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研究生:張美蘭
研究生(外文):Mei-Lan Chang
論文名稱:應用ELISPOT方法偵測B型肝炎疫苗接種者的B型肝炎表面抗原特異的記憶性B細胞
論文名稱(外文):Development of ELISPOT assay to detect the HBsAg-specific memory B cells in HB vaccinated population
指導教授:劉清泉劉清泉引用關係黎煥耀黎煥耀引用關係
指導教授(外文):Ching-Chuan LiuHuan-Yao Lei
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物及免疫學研究所
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:2005
畢業學年度:93
語文別:中文
論文頁數:70
中文關鍵詞:記憶性B細胞酵素結合免疫斑點分析法B型肝炎
外文關鍵詞:HBVmemory B cellELISPOT assay
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  • 收藏至我的研究室書目清單書目收藏:1
  在1984年之前台灣是B型肝炎病毒 (HBV) 感染的高危險區域,帶原率大約為15%~20%;而且大多的感染都是來自母體的垂直傳染。有鑑於此,政府決策在1984年7月開始實施全面接種B型肝炎疫苗。接種疫苗10到15年後,B型肝炎帶原者的比例從15%~20% 降到1%~2%,疫苗成效顯著。但是接種疫苗15年之後,有一半的接種者體內測不到抗B型肝炎表面抗原的抗體 (anti-HBs) 存在,抗體的量會隨著時間而遞減的。所以是否要為那些接種過B型肝炎疫苗但目前抗體低下的人做進一步的B型肝炎疫苗追加注射是一個重要的問題。過去的研究顯示記憶性B細胞可以快速補充體內抗體再生,所以我們應用酵素結合免疫斑點分析法 (ELISPOT) 評估這些疫苗接種者體內的B型肝炎表面抗原 (HBsAg) 特異的記憶性B細胞。首先我們將周邊血液單核球細胞 (PBMC) 培養六天,發現PBMC可以被裂殖原活化分泌免疫球蛋白,而且細胞也表現了較高量的記憶性B細胞活化指標,CD27。我們用競爭性實驗確認了在ELISPOT中所產生的免疫斑點是B型肝炎表面抗原特異性的斑點。應用ELISPOT技術我們測定低抗體表現者 (<100 mIU/ml) 的HBsAg-特異的記憶性B細胞,並計算其頻率,在追加注射之前是1.49 ± 0.43% (0.9% ~ 2.3%, n=30),但是在經過一劑B肝疫苗的追加注射之後,頻率上升兩倍以上,變成3.27 ± 0.84% (2.2% ~ 4.6%, n=30)。我們如果再和另外一群原本就是高抗體者 (>1000 mIU/ml) 做比較,發現他們表現的HBsAg-特異的記憶B細胞頻率是1.72 ± 0.92% (0.9% ~ 2.8%, n=15),和低抗體的人在追加注射之前的頻率沒有統計上的差異 (P=0.582)。而且anti-HBs的抗體力價和HBsAg-特異記憶性B細胞頻率有呈線性關係 (P<0.05)。除此之外,在追加注射者,有66%的人再追加一劑之後,anti-HBs力價會上升大於1000 mIU/ml。很顯然之前注射過B型肝炎疫苗而目前是低抗體的人,體內具有B型肝炎表面抗原專一性的記憶性B細胞存在,當再次遇到HBsAg刺激時,可以立即產生保護性的高效價抗體。此外我們也發現那些感染過HBV的人,他們體內的HBsAg-特異的記憶性B細胞頻率是6.13 ± 0.19% (5.36% ~ 6.9%, n=5),比追加注射疫苗 (anti-HBs > 1000 mIU/ml) 的人要來的高。而且B型肝炎帶原者也有B型肝炎表面抗原專一性的記憶性的B細胞存在 (2.47 ± 0.5%, n=5),這顯示帶原者的體內產生的HBsAg也會刺激特異的記憶B細胞,只是他們的體內可能沒有發生作用。所以被B型肝炎病毒感染後,不論是痊癒者或仍是帶原者,體內的B細胞受HBsAg刺激都有HBsAg-特異記憶B細胞存在。這些HBsAg-特異記憶B細胞在15年後仍存在,在測不到anti-HBs者並未比有高力價者低,它們在受到HBsAg再刺激時,全立即被活化。這顯示即使在15年後,沒有anti-HBs的人可能還不需要追加一劑B型肝炎疫苗。
 A universal vaccination program to hepatitis B virus (HBV) infection has been launched in Taiwan since July 1, 1984 that resulted in a significant decline of HBV carrier rate from 15-20% to 1-2%. However, the anti-HBs titer in the vaccinated population will decay along the time. It was found that 50% of HB vaccinees do not have detectable anti-HBs (<10 mIU/ml) after 15 years of vaccination. Therefore, it is important to clarify if the HB vaccine still had a protective function for the population with low anti-HBs titers and whether we have to boost them after 15 years. It’s well known that memory B cells are responsible for the long-term humoral immunity elicited by most vaccines. Recently, an antigen-specific enzyme-linked immunospot (ELISPOT) has been developed to detect the memory B cells that can differentiate into antibody secreting cells after in vitro culture. After B cell mitogen stimulation for 6 days, the cell numbers of the PBMCs were activated to express B cell activation marker, CD27, and can secret immunoglobulins. The HBsAg-ELISPOT