妊娠期孕鼠接触SEB对子代成年大鼠T细胞的印迹效应及机制的研究
发布时间：2018-08-06 17:02
【摘要】：目的： 金黄色葡萄球菌肠毒素B（Staphylococcal enterotoxin B，SEB）既是一种毒素性物质，又是一种超抗原。SEB作为一种肠毒素少量进入人体内即可导致严重的疾病，作为一种超抗原可以刺激机体免疫系统中大量T细胞活化增殖。SEB进入机体后，初期可激活Vβ8+T细胞并诱发大量增殖，继而过度增殖的细胞发生凋亡导致克隆清除，而出现中枢或外周耐受。本实验室前期研究发现妊娠期孕鼠接触SEB导致子代新生鼠T细胞亚群比例的改变，表明妊娠期孕鼠接触SEB已在新生儿期对子代鼠细胞免疫产生影响，那么这种改变是否可保留至成年期形成印迹效应，目前国内外尚未见报道。因此，我们在前期研究基础上，研究妊娠期孕鼠接触SEB对子代成年大鼠胸腺、脾脏和血液中CD4/CD8T细胞及Vβ8.2T细胞亚群的影响；研究妊娠期孕鼠接触SEB对子代成年大鼠体外培养的脾淋巴细胞增殖应答的影响；研究子代成年大鼠细胞免疫对再次接受SEB刺激的免疫应答反应；研究子代成年大鼠外周血中IFN-γ及IL-4的水平及脾脏淋巴细胞中IFN-γ及IL-4基因的甲基化水平。其研究结果将为妊娠期接触SEB对子代细胞免疫的影响提供实验依据，也为探讨一些胎源性疾病提供理论依据。 方法： 1．选用清洁级成年SD大鼠，交配及确定受孕后，孕鼠饲养至妊娠16天随机分组，实验组（SEB组）孕鼠通过尾静脉注射15μg SEB，同时设立对照组，给予等体积的PBS（磷酸盐缓冲液），继续饲养孕鼠至自然分娩，让子代新生鼠生长至成年时期，即获得子代成年大鼠。 2．取子代成年大鼠胸腺、脾脏及外周血，制备单细胞悬液，荧光抗体（CD3FITC、CD8PE、CD4APC）染色后流式细胞仪检测CD4/CD8T细胞。 3．取子代成年大鼠的胸腺及外周血，制备单细胞悬液，荧光抗体（Vβ8.2FITC、CD8PE、CD3APC）染色后流式细胞仪检测Vβ8.2T细胞。 4．再次给予子代成年大鼠静脉注射SEB，同时设立同窝子代大鼠注射SEB作为对照组，5天后获取其胸腺、脾脏及外周血，分离其淋巴细胞，荧光抗体（CD3FITC、CD8PE、CD4APC）染色后流式细胞仪检测CD4/CD8T细胞。 5．再次给予子代成年大鼠静脉注射SEB，同时设立同窝的子代大鼠作为对照组，5天后取其胸腺及外周血，分离其淋巴细胞，染色后经流式细胞仪检测Vβ8.2T细胞。 6．无菌条件下取出子代成年大鼠的脾脏，分离淋巴细胞并进行体外培养，分别给予ConA及SEB连续刺激3天，每天取出培养的细胞检测其增殖指数及其T细胞亚群。 7．获取子代成年大鼠的血浆，ELISA检测IFN-γ及IL-4的水平。 8．获取子代成年大鼠的脾脏，通过MeDIP-qPCR检测IL-4及IFN-γ基因的甲基化水平。 结果： 1．孕鼠接触SEB对子代成年大鼠细胞免疫的影响（1）妊娠期接触SEB对子代成年大鼠胸腺、脾脏及外周血中T细胞亚群的影响：SEB组子代成年雌雄性大鼠胸腺、脾脏及外周血中CD4+CD8T细胞的比例较PBS组明显增加，而CD4CD8+T细胞的比例较PBS组明显减少；（2）妊娠期接触SEB对子代成年大鼠胸腺及外周血Vβ8.2T细胞的影响：妊娠期接触SEB可明显减少子代成年雌雄性大鼠胸腺及外周血中Vβ8.2T细胞的比例。 2．再次注射SEB对妊娠期已接触SEB孕鼠的子代成年大鼠CD4/CD8T细胞的影响妊娠期已接触SEB的子代成年雌雄性大鼠，与同窝对照组比较，再次接触SEB时可明显减少胸腺中CD4+CD8T细胞的比例，而增加CD4CD8+T细胞的比例；在子代成年雌雄性大鼠脾脏及外周血中T细胞亚群表现出与胸腺中相类似的变化。 3．再次注射SEB对妊娠期已接触SEB的子代成年大鼠Vβ8.2T细胞的影响妊娠期已接触SEB的子代成年雌雄性大鼠，再次注射SEB时对胸腺中Vβ8.2T细胞的比例与对照组比较无差异，但外周血中Vβ8.2T细胞的比例却较同窝对照组明显增加。 4．SEB刺激对体外培养的子代成年大鼠脾细胞的影响实验发现随着刺激时间增加体外培养的子代成年大鼠脾细胞的增殖能力逐渐增强，而且SEB组的子代成年雌雄性大鼠脾细胞接受SEB的刺激能力较ConA组弱；检测T细胞亚群发现，，给予SEB刺激时SEB组培养的脾细胞中CD4+CD8T细胞的比例较ConA组减弱，但对CD4CD8+T细胞的比例无明显影响。 5．妊娠期孕鼠接触SEB对子代成年大鼠外周血中细胞因子的影响ELISA检测结果发现SEB组子代成年大鼠外周血中IFN-γ及IL-4的水平分别较PBS组明显增加；在成年期再次给予SEB刺激后，其（PBS+SEB，SEB+SEB）IFN-γ及IL-4水平均较各自对照组（PBS+PBS，SEB+PBS）明显减少。 6．妊娠期孕鼠接触SEB对子代成年大鼠DNA甲基化的影响实验发现SEB组子代成年大鼠脾脏中IFN-γ及IL-4基因的甲基化水平均较PBS组明显减少。 结论： 1．妊娠期接触SEB改变了子代成年大鼠中枢及外周中CD4/CD8T细胞及Vβ8.2T细胞的比例； 2．妊娠期接触SEB改变子代成年大鼠CD4/CD8T细胞及Vβ8.2T细胞对再次SEB刺激的免疫应答反应； 3.妊娠期接触SEB减弱子代成年大鼠脾淋巴细胞体外的增殖能力，增殖能力减低可能与CD4+T细胞亚群减少有关； 4.妊娠期接触SEB增加子代成年大鼠外周血中的细胞因子IL-4及IFN-γ水平，可能与其DNA甲基化减少有关。
[Abstract]:Objective:
