本文關鍵詞： 乙型肝炎 疫苗 佐劑 胞壁酰二肽 胞壁酰二肽類似物 細胞免疫　出處：《中國藥品生物制品檢定所》2006年碩士論文　論文類型：學位論文

【摘要】：我國屬于乙型肝炎高流行地區(qū)之一，乙型肝炎表面抗原(HBsAg)攜帶者高達1億之多，其中慢性乙肝患者有3000萬人。目前對于乙肝治療尚無有效的藥物，接種疫苗是控制乙肝流行的基本措施。鋁鹽是乙肝疫苗的現行佐劑，大量研究表明，鋁佐劑主要提高體液免疫和Th2類細胞免疫應答。 1974年，Lederer E．等人發(fā)現胞壁酰二肽(MDP)是弗式完全佐劑的最小有效結構單位，是滅活分枝桿菌細胞壁的成分之一。MDP具有廣泛的生物學活性，但同時也表現出一些副作用如致熱、代謝迅速等。為了提高其活性，降低副作用，，人們對其結構加以改造，合成了許多MDP衍生物及類似物，找到了一些活性較高、副作用較低的化合物，并對其構效關系進行了深入的研究。本文采用酶聯免疫斑點分析(ELISPOT)方法對中國協(xié)和醫(yī)科大學藥物研究所合成的60種MDP類似物進行生物學活性篩選，將篩選出的2種MDP類似物(5#、17#化合物)作為HBsAg的佐劑，對其免疫原性做了進一步的研究。 首先通過ELISPOT方法，對3ug HBsAg免疫BALB／c小鼠后的細胞免疫應答動態(tài)進行研究。結果顯示，免疫后4天，產生較弱的細胞免疫應答，7天時，細胞免疫應答達到一定強度，14天時，達到高峰。同時研究了HBsAg免疫小鼠7天后脾淋巴細胞及CD8~+T細胞細胞免疫應答的劑量—效應關系，結果說明，隨著免疫劑量的增加，能夠增強機體的特異性細胞免疫應答強度。通過上述試驗，確定了適宜的檢測時間點和HBsAg的免疫劑量。 然后將60種化合物與HBsAg MHC Ⅰ類多肽S_(28-39)及HBsAg聯合免疫BALB／c小鼠后，ELISPOT方法檢測小鼠脾淋巴細胞經多肽S_(28-39)刺激，分泌IFN-γ的水平，來評價化合物的免疫佐劑活性。經過初篩和復篩，最終確定了2種免疫佐劑活性最高的MDP類似物：5#、17#化合物。 最后將篩選出的5#、17#化合物作為HBsAg的佐劑，對其免疫原性做了進一步的研究。在細胞免疫方面，ELISPOT檢測小鼠脾淋巴細胞IFN-γ分泌水平的結果顯示5#、17#化合物分別與HBsAg混合組均高于HBsAg組、疫苗組。通過Luminex方法對免疫后小鼠脾細胞經HBsAg刺激分泌的多細胞因子進行測定，探討了5#、17#化合物作為HBsAg佐劑對HBsAg細胞免疫的影響。通過~(51)Cr釋
[Abstract]:China belongs to one of the high prevalence areas of hepatitis B, and there are as many as 100 million carriers of hepatitis B surface antigen HBsAg, among which there are 30 million patients with chronic hepatitis B. there are no effective drugs for the treatment of hepatitis B at present. Aluminum salt is the current adjuvant of hepatitis B vaccine. A large number of studies show that aluminum adjuvant mainly improves humoral immunity and cellular immune response of Th2. In 1974, Lederer E. et al. Found that MDP) is the smallest effective structural unit of Freund complete adjuvant. It is one of the components of inactivated mycobacterium cell wall. MDP has a wide range of biological activities, but it also shows some side effects such as fever. In order to improve its activity and reduce its side effects, many MDP derivatives and analogues were synthesized, and some compounds with higher activity and lower side effects were found. The bioactivity of 60 MDP analogues synthesized by the Institute of Pharmacology of China Union Medical University was screened by Elisa. The immunogenicity of HBsAg was further studied by using two kinds of MDP analogues, named as the adjuvant of HBsAg. The dynamics of cellular immune response of BALB/c mice immunized with 3ug HBsAg were studied by ELISPOT method. The results showed that after 4 days of immunization, the cellular immune response reached a certain intensity at 14 days after 4 days of immunization, when a weak cellular immune response was produced at 7 days. At the same time, the dose-response relationship of spleen lymphocytes and CD8T cells immune response of mice immunized with HBsAg was studied after 7 days. The results showed that, with the increase of immune dose, It can enhance the specific cellular immune response. Through the above experiments, the appropriate detection time point and the immune dose of HBsAg were determined. Then, 60 compounds were immunized with HBsAg MHC class I polypeptides (SZ28-39) and BALB/c mice were immunized with HBsAg. The levels of IFN- 緯 secreted from spleen lymphocytes of mice were detected by Elisa method after immunizing BALB/c mice by SSP 28-39), to evaluate the immuno-adjuvant activity of the compounds, and to evaluate the immunoadjuvant activity of the compounds by screening and screening again. Finally, two MDP analogues with the highest immunological adjuvant activity were identified. Finally, the immunogenicity of the selected 5 #n17# compound as the adjuvant of HBsAg was further studied. In cellular immunity, the level of IFN- 緯 secretion in spleen lymphocytes of mice was detected by ELISPOT. The results showed that the level of IFN- 緯 secretion in the mixed group of 5 #n17# and HBsAg was higher than that in the HBsAg group. In the vaccine group, the polycytokines stimulated by HBsAg in the spleen cells of mice after immunization were determined by Luminex method, and the effect of 5 #N 17# compound as an adjuvant of HBsAg on the cellular immunity of HBsAg was investigated.
【學位授予單位】：中國藥品生物制品檢定所
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

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