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研究生:盧冠霖
論文名稱:EB病毒LMP-1蛋白對人類上皮細胞生長及對該細胞受EB病毒感染之影響
論文名稱(外文):Effects of EBV LMP-1 on Human Epithelial Cell Growth and on EBV Infection in the Cells
指導教授:楊照雄楊照雄引用關係
指導教授(外文):Ling, Der-Lin
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:186
中文關鍵詞:EB病毒上皮細胞LMP-1蛋白
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Malignant neoplasms have been the first leading cause of death in the Taiwan area since 1982. Among the ten leading cancer deaths, NPC holds the 7th rank cancer in 1994, with death rate of 3.78 per 100,000 population. Therefore, it is important to understand the pathogenesis of NPC so as to prevent and control the disease.
The goal of this study is to investigate the effects of EBV on huamn epithelial cell growth and to further understand the role of EBV on the pathogenesis of NPC. Firstly, we studied the effects of EBV LMP-1 protein on human epithelial cell growth. The EBV LMP-1 gene was transfected into a human epithelial cell line (RHEK-1) by electroporation. The results showed that the morphology of the transfected cells had been changed to a more large and spindle-like appearance than the parental RHEK-1 cells. A novel finding was that high level expression of LMP-1 gene in a human epithelial cell line (RHEK-1) induced apoptosis, which was characterized by chromosomal DNA fragmentation of the transfected cells. In particular, such an effect was more apparent under serum starvation. We also found that in the transfected RHEK-1 cells, LMP-1 expression neither affected bcl-2 expression nor led the cells to grow in semi-solid agar. These results indicate, therefore, that LMP-1 may participate in the development of EBV-associated epithelial malignancy in another mechanism different from that seen in B cell or fibroblast transformation.
Since we have demonstrated that the over-expression of LIVIP-1 in epithelial cells (RHEK-1 cells) induced cell apoptosis and Bcl-2 expression in LMP-1 transfected RHEK-1 cells was not detected, we transfected bcl-2 into LMF-1-containing RHEK-1 cells by electroporation and found that ectopic expression of bcl-2 specifically blocked apoptotic death induced by constitutive LMP-1 over- expression, resulting in a significant decrease of DNA fragmentation. In addition, co-expression of LMP-1 and Bcl-2 in RHEK-1 cells enabled the cells to grow unlimitedly, form foci, grow in lower serum requirement and to form colonies in semi-soft agar medium. The expression of Bcl-2 in LMP-1-positive cells also showed that Bcl-2 did not affect the cell proliferation and cell cycle progression, but just could co-operate with LMP-1 to transform human pithelial cells. In an immunohistochemistry study, co-expression of Bcl-2 and LMP-1 was also identified in some NPC tissues, that was consistent with those in vitro findings. The c-myc protein was up-regulated by LMP-1 but did not increase by Bcl-2 protein, lindicating that LMP-induced apoptosis might be associated with the action of c-myc. These results indicate that the cooperative interaction between these two proteins may play an important role in the processes of EBV-associated epithelial cell transformation.
In order to further understand EBV infection in human epithelial cells, we have cloned the EBV receptor CR2 cDNA from Burkitt''s lymphoma Raji cells. The CR2 receptor gene was transfected into RHEK-1 cells by electroporation. The CR2-expressing human epithelial cells (CR2-RHEK-1) could be infected by EBV in vitro, but the EBV was in a lytic state in this EBV-infected epithelial cells.
The LMF-1 protein was not detected and the EBV DNA disappeared gradually after 10-day infection. We further established a LMP-1-expressing CR2-RHEK-1 cells to study the effects of LMP-1 on EBV infection in human epithelial cells. The LMP-1 gene was transfected into CR-2-RHEK-1 cells by electroporation, and the LMF-1 and CR-2 double positive cells [CR-2-LMP(1)-KBGEK-1] were cloned. The EBV was infected into [CR-2-UMDP(1)-RHEK-1] cells, and the expression of EBV latent and lytic protein were assayed daily post-infectioon. In the presence of EBV LMP-1 protein, the EBV latent protein EBNA could be detected 10th day post-infection. At the 11th day post-infection, the expression of EBNA reached to the maximal level, and then decreased quickly. The lytic protein Zta could be detected in a hightest level at 8-9th day post-infection, and then decreased slowly. But the EA was detected only 3-5% after 4th day post- infection, the VCA and MA were detected only 0-2% during the course of EBV infection. The expression of cellular oncogenes and tumor suppressor genes in the EBV-infected cells were also analyzed by immunoblotting. The data showed that the expression of c-myc protein increased at 14th to 15th day post-infection. The data indicated that the EBV LMP-1 protein could enhance the persistence of EBV in EBV-infected human epithelial cells and increased the level of EBNA expression. It also suggested that LMP-1 could affect the EBNA expression and, further more, to enhance the expression of c-myc protein, leading to the transformation of epithelial cells by EBV.
