毒感染B細胞中 之調控機制及生理功能
Regulatory Mechanism and Functional Study of TIMP-1 in EBV-infected B Cells
摘要
EB病毒 (Epstein-Barr virus)是一廣泛流行的人類皰疹病毒，主要感染淋巴細胞與上皮細胞。臨床上，多種與EB病毒有高度相關性的疾病都具有淋巴球浸潤以及病變組織細胞含有大量細胞激素的特徵。已知，EB病毒感染細胞後可利用多種策略調控細胞激素表現量，以利於病毒基因體複製或宿主細胞存活。在本實驗室先前的cDNA微陣列分析以及細胞激素蛋白質微陣列分析中，發現受EB病毒感染之B細胞其基質金屬蛋白酶組織抑制因子 (tissue inhibitor of metalloproteinase-1，TIMP-1)表現量有明顯增加的現象。TIMP-1在許多預後較差的癌症病患檢體中可以偵測到高量表現，被認為可當作部分癌症的預後指標。因此，本研究進一步探討EB病毒感染細胞後誘發TIMP-1表現之調控機制和生物功能。 首先，以RT-Q-PCR和ELISA實驗證實，EB病毒在感染B細胞初期即可促進細胞內TIMP-1 mRNA轉錄及蛋白質分泌至細胞外，而且TIMP-1的高表現量可以持續至B細胞被EB病毒轉形成具有不斷增生能力的淋巴母芽細胞 (lymphoblastoid cell line，LCL)。而在細胞內轉染EB病毒基因後，可觀察到EB病毒溶裂期轉錄活化因子Zta在人類上皮細胞HEK293T、RHEK和多種柏金氏淋巴瘤 (Burkitt’s lymphoma)細胞株內都具有誘發TIMP-1基因表現的能力，在部分柏金氏淋巴瘤細胞株中則發現Zta和潛伏膜蛋白質LMP1可協同調控TIMP-1 mRNA表現。此外，在LCL細胞內以shRNA方式分別降解Zta和LMP1 mRNA後，TIMP-1的mRNA轉錄量皆有明顯下降情形，顯示EB病毒可藉由Zta和LMP1促進TIMP-1基因轉錄。分子機制上，經螢光酵素報導基因分析、電泳位移分析和染色質免疫沉澱法，證明Zta是藉由直接結合至TIMP-1啟動子+45 nt位置之AP-1 DNA結合位以促進TIMP-1 mRNA的轉錄活性；利用LMP1刪除圖變型質體則可知，LMP1會透過其C端活化區域CTAR1及CTAR2 domain調控TIMP-1基因表現。進一步探討TIMP-1之生物功能，利用西方墨點法分析可知，LCL內的TIMP-1可抵抗cisplatin和冷刺激誘發之細胞凋亡現象。另一方面，利用人類水溶性受體微陣列分析發現，LCL細胞內的TIMP-1經shRNA降解後會影響細胞培養液中多種水溶性受體的含量，推測TIMP-1是藉由直接影響水溶性受體表現或者藉由其基質金屬酶抑制活性間接調控水溶性受體的含量。 本研究證實，EB病毒在感染細胞後會經由Zta和LMP1協同促進TIMP-1基因轉錄，其中，Zta是藉由直接結合至TIMP-1啟動子以活化其基因表現。而EB病毒感染細胞後誘發TIMP-1生成可幫助細胞抵抗惡劣環境所誘發之細胞凋亡現象以及影響宿主細胞的水溶性受體表現。
並列摘要
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus mainly infected lymphoid and epithelial cells, and associated with diseases which are characterized by intensive lymphocytes infiltration and cytokines production. EBV infection can manipulate host cytokines production to promote virus replication and cell survival. Using cDNA microarray and cytokine antibody arrays, we found that tissue inhibitor of metalloproteinase-1 (TIMP-1) is induced in B cells after EBV infection. TIMP-1 is reported as a potential prognosis marker in numerous cancers, but its regulatory mechanism and biological function in EBV-infected cells are remaining unclear. We reveal here that TIMP-1 transcription and translation are induced immediately after EBV infection and maintain high expression level in EBV-immortalized lymphoblastoid cell line (LCL). Furthermore, EBV lytic protein, Zta, which is a key transactivator, is able to induce TIMP-1 expression in epithelial cells and B cells. Besides, EBV LMP1 can synergistic with Zta to regulate TIMP-1 production in some Burkitt’s lymphoma cell lines. Knockdown of Zta and LMP1 with shRNA both decrease TIMP-1 mRNA level in LCLs. The mechanistically underling Zta-mediated induction of TIMP-1 is through direct binding of Zta to the TIMP-1 promoter, via a consensus AP-1 binding site. Deletion of LMP1 activation domain, CTAR1 and CTAR2, abolishes LMP1-mediated induction of TIMP-1, suggesting that both CTAR domains are important for TIMP-1 induction. TIMP-1 is resistance to cisplatin and cold shock induced apoptosis in LCLs. Otherwise, the expression profiles of soluble receptors analyzed by Human soluble receptor array indicates that TIMP-1 can influence soluble receptors expressed and released by LCLs. Our data demonstrate that Zta- and LMP1-induced TIMP-1 production exhibits abilities to resist apoptosis and manipulate soluble receptors expression in EBV-infected B cells, and may contribute to the pathogenesis of EBV-associated lymphoproliferative disease.