BW006细胞免疫活性及其乙肝疫苗佐剂作用的研究

发布时间：2018-03-18 18:06

本文选题：乙型肝炎　切入点：疫苗　出处：《中国药品生物制品检定所》2008年硕士论文　论文类型：学位论文

【摘要】： 乙型肝炎(以下简称乙肝)是严重危害人类健康的疾病之一,目前尚无有效治疗慢性乙肝的药物,接种乙肝疫苗是控制乙肝流行的唯一有效措施。基因工程乙肝表面抗原(HBsAg)单独免疫免疫原性较低,需要与相应佐剂联合应用才能诱导机体产生有效的免疫应答。铝佐剂乙肝疫苗(以下简称乙肝疫苗)已大量应用多年,对乙肝病毒感染有较好的预防作用,且安全性好。然而,铝佐剂乙肝疫苗用在预防和治疗方面,都存在不足。应用于预防接种时,约10%左右的接种者呈低或无应答;且对慢性乙肝病人的疗效仍不确切。含有CpG基序的寡核苷酸(CpG-ODN)是一种新型免疫佐剂,具有增强免疫应答作用,能刺激淋巴细胞增殖、活化,诱发天然和获得性免疫应答,作为疫苗佐剂具有良好的前景。本研究应用流式细胞术、酶联免疫斑点(ELISPOT)和BranchedDNA(bDNA)等技术对一种新设计合成的CpG-ODN(BW006)的细胞免疫活性进行了探讨,并对其作为乙肝疫苗佐剂的作用进行了研究。本研究第一部分,首先通过体外刺激和体内诱导两种方式探讨BW006对小鼠脾单个核细胞(MNC)的活化作用,结果表明:(1)BW006能够有效刺激DC和B细胞表面协同刺激分子CD80、CD86的表达,且呈剂量-效应关系;(2)BW006上调NK细胞早期活化分子CD69的表达,活化NK细胞;(3)BW006可以抑制B细胞凋亡,促进B细胞增殖;(4)BW006体内诱导脾MNCs活化的最佳检测时间:DC为免疫后12h,B、NK和T细胞均为免疫后24h;(5)BW006不同免疫途径活化鼠脾B细胞作用不同:表现为肌肉免疫＞腹腔免疫＞皮下免疫;(6)磷酸氯喹可抑制BW006对DC、B和NK细胞的活化作用。应用bDNA技术,分析BW006诱导鼠脾MNCs和DC在转录水平(mRNA)表达细胞因子的影响,结果表明,IL-12b(P40)mRNA表达增加最为显著,在BW006刺激3至6h之间达峰值,较刺激前表达水平增加约26倍;IL-10 mRNA的表达水平变化趋势与IL-12b mRNA相似,其表达峰值与刺激前相比增加11.9倍;与对照组比较,BW006体外刺激3h时,鼠脾MNCs和DC表达IP-10 mRNA水平分别提高了2.8倍和7.3倍。Luminex多因子检测结果也表明,BW006体外刺激鼠脾MNCs可上调细胞因子IL-12、IL-10的表达,且呈时间效应关系:IL-12和IL-10表达水平随BW006刺激时间延长而增加,48h达峰值。IL-12是参与天然免疫应答的重要因子,BW006在转录和翻译水平早期诱导IL-12的高水平表达,表明其可以在早期诱导天然免疫应答。而IL-10的高水平表达则可能是机体反向调节IL-12作用的机制之一。此外检测应用200μg BW006经皮下免疫3h时外周血IP-10表达水平,结果表明,皮下免疫BW006 3h外周血IP-10表达水平显著提高(P＜0.01)。第二部分对BW006的佐剂作用进行了研究。以不同剂量BW006联合40μgHBsAg体外刺激DC,结果发现,BW006可以协同HBsAg上调DC表达CD80、CD86的水平,且呈剂量-效应关系。BW006(20μg)与HBsAg(2μg)联合免疫小鼠结果也表明,BW006能够协同HBsAg上调B细胞CD80、CD86和T细胞早期活化分子CD69的表达水平。为探讨BW006联合HBsAg或乙肝疫苗后诱导细胞免疫应答的剂量效应关系。以不同剂量BW006联合HBsAg(2μg)或乙肝疫苗(2μg)免疫BALB/c小鼠,7d后检测小鼠脾MNCs分泌IFN-γ和IL-2水平,结果发现:(1)BW006可以显著提高HBsAg或乙肝疫苗诱导IFN-γ和IL-2的表达水平;(2)BW006联合HBsAg诱导IFN-γ、IL-2的表达水平显著高于BW006联合铝佐剂乙肝疫苗诱导IFN-γ、IL-2水平。(3)BW006协同HBsAg或乙肝疫苗诱导IFN-γ表达的最佳剂量是0.32μg/只,而BW006协同HBsAg或铝佐剂乙肝疫苗诱导IL-2表达的最佳剂量是5μg/只。为探索BW006联合HBsAg和乙肝疫苗后诱导体液免疫应答的剂量-效应,于免疫后1、2、3和4W时分别采血检测血清中抗-HBs指标,结果表明:(1)BW006与HBsAg或乙肝疫苗联合免疫小鼠后,显著提高抗-HBs应答水平;(2)免后3和4周,BW006联合乙肝疫苗诱导抗-HBs应答水平高于BW006联合HBsAg 本课题应用题内、体外法对BW006诱导细胞免疫活性进行了分析,并从天然免疫、特异性体液免疫和特异性细胞免疫三个方面对其作为乙肝疫苗佐剂的作用进行了研究,发现BW006可以在早期诱导DC表达IL-12、有效活化免疫细胞、协同HBsAg或乙肝疫苗诱导较强的细胞和体液免疫应答,为新型预防和治疗性乙肝疫苗的研发提供了依据。
[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.

【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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