本文選題：乙型肝炎　切入點(diǎn)：疫苗　出處：《中國藥品生物制品檢定所》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 乙型肝炎(以下簡稱乙肝)是嚴(yán)重危害人類健康的疾病之一,目前尚無有效治療慢性乙肝的藥物,接種乙肝疫苗是控制乙肝流行的唯一有效措施�；蚬こ桃腋伪砻婵乖�(HBsAg)單獨(dú)免疫免疫原性較低,需要與相應(yīng)佐劑聯(lián)合應(yīng)用才能誘導(dǎo)機(jī)體產(chǎn)生有效的免疫應(yīng)答。鋁佐劑乙肝疫苗(以下簡稱乙肝疫苗)已大量應(yīng)用多年,對(duì)乙肝病毒感染有較好的預(yù)防作用,且安全性好。然而,鋁佐劑乙肝疫苗用在預(yù)防和治療方面,都存在不足。應(yīng)用于預(yù)防接種時(shí),約10%左右的接種者呈低或無應(yīng)答;且對(duì)慢性乙肝病人的療效仍不確切。 含有CpG基序的寡核苷酸(CpG-ODN)是一種新型免疫佐劑,具有增強(qiáng)免疫應(yīng)答作用,能刺激淋巴細(xì)胞增殖、活化,誘發(fā)天然和獲得性免疫應(yīng)答,作為疫苗佐劑具有良好的前景。本研究應(yīng)用流式細(xì)胞術(shù)、酶聯(lián)免疫斑點(diǎn)(ELISPOT)和BranchedDNA(bDNA)等技術(shù)對(duì)一種新設(shè)計(jì)合成的CpG-ODN(BW006)的細(xì)胞免疫活性進(jìn)行了探討,并對(duì)其作為乙肝疫苗佐劑的作用進(jìn)行了研究。 本研究第一部分,首先通過體外刺激和體內(nèi)誘導(dǎo)兩種方式探討B(tài)W006對(duì)小鼠脾單個(gè)核細(xì)胞(MNC)的活化作用,結(jié)果表明:(1)BW006能夠有效刺激DC和B細(xì)胞表面協(xié)同刺激分子CD80、CD86的表達(dá),且呈劑量-效應(yīng)關(guān)系;(2)BW006上調(diào)NK細(xì)胞早期活化分子CD69的表達(dá),活化NK細(xì)胞;(3)BW006可以抑制B細(xì)胞凋亡,促進(jìn)B細(xì)胞增殖;(4)BW006體內(nèi)誘導(dǎo)脾MNCs活化的最佳檢測時(shí)間:DC為免疫后12h,B、NK和T細(xì)胞均為免疫后24h;(5)BW006不同免疫途徑活化鼠脾B細(xì)胞作用不同:表現(xiàn)為肌肉免疫＞腹腔免疫＞皮下免疫;(6)磷酸氯喹可抑制BW006對(duì)DC、B和NK細(xì)胞的活化作用。 應(yīng)用bDNA技術(shù),分析BW006誘導(dǎo)鼠脾MNCs和DC在轉(zhuǎn)錄水平(mRNA)表達(dá)細(xì)胞因子的影響,結(jié)果表明,IL-12b(P40)mRNA表達(dá)增加最為顯著,在BW006刺激3至6h之間達(dá)峰值,較刺激前表達(dá)水平增加約26倍;IL-10 mRNA的表達(dá)水平變化趨勢與IL-12b mRNA相似,其表達(dá)峰值與刺激前相比增加11.9倍;與對(duì)照組比較,BW006體外刺激3h時(shí),鼠脾MNCs和DC表達(dá)IP-10 mRNA水平分別提高了2.8倍和7.3倍。Luminex多因子檢測結(jié)果也表明,BW006體外刺激鼠脾MNCs可上調(diào)細(xì)胞因子IL-12、IL-10的表達(dá),且呈時(shí)間效應(yīng)關(guān)系:IL-12和IL-10表達(dá)水平隨BW006刺激時(shí)間延長而增加,48h達(dá)峰值。IL-12是參與天然免疫應(yīng)答的重要因子,BW006在轉(zhuǎn)錄和翻譯水平早期誘導(dǎo)IL-12的高水平表達(dá),表明其可以在早期誘導(dǎo)天然免疫應(yīng)答。而IL-10的高水平表達(dá)則可能是機(jī)體反向調(diào)節(jié)IL-12作用的機(jī)制之一。此外檢測應(yīng)用200μg BW006經(jīng)皮下免疫3h時(shí)外周血IP-10表達(dá)水平,結(jié)果表明,皮下免疫BW006 3h外周血IP-10表達(dá)水平顯著提高(P＜0.01)。 第二部分對(duì)BW006的佐劑作用進(jìn)行了研究。以不同劑量BW006聯(lián)合40μgHBsAg體外刺激DC,結(jié)果發(fā)現(xiàn),BW006可以協(xié)同HBsAg上調(diào)DC表達(dá)CD80、CD86的水平,且呈劑量-效應(yīng)關(guān)系。BW006(20μg)與HBsAg(2μg)聯(lián)合免疫小鼠結(jié)果也表明,BW006能夠協(xié)同HBsAg上調(diào)B細(xì)胞CD80、CD86和T細(xì)胞早期活化分子CD69的表達(dá)水平。 為探討B(tài)W006聯(lián)合HBsAg或乙肝疫苗后誘導(dǎo)細(xì)胞免疫應(yīng)答的劑量效應(yīng)關(guān)系。以不同劑量BW006聯(lián)合HBsAg(2μg)或乙肝疫苗(2μg)免疫BALB/c小鼠,7d后檢測小鼠脾MNCs分泌IFN-γ和IL-2水平,結(jié)果發(fā)現(xiàn):(1)BW006可以顯著提高HBsAg或乙肝疫苗誘導(dǎo)IFN-γ和IL-2的表達(dá)水平;(2)BW006聯(lián)合HBsAg誘導(dǎo)IFN-γ、IL-2的表達(dá)水平顯著高于BW006聯(lián)合鋁佐劑乙肝疫苗誘導(dǎo)IFN-γ、IL-2水平。(3)BW006協(xié)同HBsAg或乙肝疫苗誘導(dǎo)IFN-γ表達(dá)的最佳劑量是0.32μg/只,而BW006協(xié)同HBsAg或鋁佐劑乙肝疫苗誘導(dǎo)IL-2表達(dá)的最佳劑量是5μg/只。 為探索BW006聯(lián)合HBsAg和乙肝疫苗后誘導(dǎo)體液免疫應(yīng)答的劑量-效應(yīng),于免疫后1、2、3和4W時(shí)分別采血檢測血清中抗-HBs指標(biāo),結(jié)果表明:(1)BW006與HBsAg或乙肝疫苗聯(lián)合免疫小鼠后,顯著提高抗-HBs應(yīng)答水平;(2)免后3和4周,BW006聯(lián)合乙肝疫苗誘導(dǎo)抗-HBs應(yīng)答水平高于BW006聯(lián)合HBsAg 本課題應(yīng)用題內(nèi)、體外法對(duì)BW006誘導(dǎo)細(xì)胞免疫活性進(jìn)行了分析,并從天然免疫、特異性體液免疫和特異性細(xì)胞免疫三個(gè)方面對(duì)其作為乙肝疫苗佐劑的作用進(jìn)行了研究,發(fā)現(xiàn)BW006可以在早期誘導(dǎo)DC表達(dá)IL-12、有效活化免疫細(xì)胞、協(xié)同HBsAg或乙肝疫苗誘導(dǎo)較強(qiáng)的細(xì)胞和體液免疫應(yīng)答,為新型預(yù)防和治療性乙肝疫苗的研發(fā)提供了依據(jù)。
