【摘要】： 牛乳β-乳球蛋白(β-lg)是胎兒出生后遇到的第一個(gè)外來(lái)過(guò)敏原,并且它是導(dǎo)致牛乳蛋白過(guò)敏的主要過(guò)敏原。酶水解作用可以導(dǎo)致β-lg空間三維結(jié)構(gòu)的變化,但是該酶誘導(dǎo)結(jié)構(gòu)變化對(duì)其致敏性的意義還不清楚。為了探明水解作用對(duì)β-lg致敏性或者抗原性的影響,本實(shí)驗(yàn)利用小鼠動(dòng)物模型研究了水解作用對(duì)β-lg致敏性的影響。 本研究主要從以下幾個(gè)方面探討了水解降低β-lg致敏性的機(jī)制:脾淋巴細(xì)胞增殖實(shí)驗(yàn);ELISA方法檢測(cè)血清和小腸液中的IgE水平;利用試劑盒檢測(cè)血漿中組胺水平;還檢測(cè)了脾淋巴細(xì)胞培養(yǎng)上清中各種細(xì)胞因子(IL-2,IL-4,IL-10,IFN-γ)水平,脾淋巴細(xì)胞表面抗原(CD4+,CD8+,CD3+,CD19+)。 脾細(xì)胞是主要的淋巴細(xì)胞,本試驗(yàn)通過(guò)淋巴細(xì)胞增殖試驗(yàn)來(lái)研究β-lg,和β-lg水解物在體內(nèi)免疫調(diào)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(xì)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(shí)驗(yàn)組相比有相同的趨勢(shì)。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(shí)驗(yàn)組相比,整體趨勢(shì)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(cè)了小鼠血清和小腸液IgE抗體水平,結(jié)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(shì)一致,但是整體水平要高于血清。 在本試驗(yàn)中,血漿組胺是通過(guò)ELISA試劑盒檢測(cè)的,結(jié)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(shí)驗(yàn)還檢測(cè)了脾淋巴細(xì)胞培養(yǎng)上清中Th1型細(xì)胞因子(IL-2,IFN-γ)和Th2型細(xì)胞因子(IL-4,IL-10)。在脾細(xì)胞培養(yǎng)液中加入β-lg和β-lg水解物,和對(duì)照組細(xì)胞相比,對(duì)其細(xì)胞因子分泌水平影響不大,差異不顯著。但是,在有ConA存在時(shí),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(xì)胞中IL-2的表達(dá),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(xì)胞中的表達(dá),但是對(duì)其他細(xì)胞因子的分泌沒(méi)有影響。這些結(jié)果說(shuō)明,β-lg水解物不但能夠抑制脾細(xì)胞增殖,還對(duì)細(xì)胞因子的分泌有調(diào)節(jié)作用。 創(chuàng)新點(diǎn): 對(duì)水解作用對(duì)β-lg致敏性的影響進(jìn)行了系統(tǒng)的研究。利用β-lg作為致敏原,balb/c小鼠為動(dòng)物模型,研究血清IgE,血漿組胺水平,細(xì)胞因子水平,細(xì)胞表面抗原,解釋了水解作用降低β-lg致敏性的免疫學(xué)機(jī)制,為信號(hào)轉(zhuǎn)導(dǎo)、體液免疫等進(jìn)一步研究過(guò)敏降低機(jī)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(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

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