B-LACTOGLOBULIN致敏性降低的免疫学机制

发布时间：2019-01-11 11:54

【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。
[Abstract]:Bovine milk 尾-lactoglobulin (尾-lg) is the first extraneous allergen encountered by fetus after birth, and it is the main allergen causing bovine milk protein allergy. Enzymatic hydrolysis can lead to the change of three dimensional structure of 尾-lg, but the significance of the change of enzyme induced structure to its sensitization is not clear. In order to find out the effect of hydrolysis on the sensitivities or antigenicity of 尾-lg, the effects of hydrolysis on 尾-lg sensitization were studied in an animal model of mice. In this study, the mechanism of reducing 尾-lg sensitization by hydrolysis was discussed in the following aspects: splenic lymphocyte proliferation test, ELISA method for detection of IgE in serum and small intestinal fluid, detection of histamine level in plasma by using kit; The levels of various cytokines (IL-2,IL-4,IL-10,IFN- 纬) and CD4, CD8, CD3, CD19 in the supernatant of splenic lymphocyte culture were also determined. Spleen cells are the main lymphocytes. The immunomodulatory effects of 尾-lg, and 尾-lg hydrolysates in vivo were studied by lymphocyte proliferation test. The results of no ConA group showed that, compared with 尾-lg hydrolysates and OVA groups, the immune regulation of 尾-lg, and 尾-lg hydrolysates in vivo was studied. 尾-lg significantly stimulated splenic lymphocyte proliferation (p0. 01, p0. 001), and 尾-lg hydrolysates were significantly different from OVA (p0. 002). In addition, the groups with ConA had the same trend compared with the experimental group without ConA. For example, there were significant differences among 尾-lg group, 尾-lg hydrolysate group and OVA group. In addition, compared with the experimental group without ConA, the overall trend was that the group without ConA was more stimulative than the group without ConA (p0. 508 尾-lg vs 尾-lg ConA). The levels of IgE antibody in serum and intestinal fluid of mice were detected by ELISA method. The results showed that the level of serum IgE in 尾-lg group was higher than that in 尾-lg hydrolysate group, while the level of IgE in small intestine fluid and the level of serum IgE in small intestine fluid were the same as those in 尾-lg hydrolysate group. But the overall level is higher than the serum. In this experiment, plasma histamine was detected by ELISA kit. The results showed that the level of plasma histamine in 尾-lg group was significantly higher than that in 尾-lg group and OVA group (p0. 002, p0. 000). The plasma histamine level in 尾-lg group was significantly higher than that in OVA group (p0. 007). Th1 type cytokines (IL-2,IFN- 纬) and Th2 type cytokines (IL-4,IL-10) in the supernatant of splenic lymphocyte culture were also detected. The addition of 尾-lg and 尾-lg hydrolysates into the splenocyte culture medium had little effect on cytokine secretion level compared with the control group, but the difference was not significant. However, in the presence of ConA, 尾-lg significantly stimulated the secretion of IL-4,IL-10,IFN- 纬. In addition, 尾-lg inhibited the expression of IL-2 in ConA stimulated splenocytes, but there was no significant difference compared with the control group. On the other hand, 尾-lg hydrolysates increased the expression of IFN- 纬 in ConA stimulated splenocytes, but had no effect on the secretion of other cytokines. These results suggest that 尾-lg hydrolysates can not only inhibit the proliferation of splenocytes, but also regulate the secretion of cytokines. Innovation: the effect of hydrolysis on the sensitization of 尾-lg was studied systematically. Using 尾-lg as allergen and balb/c mice as animal model, the plasma histamine level, cytokine level and cell surface antigen of serum IgE, were studied. Humoral immunity provides a reference for further study on the mechanism of allergy reduction.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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