【作者】 段翠翠；

【导师】 霍贵成；

【作者基本信息】 东北农业大学 ， 食品科学， 2010， 硕士

【摘要】 牛乳β-乳球蛋白(β-lg)是胎儿出生后遇到的第一个外来过敏原,并且它是导致牛乳蛋白过敏的主要过敏原。酶水解作用可以导致β-lg空间三维结构的变化,但是该酶诱导结构变化对其致敏性的意义还不清楚。为了探明水解作用对β-lg致敏性或者抗原性的影响,本实验利用小鼠动物模型研究了水解作用对β-lg致敏性的影响。本研究主要从以下几个方面探讨了水解降低β-lg致敏性的机制:脾淋巴细胞增殖实验;ELISA方法检测血清和小肠液中的IgE水平;利用试剂盒检测血浆中组胺水平;还检测了脾淋巴细胞培养上清中各种细胞因子(IL-2,IL-4,IL-10,IFN-γ)水平,脾淋巴细胞表面抗原(CD4+,CD8+,CD3+,CD19+)。脾细胞是主要的淋巴细胞,本试验通过淋巴细胞增殖试验来研究β-lg,和β-lg水解物在体内免疫调节作用,不加ConA组数据表明,和β-lg水解物以及OVA组相比,β-lg能够显著刺激脾淋巴细胞增殖(分别为p=0.01, p=0.001),β-lg水解物和OVA相比,差异也是极显著的。(p=0.002)。并且,加ConA的各组与不加ConA的实验组相比有相同的趋势。例如β-lg组和酶β-lg水解物组以及OVA组相比,差异都显著(分别为p=0.007,p=0.001)。除此之外,添加ConA和不添加ConA实验组相比,整体趋势是不添加ConA组要比添加ConA组有较大的刺激性(p=0.508β-lg vsβ-lg+ConA)。使用ELISA方法检测了小鼠血清和小肠液IgE抗体水平,结果表明β-lg组小鼠血清IgE水平要高于β-lg水解物组小鼠,而小肠液中IgE水平和血清IgE水平变化趋势一致,但是整体水平要高于血清。在本试验中,血浆组胺是通过ELISA试剂盒检测的,结果表明,灌胃β-lg组小鼠血浆组胺水平显著高于酶解β-lg组和OVA组(分别为p=0.002,p=0.000)。酶解β-lg组血浆组胺水平也显著高于OVA组(p=0.007)。本实验还检测了脾淋巴细胞培养上清中Th1型细胞因子(IL-2,IFN-γ)和Th2型细胞因子(IL-4,IL-10)。在脾细胞培养液中加入β-lg和β-lg水解物,和对照组细胞相比,对其细胞因子分泌水平影响不大,差异不显著。但是,在有ConA存在时,β-lg显著的刺激了IL-4,IL-10,IFN-γ的分泌。除此之外,由于β-lg的存在,抑制了ConA刺激的脾细胞中IL-2的表达,但是和空白组相比差异不显著。另一方面,β-lg水解物增加了IFN-γ在ConA刺激的脾细胞中的表达,但是对其他细胞因子的分泌没有影响。这些结果说明,β-lg水解物不但能够抑制脾细胞增殖,还对细胞因子的分泌有调节作用。创新点:对水解作用对β-lg致敏性的影响进行了系统的研究。利用β-lg作为致敏原,balb/c小鼠为动物模型,研究血清IgE,血浆组胺水平,细胞因子水平,细胞表面抗原,解释了水解作用降低β-lg致敏性的免疫学机制,为信号转导、体液免疫等进一步研究过敏降低机制提供了参考。更多还原

【Abstract】 Bovineβ-lactoglobulin (β-lg) is one of the first foreign antigens encountered by a newborn child, and it is the major allergen causing cow’s milk allergy. Hydrolysis can cause changes to the protein structure, but the significance of hydrolysis-induced changes for immunogenicity or allergenicity is not known. To clarify how hydrolysis affects allergenicity and immunogenicity, the sensitization ofβ-lg by enzymatic hydrolysis was evaluated in this paper using the mouse model.This study explored the mechanism to reduce the sensitization of hydrolysisβ-lg by the following aspects: spleens lymphocyte proliferation test; IgE levels in serum and intestinal tracts by ELISA method; determination of plasma histamine levels using kits; various cytokines in the supernatants of spleen cells (IL-2, IL-4, IL-10, IFN-γ), spleen lymphocyte surface antigen (CD4+, CD8+, CD3+, CD19+).Spleens were the main lymphocyte, and this article researched immunomodulating effect ofβ-lg and enzymatic hydrolysisβ-lg in vitro by measuring their impacts on lymphocyte proliferation which were expressed as a stimulation index (SI) and was evaluated with and without suboptimal concentration (0.5 mg/ml) of ConA (Fig 2). The results of absence ConA showed thatβ-lg could significantly stimulate lymphocyte proliferation than enzymatic hydrolysisβ-lg as well as OVA (p=0.01, p=0.000 respectively), the result of comparing enzymatic hydrolysisβ-lg to OVA also showed obvious difference (p=0.002). Furthermore, the ConA present group had the same trend with the absence group. Theβ-lg group showed significant difference to enzymatic hydrolysisβ-lg as well as OVA (p=0.007, p=0.001, respectively) but without obvious difference compared to enzymatic hydrolysisβ-lg (p=0.061). Besides, the whole trend between absence ConA and present ConA was that the absence ConA group showed a lower stimulation than present ConA group, but the difference was not significant (p=0.508β-lg vsβ-lg+ConA).Enzyme-linked immunosorbent assay (ELISA) methods were developed to quantify the antibody level in balb/c mouse by gavage ofβ-lg and enzymatic hydrolysisβ-lg with dosages of 10mg/ml, 10mg/ml, respectively. The serum IgE level inβ-lg group and intestinal fluid samples were higher than those in enzymatic hydrolysis group, but the total IgE level in intestinal fluid samples is higher than serum IgE.Plasma histamine levels were determined by an ELISA kit and the results indicated that the histamine level of mice challenged intragastrically withβ-lg showed significant high level than enzymatic hydrolysisβ-lg and OVA (p=0.002, p=0.000, respectively) (Fig 4). The histamine levels of enzymatic hydrolysisβ-lg also showed significant high level than OVA (p=0.007). This paper also examined the secretion of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines by resting splenocytes, and the results indicated that nativeβ-lg and enzymatic hydrolysisβ-lg had no effect on the secretion of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines, while nativeβ-lg significantly stimulated the secretion of IL-2 and IFN-γwith ConA-induced spleens, whereas enzymatic hydrolysisβ-lg significantly increased IFN-γsecretion.These results suggest that hydrolysing theβ-lg not only eliminates its inhibiting effect on the proliferation of splenocytes in the presence of ConA but also modifies its effect on cytokine secretion.InnovationThis paper systemically detected the serum IgE, plasma histamine levels, cytokine levels and cell surface antigens, usingβ-lg as allergens, balb/c mice as animal model, firstly from the perspective of immunology, and explained the immunological mechanism of lower sensitization of hydrolysisβ-lg, which provided references for the signal transduction, humoral immunity etc.更多还原