臺灣博碩士論文加值系統

中文摘要
C型肝炎病毒已被證實為大部分輸血性非A非B型肝炎的病原，其可持續感染宿主相當長的時間，並經由未知的致病機轉發展為慢性肝炎、肝硬化及肝癌。目前研究證實C型肝炎病毒核心蛋白是一功能多樣性的蛋白，除構成病毒顆粒的蛋白質外鞘外，對於許多細胞內或其它病毒的啟動子具有調控作用，並與許多細胞內的蛋白有交互作用，因此推測C型肝炎病毒的致病機轉可能經由核心蛋白與細胞因子交互作用而達成，但其分子機制尚待釐清。
人類巨細胞病毒為一隨處可見的病毒，在人口中的盛行率相當高，感染後通常造成終身帶原，其感染或復發只在胎兒或免疫功能不全的病人造成嚴重甚至致命的疾病。人類巨細胞病毒在宿主細胞中能否表現病毒基因及複製遺傳物質，與迅早期基因能否被活化有重要的關係，迅早期基因的活化是由人類巨細胞病毒MIE啟動子所調控，因此對於MIE啟動子表現活性及其調控機制的研究，有助於人類巨細胞病毒的潛伏感染及再活化之瞭解。
分析C型肝炎核心蛋白對人類巨細胞病毒MIE啟動子之影響有二層意義，其一是利用MIE啟動子上眾多轉錄因子的結合位置，研究核心蛋白調控細胞內或其它病毒啟動子之可能分子機轉，藉以了解核心蛋白與細胞因子交互作用的情形；其二是流行病學的調查顯示此二種病毒在器官移植後，一再復發是造成器官移植病人死亡的危險因子，若同時存在可能造成更嚴重的後果，而研究這二種病毒之病毒因子間交互作用的分子機制，將有助於預防及治療的參考。
在本論文中利用報導基因分析C型肝炎病毒核心蛋白在HuH-7細胞中對人類巨細胞病毒MIE啟動子(-760~+3)之影響，發現表現核心蛋白之質體比率較高時，對報導基因有抑制作用，表現核心蛋白之質體比率較低時對報導基因則有明顯活化作用(1.6~4.3倍)，因此核心蛋白對於MIE啟動子具有顯著的雙向調控作用。此外，於HuH-7細胞中核心蛋白對四個刪除突變MIE啟動子(分別包含長度片段-736~+2、-541~+2、-246~+2及-54~+2)仍保有活化的作用；而於HeLa細胞中除對pMIEP-54/2無影響外，對其餘刪除突變之MIE啟動子有些微的活化作用。此實驗並無法定出核心蛋白活化MIE啟動子必要之區域，但在此二種細胞中，核心蛋白對於四個刪除突變之MIE啟動子活化作用之形式並不相同。若以具有持續表現核心蛋白之HuH-7/C190及HeLa/C190細胞進行實驗，則發現核心蛋白對於刪除突變之MIE啟動子，除HeLa/C190細胞中對pMIEP-54/2無影響外，都具有明顯抑制作用。在此同樣也可以發現這二種細胞中，核心蛋白對於四個刪除突變之MIE啟動子抑制作用形式不同，因此可推測其在不同細胞株中，核心蛋白對於MIE啟動子之調控具有細胞專一性(cell-type specificity)。而值得一提的是，在短暫細胞轉染及持續表現細胞株轉染實驗所得到的結果並不一致。
以EMSA實驗結果證實核心蛋白對轉錄因子YY1、Sp1及CREB之DNA辨識序列結合能力會有影響，在HuH-7/C190細胞中這三個因子的DNA-蛋白質複合物有明顯增加的情形，在HeLa/C190細胞中則為減弱(YY1及CREB)或無影響(Sp1)，且許多證據顯示這些DNA-蛋白質複合物可能是許多轉錄因子藉由蛋白質間互相作用而形成的巨大複合物。將EMSA實驗結果與西方點墨法互相比較，在大部分的情形下對DNA-蛋白質複合物增加或減弱並不是藉由影響轉錄因子YY1、Sp1及CREB在細胞中表現的含量所造成，但對CREB在HuH-7/C190細胞中而言則不能排除此可能性。
綜合上述結果顯示，C型肝炎病毒核心蛋白具有調控人類巨細胞病毒MIE啟動子之活性，且此種活性具有細胞專一性；此外，C型肝炎病毒核心蛋白影響某些轉錄因子在細胞中的含量，並可能影響細胞內轉錄因子複合物的形成，這些作用同樣具有細胞專一性。因此核心蛋白在C型肝炎病毒致病機轉之分子機制中，扮演相當重要的角色。

Abstract
HCV (Hepatitis C virus) has been proven as being the major cause of trans-fusion-associated non-A, non-B hepatitis, and the prolonged, persistent infec-tion of HCV often progress to chronic hepatitis, cirrhosis, and hepatocellular carcinoma by unknown molecular pathogenesis. Several studies have demon-strated that the HCV core protein has a pleiotropic function and may play a major role in the pathogenesis of HCV infection, but the molecular mechanism was not well defined. Human cytomegalovirus (HCMV) is ubiquitous with very high prevalence in population. Co-infection with HCV and HCMV is therefore possible. Since the activation of the MIE promoter-enhancer is ex-pected to play pivotal roles in HCMV infection, a detailed understanding of the regulation of its expression activity may help us to gain important insights about latency and reactivation of HCMV infection.
