卵清蛋白免疫耐受因子免疫學(xué)活性的研究
發(fā)布時間：2018-08-15 12:45
【摘要】： 免疫耐受作為機體免疫調(diào)節(jié)的重要組成部分之一,在免疫系統(tǒng)識別“己”和“非己”的過程中發(fā)揮著關(guān)鍵作用。其中,外周免疫耐受的形成和維持與臨床上自身免疫性疫病、過敏反應(yīng)以及抗移植物排斥等人類重大疾病的治療密切相關(guān)。因此,闡明外周耐受產(chǎn)生和維持的分子機制不僅有利于進一步完善免疫學(xué)理論,對于臨床上免疫耐受相關(guān)疾病的治療也具有十分重要的指導(dǎo)意義。根據(jù)外周耐受能夠通過人工轉(zhuǎn)移其效應(yīng)細(xì)胞(或分子)在個體間進行傳遞的特點,人們發(fā)現(xiàn),CD4~+CD25~+等調(diào)節(jié)性T細(xì)胞是外周耐受產(chǎn)生的主要介導(dǎo)者。此外,還有一些報道指出,某些大分子物質(zhì)也能夠轉(zhuǎn)移特異性外周耐受給個正常個體。然而,小分子的非細(xì)胞成分是否也能夠攜帶并傳遞特異性免疫耐受信息,至今未見相關(guān)報道。本研究就這一問題進行了初步探討。 本研究首先通過多次尾靜脈注射卵清蛋白(ovalbumin, OVA)建立了對OVA免疫耐受的BALB/c小鼠模型,并對其耐受效果進行了鑒定。在此基礎(chǔ)上制備了3個不同分子量范圍的耐受小鼠脾淋巴細(xì)胞裂解液,通過對其轉(zhuǎn)移耐受的效果進行評估,確定了轉(zhuǎn)移OVA耐受的主要效應(yīng)分子所在的分子量范圍,并將這一分子量范圍的細(xì)胞裂解液稱為(OVA immune tolerance factors,OVA ITFs)。為進一步探討OVA ITFs轉(zhuǎn)移耐受的免疫學(xué)活性,本研究以O(shè)VA耐受鼠脾淋巴細(xì)胞為陽性對照,研究了OVA ITFs對正常BALB/c受體小鼠外周CD4~+CD25~+ T細(xì)胞亞群比例、DTH反應(yīng)、OVA特異性T淋巴細(xì)胞增殖以及抑制性細(xì)胞因子IL-10、TGF-β1分泌水平的影響�，F(xiàn)將研究結(jié)果報道如下: 1. BALB/c小鼠多次尾靜脈注射OVA(5μg/只)后,特異性T淋巴細(xì)胞增殖刺激指數(shù)(Stimulation Index ,SI)增殖的SI值為0.721±0.03,顯著低于對照組(1.676±0.07);CD4~+CD25~+ T細(xì)胞亞群在總CD4+T細(xì)胞的比例為15.37±0.22%,較對照組小鼠顯著升高(11.23±0.41%)。結(jié)果表明,多次注射小劑量OVA可建立BALB/c小鼠對OVA的免疫耐受。 2.分別制備分子量小于3ku、3~5ku和大于5ku三個分子量范圍的耐受小鼠脾淋巴細(xì)胞裂解液。體外試驗顯示,向淋巴細(xì)胞培養(yǎng)系統(tǒng)中加入2倍稀釋的各分子量范圍細(xì)胞裂解液,對OVA特異性T細(xì)胞增殖的抑制率分別為40.4%、18.4%和7.9%;體內(nèi)試驗顯示,轉(zhuǎn)移各分子量范圍的細(xì)胞裂解液給正常小鼠后,受體鼠OVA特異性T細(xì)胞增殖的SI值分別為0.557±0.02、0.954±0.07和1.094±0.01,與空白組(1.110±0.09)相比,只有分子量小于3ku的裂解液產(chǎn)生的具有顯著的抑制效果。此外,各分子量范圍的細(xì)胞裂解液還產(chǎn)生了一定的非特異性抑制作用。結(jié)果表明,分子量小于3ku的耐受小鼠脾淋巴細(xì)胞裂解液能夠顯著抑制OVA特異性T細(xì)胞的增殖。 3.轉(zhuǎn)移OVA ITFs或OVA耐受鼠脾淋巴細(xì)胞后,受體鼠脾臟CD4~+CD25~+ T細(xì)胞亞群在總CD4+ T細(xì)胞的比例逐漸上升,分別于轉(zhuǎn)移后7天和3天達到峰值,為15.32±1.03%和15.35±0.62%;外周血中CD4~+CD25~+ T細(xì)胞亞群的比例也分別于轉(zhuǎn)移后7天和3天達到峰值,為10.10±0.21%和10.07±0.07%,均較轉(zhuǎn)移前(9.97±1.38% and 7.28±0.12%)顯著上升。