Staphylococcus aureus enterotoxin B (Staphylococcal enterotoxin B, SEB) is not only a toxin substance, but also a kind of superantigen.SEB as a small amount of enterotoxin into the human body and can cause serious disease. As a superantigen, it can stimulate the large amount of T cells in the immune system to enter the body after the activation and proliferation of T cells, and can be stimulated at the initial stage. Living V beta 8+T cells and inducing a large number of proliferation, and then excessive proliferation of cell apoptosis leading to clone scavenging, and the emergence of central or peripheral tolerance. Previous studies in this laboratory found that pregnant mice exposed to SEB led to the change in the proportion of T cell subsets in newborn rats, indicating that pregnant mice exposed to SEB had been immune to the progeny of offspring rats during neonatal period. The effect of pestilence on whether this change can be retained to adulthood to form an imprinting effect has not yet been reported at home and abroad. Therefore, on the basis of previous studies, we studied the effects of SEB on the thymus, the spleen and blood CD4/CD8T cells and the V beta 8.2T cell subsets in the adult rats of pregnancy. The effect of SEB on the proliferation response of splenic lymphocyte cultured in vitro of adult rats; study the immune response response to SEB stimulation in adult adult rat cells; study the level of IFN- gamma and IL-4 in the peripheral blood of adult adult rats and the methylation level of IFN- gamma and IL-4 genes in the spleen lymphocytes. The results will provide experimental basis for the effect of SEB exposure during pregnancy on the cellular immunity of offspring, and also provide theoretical basis for exploring some fetal diseases.
Method:
1. with clean grade adult SD rats, after mating and determining pregnancy, pregnant rats were randomly divided into 16 days of pregnancy, and the experimental group (group SEB) was injected 15 mu g SEB through the tail vein. At the same time, the control group was set up to give the equal volume of PBS (phosphate buffer), continue to feed pregnant rats to natural childbirth, and let the offspring of the newborn rats grow into adulthood. Adult rats of the offspring.
2. the adult rat thymus, spleen and peripheral blood were taken to prepare single cell suspension, and CD4/CD8T cells were detected by flow cytometry after fluorescent antibody (CD3FITC, CD8PE, CD4APC) staining.
3. Single cell suspension was prepared from thymus and peripheral blood of offspring adult rats and stained with fluorescent antibodies (Vbeta 8.2 FITC, CD8PE, CD3APC) and detected by flow cytometry.
4. the adult rats were given intravenously SEB again. At the same time, the rats were injected with SEB as the control group. After 5 days, the thymus, spleen and peripheral blood were obtained, their lymphocytes were separated and the fluorescent antibodies (CD3FITC, CD8PE, CD4APC) were stained by flow cytometry to detect CD4/ CD8T cells.
