【摘要】： 牛乳β-乳球蛋白(β-lg)是胎兒出生后遇到的第一個外來過敏原,并且它是導致牛乳蛋白過敏的主要過敏原。酶水解作用可以導致β-lg空間三維結構的變化,但是該酶誘導結構變化對其致敏性的意義還不清楚。為了探明水解作用對β-lg致敏性或者抗原性的影響,本實驗利用小鼠動物模型研究了水解作用對β-lg致敏性的影響。 本研究主要從以下幾個方面探討了水解降低β-lg致敏性的機制:脾淋巴細胞增殖實驗;ELISA方法檢測血清和小腸液中的IgE水平;利用試劑盒檢測血漿中組胺水平;還檢測了脾淋巴細胞培養(yǎng)上清中各種細胞因子(IL-2,IL-4,IL-10,IFN-γ)水平,脾淋巴細胞表面抗原(CD4+,CD8+,CD3+,CD19+)。 脾細胞是主要的淋巴細胞,本試驗通過淋巴細胞增殖試驗來研究β-lg,和β-lg水解物在體內免疫調節(jié)作用,不加ConA組數(shù)據(jù)表明,和β-lg水解物以及OVA組相比,β-lg能夠顯著刺激脾淋巴細胞增殖(分別為p=0.01, p=0.001),β-lg水解物和OVA相比,差異也是極顯著的。(p=0.002)。并且,加ConA的各組與不加ConA的實驗組相比有相同的趨勢。例如β-lg組和酶β-lg水解物組以及OVA組相比,差異都顯著(分別為p=0.007,p=0.001)。除此之外,添加ConA和不添加ConA實驗組相比,整體趨勢是不添加ConA組要比添加ConA組有較大的刺激性(p=0.508β-lg vsβ-lg+ConA)。 使用ELISA方法檢測了小鼠血清和小腸液IgE抗體水平,結果表明β-lg組小鼠血清IgE水平要高于β-lg水解物組小鼠,而小腸液中IgE水平和血清IgE水平變化趨勢一致,但是整體水平要高于血清。 在本試驗中,血漿組胺是通過ELISA試劑盒檢測的,結果表明,灌胃β-lg組小鼠血漿組胺水平顯著高于酶解β-lg組和OVA組(分別為p=0.002,p=0.000)。酶解β-lg組血漿組胺水平也顯著高于OVA組(p=0.007)。 本實驗還檢測了脾淋巴細胞培養(yǎng)上清中Th1型細胞因子(IL-2,IFN-γ)和Th2型細胞因子(IL-4,IL-10)。在脾細胞培養(yǎng)液中加入β-lg和β-lg水解物,和對照組細胞相比,對其細胞因子分泌水平影響不大,差異不顯著。但是,在有ConA存在時,β-lg顯著的刺激了IL-4,IL-10,IFN-γ的分泌。除此之外,由于β-lg的存在,抑制了ConA刺激的脾細胞中IL-2的表達,但是和空白組相比差異不顯著。另一方面,β-lg水解物增加了IFN-γ在ConA刺激的脾細胞中的表達,但是對其他細胞因子的分泌沒有影響。這些結果說明,β-lg水解物不但能夠抑制脾細胞增殖,還對細胞因子的分泌有調節(jié)作用。 創(chuàng)新點: 對水解作用對β-lg致敏性的影響進行了系統(tǒng)的研究。利用β-lg作為致敏原,balb/c小鼠為動物模型,研究血清IgE,血漿組胺水平,細胞因子水平,細胞表面抗原,解釋了水解作用降低β-lg致敏性的免疫學機制,為信號轉導、體液免疫等進一步研究過敏降低機制提供了參考。
[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- 緯) 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- 緯) 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- 緯. 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- 緯 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.
【學位授予單位】：東北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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