Analysis of the effect of HCV core protein on HCMV promoter activity has two significances. First, utilizing binding sites of various transcription factors on HCMV MIE promoter may help us to elucidate the possible molecular mechanism for the regulatory activity of HCV core protein. Second, recurrence of HCV or HCMV infection or both after allograft transplant is risk factor for transplanted patients, investigation of the interaction between these two viral factors may provide important insights for prophylaxis and treatment.
In our results, the full-length HCV core protein (c195) has biphasic effects on HCMV MIE promoter in HuH-7 cells in transient transfection experiment, while the deletion mutants of core protein have slight (c122) or no (c101) effect. With lower expression level, the full-length HCV core protein has trans-activa-tion ability on HCMV MIE promoter (containing promoter region -760~+3), while it has trans-suppression effect with the higher expression level. Additionally, the full-length HCV core protein also has trans-activation ability on four deletion mutants of HCMV MIE promoter (containing promoter region -736~+2, -541~+2, -246~+2, and -54~+2, respectively) in HuH-7 cells. Apart from the MIE promoter region spanning -54 to +2 (pMIEP-54/2), similar re-sults were found in HeLa cells, but differ from that of HuH-7 cells in activation pattern. Interestingly, an opposite, significant suppression of HCMV promoter activity was observed in HuH-7 and HeLa core protein-producing cell lines (HuH-7 /C190 and HeLa/C190, respectively) except pMIEP-54/2 in HeLa/C190 cells. Thus, the regulatory activity of the HCV core protein on HCMV MIE promoter possesses cell-type specificity.
Using electrophoretic mobility shift assay (EMSA) I found that the expres-sion of HCV core protein could enhance the DNA binding activity of YY1, Sp1, and CREB in HuH-7/C190 cells. In HeLa/C190 cells, the DNA binding activity of YY1 and CREB was reduced by core protein, but no effect were found in Sp1-binding activity. The results also suggest that these DNA-protein complex-es are large complexes composed of different transcription factors through protein-protein interaction. Furthermore, Western bolt analysis indicated that these effects on DNA binding activity did not result from the alteration of ex-pression levels of YY1, Sp1, and CREB by the HCV core protein under most conditions, except in the case of CREB in HuH-7/C190 cells.
Taken together, the results demonstrate that the HCV core protein possesses transcriptional regulatory ability on HCMV MIE promoter. Additionally, core protein may alter the expression level of some transcription factors and en-hance or suppress the transcriptional complex formation in a cell-type specific manner. Therefore, our findings imply that HCV infection may alter the HCMV life cycle. Moreover, these features of core protein may at least par-tially account for its pleiotropic effects on regulation of gene expression and the molecular mechanism of HCV pathogenesis.