5. the adult rats were given intravenously SEB again. At the same time, the offspring rats with the same nest were set up as the control group. After 5 days, the thymus and peripheral blood were taken and their lymphocytes were separated. The V beta 8.2T cells were detected by flow cytometry after staining.
6. under aseptic conditions, the spleen of adult rats was removed and lymphocytes were isolated and cultured in vitro. ConA and SEB were given for 3 days respectively. The proliferation index and T cell subgroup were detected every day.
7. the plasma of adult rats was obtained, and the levels of IFN- and IL-4 were detected by ELISA.
8. the spleen of adult offspring rats was obtained, and the methylation level of IL-4 and IFN- gamma genes was detected by MeDIP-qPCR.
Result:
Effect of 1. pregnant mice exposed to SEB on the cell immunity of adult rats (1) the effect of exposure to SEB on the thymus, spleen and peripheral blood T cell subsets in the adult rats during pregnancy: the proportion of CD4+CD8T cells in the thymus, spleen and peripheral blood of the offspring of group SEB was significantly higher than that of the PBS group, while the proportion of CD4CD8+T cells was more than that of the PBS group. (2) the effect of exposure to SEB on the thymus and peripheral blood V beta 8.2T cells in the adult rats during pregnancy: the exposure to SEB during pregnancy can significantly reduce the proportion of V beta 8.2T cells in the thymus and peripheral blood of the adult male and female offspring.
2. the effect of the second injection of SEB on the CD4/CD8T cells of the offspring of SEB pregnant rats during pregnancy, the adult male and female rats were exposed to the offspring of SEB in pregnancy. Compared with the same nest control group, the proportion of CD4+CD8T cells in the thymus could be significantly reduced and the proportion of CD4CD8 +T cells was increased, and the adult male and female sex of the progeny was larger than that of the control group of the same nest. The T cell subsets in rat spleen and peripheral blood showed similar changes with that in the thymus.
3. the effect of the second injection of SEB on the V beta 8.2T cells of the adult rats exposed to SEB during pregnancy. The proportion of V beta 8.2T cells in the thymus was not different from that of the control group, but the ratio of V beta 8.2T cells in the peripheral blood was significantly higher than that in the control group.
The effect of 4.SEB stimulation on the splenocytes of adult adult rats in vitro showed that the proliferation ability of spleen cells increased gradually with the increase of stimulation time, and the stimulation ability of spleen cells in the adult male and female rats of SEB group was weaker than that of the ConA group, and the detection of T cell subgroups was found to be given to SEB. The percentage of CD4+CD8T cells in SEB group was lower than that in ConA group, but there was no significant effect on the percentage of CD4CD8+T cells.
5. the effects of SEB on the cytokine in the peripheral blood of the offspring of the offspring of the pregnant rats ELISA detection results showed that the level of IFN- gamma and IL-4 in the peripheral blood of the SEB group was significantly higher than that of the PBS group, and the level of PBS+SEB, SEB+SEB IFN- gamma and IL-4 was compared with the control group (PBS+PBS, PBS+PBS,) after the adult stage was given SEB stimulation again. SEB+PBS) obviously decreased.
6. the effects of exposure to SEB on DNA methylation of adult rats in pregnancy showed that the level of methylation of IFN- gamma and IL-4 genes in the spleen of group SEB was significantly lower than that of the PBS group.
Conclusion:
1. exposure to SEB during pregnancy changed the proportion of CD4/CD8T cells and V beta 8.2T cells in the central and peripheral parts of adult rats.
2. exposure to SEB during pregnancy altered the immune responses of CD4/CD8T cells and V beta 8.2T cells of offspring rats to re SEB stimulation.
3. Exposure to SEB during pregnancy decreases the proliferation of splenic lymphocytes in vitro, which may be related to the decrease of CD4+T cell subsets.
4. SEB exposure during pregnancy increases the levels of cytokines IL-4 and IFN-gamma in peripheral blood of offspring adult rats, which may be related to the decrease of DNA methylation.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.25
本文编号：2168375
[Abstract]:Objective:
Staphylococcus aureus enterotoxin B (Staphylococcal enterotoxin B, SEB) is not only a toxin substance, but also a kind of superantigen.SEB as a small amount of enterotoxin into the human body and can cause serious disease. As a superantigen, it can stimulate the large amount of T cells in the immune system to enter the body after the activation and proliferation of T cells, and can be stimulated at the initial stage. Living V beta 8+T cells and inducing a large number of proliferation, and then excessive proliferation of cell apoptosis leading to clone scavenging, and the emergence of central or peripheral tolerance. Previous studies in this laboratory found that pregnant mice exposed to SEB led to the change in the proportion of T cell subsets in newborn rats, indicating that pregnant mice exposed to SEB had been immune to the progeny of offspring rats during neonatal period. The effect of pestilence on whether this change can be retained to adulthood to form an imprinting effect has not yet been reported at home and abroad. Therefore, on the basis of previous studies, we studied the effects of SEB on the thymus, the spleen and blood CD4/CD8T cells and the V beta 8.2T cell subsets in the adult rats of pregnancy. The effect of SEB on the proliferation response of splenic lymphocyte cultured in vitro of adult rats; study the immune response response to SEB stimulation in adult adult rat cells; study the level of IFN- gamma and IL-4 in the peripheral blood of adult adult rats and the methylation level of IFN- gamma and IL-4 genes in the spleen lymphocytes. The results will provide experimental basis for the effect of SEB exposure during pregnancy on the cellular immunity of offspring, and also provide theoretical basis for exploring some fetal diseases.