[Abstract]:Hepatitis B (hereinafter referred to as b) is a serious harm to human health, there is no effective drug for the treatment of chronic hepatitis B, hepatitis B vaccination is the only effective measures to control hepatitis B epidemic. Genetic engineering hepatitis B surface antigen (HBsAg) immunized with low immunogenicity, so adjuvant is needed to induce immune effective response. Aluminum adjuvant hepatitis B vaccine (hereinafter referred to as the hepatitis B vaccine) has been used for many years, has a good preventive effect on hepatitis B virus infection, and the safety is good. However, aluminum adjuvant hepatitis B vaccine used in the prevention and treatment are insufficient. Applied to vaccination, with about 10% a low or no response; and the curative effect of chronic hepatitis B patients is still not clear.
Oligonucleotides containing CpG motif (CpG-ODN) is a new type of immune adjuvant can enhance the immune response, can stimulate cell proliferation, activation, induced by natural and acquired immune response, as vaccine adjuvant has good prospects. The research and application of flow cytometry, enzyme-linked immunospot assay (ELISPOT) and BranchedDNA (bDNA) technology of a new design and synthesis of CpG-ODN (BW006) cell immune activity were discussed, and the adjuvant of hepatitis B vaccine was studied.
The first part of this study, first by in vitro stimulation and in vivo induced by two ways to discuss the effects of BW006 on mice spleen mononuclear cells (MNC) activation, results showed that: (1) BW006 can effectively stimulate DC and B cell surface costimulatory molecules CD80, CD86 expression, and showed a dose effect relationship; (2 BW006) expression of NK cells by regulating the early activation of CD69, activation of NK cells; (3) BW006 can inhibit the apoptosis of B cells, promote the proliferation of B cells; (4) the optimal time of detection of splenic MNCs activation induced by BW006 in vivo: DC 12h after immunization B, NK and T cells were 24h after immunization (; 5) BW006 different immune pathway activation of mouse spleen B cells: effect of different manifestations of muscle, abdominal subcutaneous immunization, immune immunity; (6) BW006 of chloroquine phosphate can inhibit the activation of DC, B and NK cells.