結(jié)果表明,OVA ITFs能夠誘導(dǎo)外周CD4~+CD25~+ T細(xì)胞的產(chǎn)生,但與耐受鼠淋巴細(xì)胞相比,其誘導(dǎo)外周CD4~+CD25~+ T細(xì)胞亞群達到峰值需要更長的時間。 4.轉(zhuǎn)移OVA ITFs或OVA耐受鼠脾淋巴細(xì)胞后免疫OVA并激發(fā)DTH,檢測其對OVA特異性免疫反應(yīng)的抑制效果。結(jié)果表明,OVA ITFs能夠有效抑制受體小鼠的DTH反應(yīng),其反應(yīng)部位的皮膚增厚下降了60%。 5.轉(zhuǎn)移OVA ITFs或OVA耐受鼠脾淋巴細(xì)胞后免疫OVA,受體鼠特異性淋巴細(xì)胞增殖的SI值分別為0.699±0.05和0.704±0.03,與對照組(1.356±0.07)相比明顯受到抑制;培養(yǎng)上清中TGF-β1的分泌量分別為129.15±6.14和106.71±2.89 pg/mL,顯著高于對照組(52.82±3.68 pg/mL), IL-10的分泌量未檢出。結(jié)果表明,OVA ITFs能有效抑制OVA特異性T細(xì)胞的增殖并促進抑制性細(xì)胞因子TGF-β1的的產(chǎn)生。 6.轉(zhuǎn)移空白小鼠的脾淋巴細(xì)胞裂解液(OVA ITFs control)后,受體小鼠的外周CD4~+CD25~+ T細(xì)胞比例、OVA特異性淋巴細(xì)胞增殖及TGF-β1、IL-10分泌量均無顯著變化。說明OVA ITFs并非機體的固有成分,而是由免疫耐受個體獲得性產(chǎn)生的。以上研究結(jié)果表明,本研究所制備的OVA ITFs具有將OVA特異性免疫耐受傳遞給正常受體小鼠的特性。這一結(jié)果首次證明了小分子物質(zhì)也能夠攜帶并傳遞特異性免疫耐受信息,這對進一步闡明外周耐受的分子機制以及指導(dǎo)臨床上自身免疫病、抗移植物排斥等免疫耐受相關(guān)疾病的治療具有重要的參考價值。
[Abstract]:Immune tolerance, as one of the important components of immune regulation, plays a key role in the process of recognizing "self" and "non-self" by the immune system. Therefore, elucidating the molecular mechanisms underlying the production and maintenance of peripheral tolerance is not only conducive to further improving immunological theory, but also of great significance in the treatment of immunotolerance-related diseases. In addition, some reports indicate that some macromolecules can also transfer specific peripheral tolerance to a normal individual. However, whether small molecules of non-cellular components can also carry and transmit specific immune tolerance information has not been reported so far. This study discussed the problem preliminarily.