specificity was verified by addition of excess HBsAg to block the binding in the assay. The frequency of HBsAg-specific memory B cells per total IgG+ memory B cells in persons with anti-HBs titer below 100 mIU/ml was increased from 1.49 ± 0.43% (0.9% ~ 2.3%, n=30) to 3.27 ± 0.84% (2.2% ~ 4.6%, n=30) (P<0.001), after one dose of HB booster vaccination. There is positive correlation between anti-HBs titer and HBsAg-specific memory B cell frequency. Furthermore, the frequency of HBsAg-specific memory B cells per total IgG+ memory B cells was 1.72 ± 0.92% (0.9% ~ 2.8%, n=15) in the population of anti-HBs titer above 1000 mIU/ml, which was not different from those of low anti-HBs titer (<100 mIU/ml). In the boosting population of low anti-HBs titer, 66% of boosters increased their anti-HBs titer above 1000 mIU/ml after one dose of booster vaccination. We also found that naturally HBV infected individuals (anti-HBc +) had HBsAg-specific memory B cells in their blood circulation. The frequency of HBsAg-specific memory B cells was (6.13 ± 0.19%, n=5) higher than in HBsAg(+) carriers (2.47 ± 0.5%, n=5). In conclusion, our study shows that it is not necessary to boost the low anti-HBs titer population, because their memory B cell frequency was similar to those with high anti-HBs titer. Therefore, the frequency of memory B cells will also be a judgment more than anti-HBs titers to evaluate whether who needs to have the HB vaccine boost or not. Either HB vaccine or HBV infection would stimulate HBsAg-specific memory B cells. These memory B cells still exist after 15 years. The frequency of HBsAg-specific memory B cells in low anti-HBs titer population was not different from that of high anti-HBs titer ones. They can be activated to produce anti-HBs after additional boost. This implies that additional HB vaccine boost might not be necessary even after 15 years of HB vaccination.
誌謝 Ⅰ
中文摘要 Ⅱ
英文摘要 Ⅳ
目錄 Ⅴ
圖目錄 Ⅷ
表目錄 Ⅸ
壹、緒論
1. B型肝炎病毒 1
1.1 B型肝炎病毒概論 1
1.2 B型肝炎病毒複製與傳染 2
1.3 B型肝炎病毒感染的病程發展 2
1.4 B型肝炎病毒與肝癌的相關性 4
1.5 B型肝炎病毒感染的流行病學分佈 5
1.6 實行B型肝炎疫苗施打 5
1.7 B型肝炎疫苗施打成效 6
2. B細胞免疫系統 7
3. 酵素結合免疫斑點分析法 (ELISPOT assay) 9
4. 研究動機 10
貳、材料與方法 12
1. 材料 12
1.1 檢體收集 13
1.2 細胞培養 14
1.3 流式細胞儀 14
1.4 蛋白質電泳 (SDS-PAGE) 16
1.5 銀染 (silver stain) 16
1.6 酵素結合免疫吸附分析法套組 (ELISA kit) 17
1.7 酵素結合免疫斑點分析法 (ELISPOT assay) 18
2. 方法 18
2.1 細胞繼代培養、冷凍、解凍 18
2.2 純化B型肝炎表面抗原 19
2.3 蛋白質電泳 (SDS-PAGE) 20
2.4 銀染 (silver stain) 20
2.5 利用酵素結合免疫吸附法偵測HBsAg濃度 21
2.6 人類週邊血液單核細胞 (PBMC) 的分離與培養 21
2.7 冷凍PBMC 22
2.8 利用流式細胞儀分析免疫細胞表面標定的表現 22
2.9 利用ELISA的分法偵測PBMC所分泌出的免疫球蛋白 (Immunoglobulin) 23
2.10 酵素結合免疫斑點分析法 (ELISPOT assay) 24
2.11 酵素結合免疫斑點分析儀器 (ELISPOT analyzer) 25
2.12 計算專一性的記憶性B細胞佔所有會產生IgG的B細胞百分比 26
叁、結果
一、 記憶性B細胞分化成漿細胞 28
A. 建立酵素結合免疫斑點法 28
B. B cell polyclonal activators 29
C. 刺激記憶性B細胞分化成漿細胞 30
二、 計算HBsAg-specific memory B cell frequency 32
A. Specific spots for HBsAg 32
B. Memory B cell frequencies in total IgG+ memory B cells 33
肆、討論 36
伍、結論 44
陸、參考文獻 45
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