The application of bDNA technology, analysis of BW006 induced rat spleen MNCs and DC at the transcriptional level (mRNA), the results showed that the expression of cytokines, IL-12b (P40) mRNA expression increased most significantly, in response to BW006 between 3 and 6h reached the peak, compared with before stimulation increased the expression level of about 26 times; the expression level change trend with IL-12b mRNA IL-10 mRNA is similar, the expression peak and before stimulation compared to an increase of 11.9 times; compared with the control group, BW006 stimulated in vitro 3h, mouse spleen MNCs and DC expression level of mRNA IP-10 was increased by 2.8 times and 7.3 times the.Luminex multi factor test results also show that, BW006 in vitro stimulation of mouse spleen MNCs upregulates cytokine IL-12, the expression of IL-10, and showed the time effect relationship. The expression level of IL-12 and IL-10 with BW006 stimulation time increased, and reached the peak at 48h.IL-12 is an important factor involved in the innate immune response, BW006 transcription and translation level in the early induction of IL-12 high The level of expression that can induce innate immune responses in the early and high levels of IL-10 expression may be one of the mechanisms of body reverse regulation of IL-12. In addition the detection using 200 g BW006 subcutaneously immunized with 3H IP-10 expression level in peripheral blood, the results show that the subcutaneous BW006 of peripheral blood 3H IP-10 expression significantly increase (P < 0.01).
The second part of the adjuvant effects of BW006 were studied. The different dosages of BW006 combined with 40 gHBsAg in vitro stimulation DC, results show that BW006 can upregulate the expression of DC CD80 collaborative HBsAg, CD86 level, and showed a dose effect relationship of.BW006 (20 g) and HBsAg (2 g) combined with immune mice results show that BW006 can cooperate with HBsAg up regulation of B CD80 cells, the expression level of CD86 and T cells early activation of CD69.
In order to investigate the dose effect relationship of BW006 combined with HBsAg or hepatitis B vaccine induced cellular immune responses. The different dosages of BW006 combined with HBsAg (2 g) or hepatitis B vaccine (2 g) BALB/c mice were immunized with 7d, found after the detection of IFN- and IL-2 levels, the secretion of mouse spleen MNCs: (1) BW006 can significantly improve the expression level of HBsAg or hepatitis B vaccine induced by IFN- gamma and IL-2; (2) BW006 and HBsAg induced by IFN- gamma, the expression level of IL-2 was significantly higher than that of IFN-, induced by BW006 and Al adjuvant of hepatitis B vaccine IL-2 level. (3) BW006 Co HBsAg or hepatitis B vaccine induced by IFN- gamma table of optimal dose is 0.32 g/ only BW006, and the optimal dosage of HBsAg or co aluminum adjuvant hepatitis B vaccine induced the expression of IL-2 is 5 g/.
In order to explore the combination of BW006 HBsAg and hepatitis B vaccine induced humoral immune response after immunization in a dose effect, anti -HBs index, 1,2,3 and 4W were measured in serum showed that: (1) BW006 and HBsAg combined with hepatitis B vaccine or immune mice, significantly improve the anti -HBs response level; (2) after immunization 3 and 4 weeks, BW006 combined with hepatitis B vaccine induced anti -HBs response level is higher than that of BW006 combined with HBsAg
This paper applied problems, analyzes the BW006 induced cellular immune activity in vitro, and from the natural immune adjuvant of hepatitis B vaccine were studied on three aspects as the specific humoral and cellular immune specificity, BW006 can be found in the early induction of DC expression by IL-12, the effective activation of immune cells, Co HBsAg or hepatitis B vaccine induced humoral and cellular immune response is strong, provide the basis for the development of new prevention and treatment of hepatitis B vaccine.

【學(xué)位授予單位】：中國藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 歐強(qiáng);陳良;孫洪清;唐徐英;;慢性HBV感染者外周血T淋巴細(xì)胞亞群和NK細(xì)胞活性變化[J];臨床肝膽病雜志;2006年04期

2 邢利和,王福生,劉明旭,朱傳琳,李捍衛(wèi),雷周云,王慧芬,張冰,金磊;慢性乙型肝炎患者樹突狀細(xì)胞的培養(yǎng)鑒定和功能特點(diǎn)[J];中華傳染病雜志;2001年06期

3 劉樹人 ,彭齊榮 ,李灼亮 ,余宙耀 ,張宜俊;慢性乙肝患者樹突狀細(xì)胞表型和抗原提呈能力的測定[J];中國現(xiàn)代醫(yī)學(xué)雜志;2003年09期

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本文編號(hào)：1630703