卵清蛋白免疫耐受因子免疫学活性的研究
发布时间：2018-08-15 12:45
【摘要】： 免疫耐受作为机体免疫调节的重要组成部分之一,在免疫系统识别“己”和“非己”的过程中发挥着关键作用。其中,外周免疫耐受的形成和维持与临床上自身免疫性疫病、过敏反应以及抗移植物排斥等人类重大疾病的治疗密切相关。因此,阐明外周耐受产生和维持的分子机制不仅有利于进一步完善免疫学理论,对于临床上免疫耐受相关疾病的治疗也具有十分重要的指导意义。根据外周耐受能够通过人工转移其效应细胞(或分子)在个体间进行传递的特点,人们发现,CD4~+CD25~+等调节性T细胞是外周耐受产生的主要介导者。此外,还有一些报道指出,某些大分子物质也能够转移特异性外周耐受给个正常个体。然而,小分子的非细胞成分是否也能够携带并传递特异性免疫耐受信息,至今未见相关报道。本研究就这一问题进行了初步探讨。 本研究首先通过多次尾静脉注射卵清蛋白(ovalbumin, OVA)建立了对OVA免疫耐受的BALB/c小鼠模型,并对其耐受效果进行了鉴定。在此基础上制备了3个不同分子量范围的耐受小鼠脾淋巴细胞裂解液,通过对其转移耐受的效果进行评估,确定了转移OVA耐受的主要效应分子所在的分子量范围,并将这一分子量范围的细胞裂解液称为(OVA immune tolerance factors,OVA ITFs)。为进一步探讨OVA ITFs转移耐受的免疫学活性,本研究以OVA耐受鼠脾淋巴细胞为阳性对照,研究了OVA ITFs对正常BALB/c受体小鼠外周CD4~+CD25~+ T细胞亚群比例、DTH反应、OVA特异性T淋巴细胞增殖以及抑制性细胞因子IL-10、TGF-β1分泌水平的影响。现将研究结果报道如下: 1. BALB/c小鼠多次尾静脉注射OVA(5μg/只)后,特异性T淋巴细胞增殖刺激指数(Stimulation Index ,SI)增殖的SI值为0.721±0.03,显著低于对照组(1.676±0.07);CD4~+CD25~+ T细胞亚群在总CD4+T细胞的比例为15.37±0.22%,较对照组小鼠显著升高(11.23±0.41%)。结果表明,多次注射小剂量OVA可建立BALB/c小鼠对OVA的免疫耐受。 2.分别制备分子量小于3ku、3~5ku和大于5ku三个分子量范围的耐受小鼠脾淋巴细胞裂解液。体外试验显示,向淋巴细胞培养系统中加入2倍稀释的各分子量范围细胞裂解液,对OVA特异性T细胞增殖的抑制率分别为40.4%、18.4%和7.9%;体内试验显示,转移各分子量范围的细胞裂解液给正常小鼠后,受体鼠OVA特异性T细胞增殖的SI值分别为0.557±0.02、0.954±0.07和1.094±0.01,与空白组(1.110±0.09)相比,只有分子量小于3ku的裂解液产生的具有显著的抑制效果。此外,各分子量范围的细胞裂解液还产生了一定的非特异性抑制作用。结果表明,分子量小于3ku的耐受小鼠脾淋巴细胞裂解液能够显著抑制OVA特异性T细胞的增殖。 3.转移OVA ITFs或OVA耐受鼠脾淋巴细胞后,受体鼠脾脏CD4~+CD25~+ T细胞亚群在总CD4+ T细胞的比例逐渐上升,分别于转移后7天和3天达到峰值,为15.32±1.03%和15.35±0.62%;外周血中CD4~+CD25~+ T细胞亚群的比例也分别于转移后7天和3天达到峰值,为10.10±0.21%和10.07±0.07%,均较转移前(9.97±1.38% and 7.28±0.12%)显著上升。结果表明,OVA ITFs能够诱导外周CD4~+CD25~+ T细胞的产生,但与耐受鼠淋巴细胞相比,其诱导外周CD4~+CD25~+ T细胞亚群达到峰值需要更长的时间。 4.转移OVA ITFs或OVA耐受鼠脾淋巴细胞后免疫OVA并激发DTH,检测其对OVA特异性免疫反应的抑制效果。结果表明,OVA ITFs能够有效抑制受体小鼠的DTH反应,其反应部位的皮肤增厚下降了60%。 5.转移OVA ITFs或OVA耐受鼠脾淋巴细胞后免疫OVA,受体鼠特异性淋巴细胞增殖的SI值分别为0.699±0.05和0.704±0.03,与对照组(1.356±0.07)相比明显受到抑制;培养上清中TGF-β1的分泌量分别为129.15±6.14和106.71±2.89 pg/mL,显著高于对照组(52.82±3.68 pg/mL), IL-10的分泌量未检出。结果表明,OVA ITFs能有效抑制OVA特异性T细胞的增殖并促进抑制性细胞因子TGF-β1的的产生。 6.转移空白小鼠的脾淋巴细胞裂解液(OVA ITFs control)后,受体小鼠的外周CD4~+CD25~+ T细胞比例、OVA特异性淋巴细胞增殖及TGF-β1、IL-10分泌量均无显著变化。说明OVA ITFs并非机体的固有成分,而是由免疫耐受个体获得性产生的。以上研究结果表明,本研究所制备的OVA ITFs具有将OVA特异性免疫耐受传递给正常受体小鼠的特性。这一结果首次证明了小分子物质也能够携带并传递特异性免疫耐受信息,这对进一步阐明外周耐受的分子机制以及指导临床上自身免疫病、抗移植物排斥等免疫耐受相关疾病的治疗具有重要的参考价值。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
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