Chinese Abstract
English Abstract
Acknowlegement
Contents
Abbreviation
List of Figures and Tables
I Introduction
1.1. Introduction to Epstein-Barr virus (EBV)
I.2. Primary EBV infection in B lymphocytes
1.3. EBV proteins expressed in latently infected B-lymphocytes
I.3.1. EBV nuclear antigen I (EBNAI)
1.3.2. EBV nuclear antigen 2 (EBNA2)
1.3.3. EBV nuclear antigens 3A, 3B and 3C (EBNAs 3A, 3B and 3C)
1.3.4. EBV nuclear antigen leader protein (EBNA-LP)
1.3.5. EBV latent membrane protein I (LMP-I)
1.3.6. EBV latent membrane protein 2A and 2B (LMP-2A, 2B)
1.4. EBV RNAs expressed in latently infected cells
1.4.1. EBV-encoded small nonpolyadenylated RNAs (EBERs)
1.4.2. BHRFI transcripts
1.4.3. BARFO transcripts
1.5. EBV persistence
1.6. Lytic cycle of EBV infection
1.6.1. Immediate early genes: BZLFI and BRLFI
I.6.2. Early proteins: BALF2 and BHRFI
1.6.3, EBV DNA replication associated early proteins:BALF5 (DNA polymerase), BALF2 (major DNA binding protein)BORF2 and BARFI (ribonucleotide reductase), BXLFI (thymidinekinase) and BGLF5 (alkaline exonuclease)
1.6.4. Late proteins in EBV lytic cycle
1.7. The types of EBV
1.8. EBV and associated human diseases
1.8.1. Association of nasopharyngeal carcinoma (NPC) with EBV
1.9. Introduction to apoptosis
II. Effects of EBV LMP-I and Bcl-2 on Human Epithelial Cell Growth
II.I. Introduction to part II
11.2. Materials and Methods
II.2.1. Cells
II.2.1.1. RHEK-I ceUs
11.2.1.2. LMP-l-expressing RHEK-I cells
II.2.1.3. Bcl-2/LMP-l-co-expressing RHEK-I ceUs
II.2.2. Immunoblotting
II.2.3. Indirect immunofluorescence assay
II.2.4. In vitro growth assays and determination of cell viability
II.2.5. Fluorescent intensity, fragmented DNA and cell cycle analysisby flow cytometry
II.2.6. DNA fragmentation analysis by agarose gel electrophoresis
II.2.7. Anchorage-independent growth assay
II.2.8. Nuclear morphology observation of apoptotic cells stained withacridine orange
II.2.9. Immunohistochemistry
II.2.10. Tumorigenicity test
II.3. RESULTS
11.3.1. Expression of transfected LMP-I in human epithelial RHEK-I cells
II.3.2. Increased ceil death caused by LMP-I expression
II.3.3. Effects of expression of LMP-I on susceptibility to apoptosisand on cell cycle progression
II.3.4. Bcl-2 expression in LMP-I transfected epithelial cells
II.3.5. Test for LMP-I transfected epithelial cells to form coloniesin semi-solid agar
II.3.6. Over-expression of Bcl-2 and LMP-I in RHEK-I cells
II.3.7. Inhibition of LMP-l-induced apoptotic cell death by ectopic Bcl-2co-expression in epithelial cells
II.3.8. Effects of interaction between Bcl-2 and LMP-I on the distributionand progression of cell cycle
II.3.9. Transformed morphology and anchorage-independent growth ofBcl-2 and LMP-I co-expressed human epithelial cells
II.3.10. LMP-I and Bcl-2 expression in NPC tissues
II.3.11. Expression of oncogenes and tumor suppressor genes in Bcl-2and LMP-I co-expressed human epithelial cells
II4. CONCLUSIONS and DISCUSSIONS
11.4.1. Induction of Human Epithelial Cells Apoptosis by EBV LMP-I Protein
II.4.2. Cooperation between LMP-I and Bcl-2 in the Transformationof Human Epithelial Cells
III. Effects of EBV LMP-I protein on the EBV infection in humanepithelial cells
III.1. Introduction to part HI
III.2. Materials and Methods
III.2.1. Preparation ofB95-8 virus
III.2.2. CR2-expressing RHEK-I ceUs
III.2.3. CR2/LMP-l-co-expressing RHEK-I ceUs
III.2.4. Virus infection
III.2.5. Immunofluorescence staining and flow cytometric analysis
III.2.5.1. Immunofluorescence on fixed-cell smear
III.2.5.2. Double staining for LMP-I and CR2 in CR2-LMP(1)-RHEK-1 cells
III.2.5.3. Cytometric analysis for LMP-I and CR2 in CR2-LMP(1)-RHEK-1 ceUs
III.2.5.4. Immunoblotting
III.3. Results
III.3.1. Establishment of CR2-expressing RHEK-I (CR2-RHEK-1),mock-transfected CR2-RHEK-1 (Hyg/CR2-RHEK-l) and LMP-I-expressmg CR2-RHEK-1 ceU lines [CR2-LMP(1)-RHEK-1]
III.3.2. Expression of EBV latent proteins in the EBV-infectedCR2-LMP(1)-RHEK-1 epithelial ceUs
III.3.3. Expression of EBV lytic proteins in the EBV-infectedCR2-LMP(1)-RHEK-1 epithelial ceUs
III.3.4. Expression of EBV latent proteins in the EBV-infectedHyg/CR2-RHEK-l epithelial ceUs
III.3.5. Expression of EBV lytic proteins in the EBV-infectedHyg/CR2-RHEK-l epithelial ceUs
III.3.6. Expression of EBV latent proteins in the EBV-infected CR2-RHEK-1 epithelial ceUs
III.3.7. The expression ofEBV lytic proteins in the EBV-infectedCR2-LMP(1)-RHEK-1 epithelial cells
III.3.8. Expression of cellular oncogenes and tumor suppressorgenes in EBV-infected human epithelial cells
III.4. DISCUSSIONS
IV. Figures and tables
VI. References
VIL Appendix
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