In this study, ovalbumin (OVA) was injected into the tail vein several times to establish the BALB/c mice model of OVA immune tolerance, and the tolerance effect was identified. On this basis, the splenic lymphocyte lysates of three tolerant mice with different molecular weight ranges were prepared, and the effect of metastasis tolerance was evaluated. The molecular weight range of the main effectors of OVA tolerance was determined and the cell lysate was called (OVA immune tolerance factors, OVA ITFs). In order to further explore the immunological activity of OVA ITFs in metastasis tolerance, we studied the effect of OVA ITFs on normal BALB/c in spleen lymphocytes of OVA tolerant mice as a positive control. The effects of peripheral CD4~+CD25~+ T cell subsets, DTH response, OVA-specific T lymphocyte proliferation and the secretion of inhibitory cytokines IL-10 and TGF-beta 1 in recipient mice are reported as follows:
1. The SI value of specific T lymphocyte proliferation stimulation index (SI) in BALB/c mice after repeated tail vein injection of OVA (5 ug/mouse) was 0.721.03, significantly lower than that in control group (1.676.07); the proportion of CD4 +CD25 + T cell subsets in total CD4 + T cells was 15.37.22%, significantly higher than that in control group (11.23.41%). It shows that multiple doses of OVA can establish BALB/c mice immune tolerance to OVA.
2. Splenic lymphocyte lysates of tolerant mice with molecular weights less than 3 ku, 3-5 Ku and 5 Ku were prepared respectively. The SI values of OVA-specific T cell proliferation in recipient mice were 0.557 (+ 0.02), 0.954 (+ 0.07) and 1.094 (+ 0.01) respectively after transferring cell lysates with different molecular weight ranges to normal mice. Compared with the blank group (1.110 (+ 0.09), only lysates with molecular weight less than 3 Ku produced significant inhibitory effects. The results showed that the splenic lymphocyte lysate with molecular weight less than 3 Ku significantly inhibited the proliferation of OVA-specific T cells.
3. After transferring OVA ITFs or OVA-tolerant splenic lymphocytes, the percentage of CD4~+CD25~+ T cell subsets in recipient mice spleen gradually increased, reaching the peak at 7 days and 3 days respectively, reaching 15.32 (+ 1.03%) and 15.35 (+ 0.62%). The percentage of CD4~+CD25~+ T cell subsets in peripheral blood also reached the peak at 7 days and 3 days after metastasis, reaching 10.32 (+ 1.03%). The results showed that OVA ITFs could induce the production of peripheral CD4~+CD25~+ T cells, but it took longer for OVA ITFs to induce the subpopulations of peripheral CD4~+CD25~+ T cells to reach the peak value than the tolerant mice.
4. Immune OVA and stimulate DTH after transferring OVA ITFs or OVA-tolerant spleen lymphocytes to detect its inhibitory effect on OVA-specific immune response. The results showed that OVA ITFs could effectively inhibit the DTH response of recipient mice, and the skin thickening of the reaction site was reduced by 60%.
5. The SI values of specific lymphocyte proliferation in recipient mice were 0.699 (+0.05) and 0.704 (+0.03) respectively, which were significantly inhibited compared with the control group (1.356 (+0.07), and the secretion of TGF-beta 1 in supernatant was 129.15 (+6.14) and 106.71 (+2.89) pg/mL, respectively, which were significantly higher than those in the control group (52.82 (+3.68). The results showed that OVA ITFs could effectively inhibit the proliferation of OVA-specific T cells and promote the production of inhibitory cytokine TGF-beta 1.
6. After transferring the splenic lymphocyte lysate (OVA ITFs control) of the blank mice, the peripheral CD4~+CD25~+ T cell ratio, OVA specific lymphocyte proliferation and the secretion of TGF-beta 1 and IL-10 in the recipient mice did not change significantly. These results demonstrate for the first time that small molecule substances can also carry and transmit specific immune tolerance information, which will further elucidate the molecular mechanism of peripheral tolerance and guide clinical autoimmune diseases and anti-graft rejection. The treatment of immune tolerance related diseases is of great reference value.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
本文編號：2184242
[Abstract]:Immune tolerance, as one of the important components of immune regulation, plays a key role in the process of recognizing "self" and "non-self" by the immune system. Therefore, elucidating the molecular mechanisms underlying the production and maintenance of peripheral tolerance is not only conducive to further improving immunological theory, but also of great significance in the treatment of immunotolerance-related diseases. In addition, some reports indicate that some macromolecules can also transfer specific peripheral tolerance to a normal individual. However, whether small molecules of non-cellular components can also carry and transmit specific immune tolerance information has not been reported so far. This study discussed the problem preliminarily.
