發(fā)布時(shí)間：2018-02-16 19:09

  本文關(guān)鍵詞： 乙型肝炎 疫苗 佐劑 胞壁酰二肽 胞壁酰二肽類似物 細(xì)胞免疫　出處：《中國(guó)藥品生物制品檢定所》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：我國(guó)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(dá)1億之多，其中慢性乙肝患者有3000萬(wàn)人。目前對(duì)于乙肝治療尚無(wú)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(xiàn)行佐劑，大量研究表明，鋁佐劑主要提高體液免疫和Th2類細(xì)胞免疫應(yīng)答。 1974年，Lederer E．等人發(fā)現(xiàn)胞壁酰二肽(MDP)是弗式完全佐劑的最小有效結(jié)構(gòu)單位，是滅活分枝桿菌細(xì)胞壁的成分之一。MDP具有廣泛的生物學(xué)活性，但同時(shí)也表現(xiàn)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(duì)其結(jié)構(gòu)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(duì)其構(gòu)效關(guān)系進(jìn)行了深入的研究。本文采用酶聯(lián)免疫斑點(diǎn)分析(ELISPOT)方法對(duì)中國(guó)協(xié)和醫(yī)科大學(xué)藥物研究所合成的60種MDP類似物進(jìn)行生物學(xué)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(duì)其免疫原性做了進(jìn)一步的研究。 首先通過(guò)ELISPOT方法，對(duì)3ug HBsAg免疫BALB／c小鼠后的細(xì)胞免疫應(yīng)答動(dòng)態(tài)進(jìn)行研究。結(jié)果顯示，免疫后4天，產(chǎn)生較弱的細(xì)胞免疫應(yīng)答，7天時(shí)，細(xì)胞免疫應(yīng)答達(dá)到一定強(qiáng)度，14天時(shí)，達(dá)到高峰。同時(shí)研究了HBsAg免疫小鼠7天后脾淋巴細(xì)胞及CD8~+T細(xì)胞細(xì)胞免疫應(yīng)答的劑量—效應(yīng)關(guān)系，結(jié)果說(shuō)明，隨著免疫劑量的增加，能夠增強(qiáng)機(jī)體的特異性細(xì)胞免疫應(yīng)答強(qiáng)度。通過(guò)上述試驗(yàn)，確定了適宜的檢測(cè)時(shí)間點(diǎn)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(lián)合免疫BALB／c小鼠后，ELISPOT方法檢測(cè)小鼠脾淋巴細(xì)胞經(jīng)多肽S_(28-39)刺激，分泌IFN-γ的水平，來(lái)評(píng)價(jià)化合物的免疫佐劑活性。經(jīng)過(guò)初篩和復(fù)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(duì)其免疫原性做了進(jìn)一步的研究。在細(xì)胞免疫方面，ELISPOT檢測(cè)小鼠脾淋巴細(xì)胞IFN-γ分泌水平的結(jié)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(guò)Luminex方法對(duì)免疫后小鼠脾細(xì)胞經(jīng)HBsAg刺激分泌的多細(xì)胞因子進(jìn)行測(cè)定，探討了5#、17#化合物作為HBsAg佐劑對(duì)HBsAg細(xì)胞免疫的影響。通過(guò)~(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(xué)位授予單位】：中國(guó)藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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