2010-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658042摘要：在慢性B 型肝炎感染的病程中,病患長期處於B 型肝炎e 抗原陽性的狀態為導致肝細胞癌的重要危險因子之一,相對的,病患體內進行B 型肝炎e 抗原抗體血清轉換則被視為肝炎病毒活性明顯下降,以及病毒複製量顯著減少的重要臨床指標,同時,這也是目前臨床醫師治療慢性B 型肝炎所要達成的重要目標之一。然而,慢性B 型肝炎感染的病患發生e 抗原抗體血清轉換的年齡差異性卻是非常的分岐,究竟是由如何的機轉促使慢性B 型肝炎的病患於感染的過程中發生e 抗原抗體血清轉換至今仍然不甚明瞭。資料顯示, 大部份垂直傳染的慢性B 型肝炎病毒帶原者於青春期後發生e 抗原抗體血清轉換, 先前於男性的研究亦顯示青春期與e 抗原抗體血清轉換的相關。因此,本研究的目的在於釐清青春期與e 抗原抗體血清轉換在女性的相關, 以及於青春期會上昇之腎上腺賀爾蒙在男女與自發性e 抗原抗體血清轉換病程與病毒量的關聯性。免疫耐受期血清介白質十濃度較高之慢性B 肝帶原者於我們之前的研究顯示會有較早的e 抗原抗體血清轉換發生, 本研究將會分析不同介白質十基因型的慢性B 肝帶原者於免疫耐受期, 發炎期與不活動B 型肝炎期前驅及核心基因的突變差異, 同時, 以體外細胞培養模式探討介白質十對B 型肝炎特異性殺手T 淋巴球的影響。研究設計本研究預期收案一百個慢性B 型肝炎的B 型肝炎帶原男性病患, 與一百個慢性B型肝炎的B 型肝炎帶原女性病患, 他們初診時皆為e 抗原陽性, 初診年齡均小於十歲,每位病患都以六個月的週期規則追蹤十年以上,每次門診追蹤時都由醫師進行身體理學檢查並抽血檢驗, 有關肝臟轉胺酶與B 型肝炎病毒血清學的狀態於每次返診時均被檢驗,一旦病患被發現有異常的肝臟轉胺酶時, 他們的追蹤週期會被縮短成一到三個月,病患B 型肝炎病毒的基因型亦會在本研究中被檢驗,而這些B 型肝炎帶原者均在本院追蹤的過程中於發生e 抗原抗體血清轉換前均未接受抗病毒藥物治療。研究方法我們以初經的年齡反應女性患者青春期的早晚, 與女性患者10 歲, 20 歲, 以及初經時的病毒量與肝功能, 自發性e 抗原抗體血清轉換的年紀作分析, 病患體內B 型肝炎病毒的基因型因素都會被納入統計的考量, 於200 位病患於10 歲以及15 歲以ELISA 分析其腎上腺賀爾蒙DEHAS 並以多變數分析, 生存統計與多變數分析的統計方法處理上述的變因分析。我們將以cloning 的技術分析20 位已經e 抗原抗體血清轉換慢性B 肝帶原病患於免疫耐受期, 發炎期與不活動期於B 型肝炎期前驅及核心基因的突變差異,並分析不同介白質十基因型個體病毒突變的差異, 來探討宿主免疫與病毒變異間的互動。我們也將會以體外週邊白血球於B 型肝炎e 抗原刺激的培養系統來探討外加於培養液的介白質十與二對B 型肝炎特異性殺手T 淋巴球的數量與毒殺性的影響, 研究中以tetramer 方式來辨識B 型肝炎特異性殺手T 淋巴球的數量, 並以細胞內染色來辨識perforin 與interferon-r 於B 型肝炎特異性殺手T 淋巴球的表現來探討其對病毒的毒殺功能。預期成果慢性B 型肝炎患者於三十歲後才發生e 抗原抗體血清轉換與肝細胞癌的發生有顯著的關聯性已經於文獻中被提及,我們嘗試由本研究尋找可以預測自發性B 型肝炎e 抗原抗體血清轉換的宿主因素,藉此研究的結果,希望可以幫助我們對於B 型肝炎病毒感染後成為帶原者與否以及e 抗原抗體血清轉換的過程中各種宿主反應所扮演的角色有進一步的分析,以了解B 型肝炎病毒感染後宿主與病原菌之間的互動。<br> Abstract: Persistent hepatitis B e antigen (HBeAg) after the 3rd decade of life in chronic HBVinfected patients is regarded as an important risk factor hepatocellular carcinoma. HBeAgseroconversion is an important indicator of subsiding in disease activity and decreasing ofactive viral replication. However, the onset age of HBeAg seroconversion varies greatlybetween individuals with chronic HBV infection. The detail mechanism of this phenomenonleft largely unknown till now.The majority of vertical transmitted chronic HBsAg carriers developed spontaneousHBeAg seroconversion after puberty. Our preliminary work demonstrated the associationbetween puberty onset and onset age of spontaneous HBeAg seroconversion age in males.Hence, our study aim to clarify the possible association in chronic HBV infected femalesbetween puberty onset and HBeAg seroconversion age. Effects of DHEAS, an importantadrenal hormone, which will increase 2-3 years prior the puberty onset will also beinvestigated in this study.Increased serum interleuin-10 at immune tolerance phase was noted to associate withearlier onset of HBeAg seroconversion, we will also evaluate the relationship between hostinterleukin-10 genotype and HBV mutation rate in the HBV precore/core gene sequence. Theeffect of interleukin-10 on the amount and cytotoxicity of HBV specific cytotoxic Tlymphocytes (CTL) will also be investigated.Study DesignsSubjectsIn this study, 100 chronic HBV infected males, and another 100 chronic HBV infectedfemales with HBeAg-positivity at the first visit before 10 years of age and have beenlongitudinally followed for more than 10 years in our institute, will be included in this study.They had regular follow up for physical examination, and blood sampling is arranged at6-month interval.MethodsWe will correlate the menarche age in these 100 chronic HBV infected females with theirserial HBV viral load at 10, and 20 years of age, age of spontaneous e seroconversion andserum ALT levels in the analysis. Serial serum DHEAS levels at 10 and 15 years of age willbe determined in these 200 chronic HBV infected males and females to elucidate the possibleroles of DHEAS on HBV.To evaluate the effect of interleukin-10 on HBV precore/core gene mutation rates, we willinclude 20 chronic HBV infected subjects with spontaneous e seroconversion in our cohortwith known interleukin-10 promoter genotype (predicts different serum interleukin-10 levels).We will evaluate the difference in HBV mutation rate by serial HBV precore/core genecloning at the tolerance, inflammatory and inactive phase of these of these 20 patients withdifferent interleukin-10 genotype.The effect of interleukin-10 on the HBV specific CTL will be assessed by peripheral bloodmononuclear cell in vitro culture system. Tetramer will be applied to identify HBV specificCTL, and intracellular perforin and interferon-r staining for the cytotoxicity of HBV specificCTL.Anticipating OutcomeWe will assess possible host factors predicting the HBeAg seroconversion in this study.This may help us to understand the possible roles of host factors on the process ofspontaneous HBeAg seroconversion and the interaction between host immune system andvirus adaptation.B型肝炎病毒自發性B型肝炎e抗原抗體血清轉換介白質十Hepatitis B virusHepatitis B e antigenInterleukin-10Precore-core gene