In this study, ovalbumin (OVA) was injected into the tail vein several times to establish the BALB/c mice model of OVA immune tolerance, and the tolerance effect was identified. On this basis, the splenic lymphocyte lysates of three tolerant mice with different molecular weight ranges were prepared, and the effect of metastasis tolerance was evaluated. The molecular weight range of the main effectors of OVA tolerance was determined and the cell lysate was called (OVA immune tolerance factors, OVA ITFs). In order to further explore the immunological activity of OVA ITFs in metastasis tolerance, we studied the effect of OVA ITFs on normal BALB/c in spleen lymphocytes of OVA tolerant mice as a positive control. The effects of peripheral CD4~+CD25~+ T cell subsets, DTH response, OVA-specific T lymphocyte proliferation and the secretion of inhibitory cytokines IL-10 and TGF-beta 1 in recipient mice are reported as follows:
1. The SI value of specific T lymphocyte proliferation stimulation index (SI) in BALB/c mice after repeated tail vein injection of OVA (5 ug/mouse) was 0.721.03, significantly lower than that in control group (1.676.07); the proportion of CD4 +CD25 + T cell subsets in total CD4 + T cells was 15.37.22%, significantly higher than that in control group (11.23.41%). It shows that multiple doses of OVA can establish BALB/c mice immune tolerance to OVA.
2. Splenic lymphocyte lysates of tolerant mice with molecular weights less than 3 ku, 3-5 Ku and 5 Ku were prepared respectively. The SI values of OVA-specific T cell proliferation in recipient mice were 0.557 (+ 0.02), 0.954 (+ 0.07) and 1.094 (+ 0.01) respectively after transferring cell lysates with different molecular weight ranges to normal mice. Compared with the blank group (1.110 (+ 0.09), only lysates with molecular weight less than 3 Ku produced significant inhibitory effects. The results showed that the splenic lymphocyte lysate with molecular weight less than 3 Ku significantly inhibited the proliferation of OVA-specific T cells.
3. After transferring OVA ITFs or OVA-tolerant splenic lymphocytes, the percentage of CD4~+CD25~+ T cell subsets in recipient mice spleen gradually increased, reaching the peak at 7 days and 3 days respectively, reaching 15.32 (+ 1.03%) and 15.35 (+ 0.62%). The percentage of CD4~+CD25~+ T cell subsets in peripheral blood also reached the peak at 7 days and 3 days after metastasis, reaching 10.32 (+ 1.03%). The results showed that OVA ITFs could induce the production of peripheral CD4~+CD25~+ T cells, but it took longer for OVA ITFs to induce the subpopulations of peripheral CD4~+CD25~+ T cells to reach the peak value than the tolerant mice.
4. Immune OVA and stimulate DTH after transferring OVA ITFs or OVA-tolerant spleen lymphocytes to detect its inhibitory effect on OVA-specific immune response. The results showed that OVA ITFs could effectively inhibit the DTH response of recipient mice, and the skin thickening of the reaction site was reduced by 60%.
5. The SI values of specific lymphocyte proliferation in recipient mice were 0.699 (+0.05) and 0.704 (+0.03) respectively, which were significantly inhibited compared with the control group (1.356 (+0.07), and the secretion of TGF-beta 1 in supernatant was 129.15 (+6.14) and 106.71 (+2.89) pg/mL, respectively, which were significantly higher than those in the control group (52.82 (+3.68). The results showed that OVA ITFs could effectively inhibit the proliferation of OVA-specific T cells and promote the production of inhibitory cytokine TGF-beta 1.
6. After transferring the splenic lymphocyte lysate (OVA ITFs control) of the blank mice, the peripheral CD4~+CD25~+ T cell ratio, OVA specific lymphocyte proliferation and the secretion of TGF-beta 1 and IL-10 in the recipient mice did not change significantly. These results demonstrate for the first time that small molecule substances can also carry and transmit specific immune tolerance information, which will further elucidate the molecular mechanism of peripheral tolerance and guide clinical autoimmune diseases and anti-graft rejection. The treatment of immune tolerance related diseases is of great reference value.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
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