中文摘要--------------------------------------------------------------------------------------------------------1
英文摘要--------------------------------------------------------------------------------------------------------3
壹、緒論--------------------------------------------------------------------------------------------------------5
一、C型肝炎病毒(Hepatits C Virus, HCV)-------------------------------------------------------5
1. 概述----------------------------------------------------------------------------------------------5
2. C型肝炎病毒核心蛋白----------------------------------------------------------------------6
3. C型肝炎病毒核心蛋白與宿主細胞間的交互作用-----------------------------------7
二、人類巨細胞病毒(Human Cytomegalovirus, HCMV)--------------------------------------8
1. 概述-----------------------------------------------------------------------------------------------8
2. 人類巨細胞病毒之感染複製及基因表現-----------------------------------------------9
3. 人類巨細胞病毒MIE啟動子---------------------------------------------------------------11
三、研究目的及策略---------------------------------------------------------------------------------11
貳、實驗材料與方法---------------------------------------------------------------------------------------13
一、實驗材料：---------------------------------------------------------------------------------------13
1. 菌株---------------------------------------------------------------------------------------------13
2. 細胞株------------------------------------------------------------------------------------------13
3. 培養基與培養液-----------------------------------------------------------------------------13
4. 質體---------------------------------------------------------------------------------------------14
5. 溶液---------------------------------------------------------------------------------------------15
6. 化學藥品---------------------------------------------------------------------------------------16
7. 酵素---------------------------------------------------------------------------------------------16
8. 引子(Primer)-----------------------------------------------------------------------------------16
9. 放射性同位素--------------------------------------------------------------------------------17
10. 抗體--------------------------------------------------------------------------------------------17
二、實驗方法：---------------------------------------------------------------------------------------17
1. 大腸桿菌質體之轉形(Transformation)與抽取----------------------------------------17
2. 氯化銫(CsCl)密度梯度離心之質體製備-----------------------------------------------17
3. 聚合鏈鎖反應(Polymerase chain reaction, PCR)-----------------------------------18
4. 細胞培養---------------------------------------------------------------------------------------18
5. 細胞轉染(Transfection)---------------------------------------------------------------------18
6. Luciferase酵素活性分析-------------------------------------------------------------------19
7. 32P標記DNA探針之製備------------------------------------------------------------------19
8. Electrophoretic mobility shift assay (EMSA)---------------------------------------------19
9. SDS-聚丙烯醯胺凝膠電泳(Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, SDS-PAGE)-----------------------------------------------------------------20
10. 西方點墨法(Western blot)-----------------------------------------------------------------20
參、實驗結果-------------------------------------------------------------------------------------------------22
一、C型肝炎病毒核心蛋白對人類巨細胞病毒MIE啟動子之影響-----------------------22
二、C型肝炎病毒核心蛋白對刪除突變之人類巨細胞病毒MIE啟動子之影響-------23
1. 選殖人類巨細胞病毒MIE啟動子之刪除突變報導質體----------------------------23
2. Transient細胞轉染實驗----------------------------------------------------------------------24
3. C型肝炎病毒核心蛋白持續表現細胞株轉染實驗-----------------------------------24
三、以EMSA實驗觀察核心蛋白對轉錄因子DNA辨識序列結合能力之影響---------25
1. YY1探針之EMSA實驗-----------------------------------------------------------------------25
2. Sp1探針之EMSA實驗------------------------------------------------------------------------26
3. CREB探針之EMSA實驗---------------------------------------------------------------------26
四、轉錄因子YY1、Sp1及CREB在核心蛋白持續表現細胞中之含量-------------------27
肆、討論---------------------------------------------------------------------------------------------------------29
一、C型肝炎病毒核心蛋白對人類巨細胞病毒MIE啟動子具有雙向調控作用--------29
二、核心蛋白對刪除突變之MIE啟動子在不同細胞株中活化作用之形式不同-------30
三、短暫細胞轉染及持續表現細胞株轉染實驗所得到的結果並不一致----------------30
四、核心蛋白影響轉錄因子複合物的形成------------------------------------------------------32
伍、參考文獻---------------------------------------------------------------------------------------------------34
圖表----------------------------------------------------------------------------------------------------------------43

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