Method:
1. with clean grade adult SD rats, after mating and determining pregnancy, pregnant rats were randomly divided into 16 days of pregnancy, and the experimental group (group SEB) was injected 15 mu g SEB through the tail vein. At the same time, the control group was set up to give the equal volume of PBS (phosphate buffer), continue to feed pregnant rats to natural childbirth, and let the offspring of the newborn rats grow into adulthood. Adult rats of the offspring.
2. the adult rat thymus, spleen and peripheral blood were taken to prepare single cell suspension, and CD4/CD8T cells were detected by flow cytometry after fluorescent antibody (CD3FITC, CD8PE, CD4APC) staining.
3. Single cell suspension was prepared from thymus and peripheral blood of offspring adult rats and stained with fluorescent antibodies (Vbeta 8.2 FITC, CD8PE, CD3APC) and detected by flow cytometry.
4. the adult rats were given intravenously SEB again. At the same time, the rats were injected with SEB as the control group. After 5 days, the thymus, spleen and peripheral blood were obtained, their lymphocytes were separated and the fluorescent antibodies (CD3FITC, CD8PE, CD4APC) were stained by flow cytometry to detect CD4/ CD8T cells.
5. the adult rats were given intravenously SEB again. At the same time, the offspring rats with the same nest were set up as the control group. After 5 days, the thymus and peripheral blood were taken and their lymphocytes were separated. The V beta 8.2T cells were detected by flow cytometry after staining.
6. under aseptic conditions, the spleen of adult rats was removed and lymphocytes were isolated and cultured in vitro. ConA and SEB were given for 3 days respectively. The proliferation index and T cell subgroup were detected every day.
7. the plasma of adult rats was obtained, and the levels of IFN- and IL-4 were detected by ELISA.
8. the spleen of adult offspring rats was obtained, and the methylation level of IL-4 and IFN- gamma genes was detected by MeDIP-qPCR.
Result:
Effect of 1. pregnant mice exposed to SEB on the cell immunity of adult rats (1) the effect of exposure to SEB on the thymus, spleen and peripheral blood T cell subsets in the adult rats during pregnancy: the proportion of CD4+CD8T cells in the thymus, spleen and peripheral blood of the offspring of group SEB was significantly higher than that of the PBS group, while the proportion of CD4CD8+T cells was more than that of the PBS group. (2) the effect of exposure to SEB on the thymus and peripheral blood V beta 8.2T cells in the adult rats during pregnancy: the exposure to SEB during pregnancy can significantly reduce the proportion of V beta 8.2T cells in the thymus and peripheral blood of the adult male and female offspring.
2. the effect of the second injection of SEB on the CD4/CD8T cells of the offspring of SEB pregnant rats during pregnancy, the adult male and female rats were exposed to the offspring of SEB in pregnancy. Compared with the same nest control group, the proportion of CD4+CD8T cells in the thymus could be significantly reduced and the proportion of CD4CD8 +T cells was increased, and the adult male and female sex of the progeny was larger than that of the control group of the same nest. The T cell subsets in rat spleen and peripheral blood showed similar changes with that in the thymus.
3. the effect of the second injection of SEB on the V beta 8.2T cells of the adult rats exposed to SEB during pregnancy. The proportion of V beta 8.2T cells in the thymus was not different from that of the control group, but the ratio of V beta 8.2T cells in the peripheral blood was significantly higher than that in the control group.
The effect of 4.SEB stimulation on the splenocytes of adult adult rats in vitro showed that the proliferation ability of spleen cells increased gradually with the increase of stimulation time, and the stimulation ability of spleen cells in the adult male and female rats of SEB group was weaker than that of the ConA group, and the detection of T cell subgroups was found to be given to SEB. The percentage of CD4+CD8T cells in SEB group was lower than that in ConA group, but there was no significant effect on the percentage of CD4CD8+T cells.
5. the effects of SEB on the cytokine in the peripheral blood of the offspring of the offspring of the pregnant rats ELISA detection results showed that the level of IFN- gamma and IL-4 in the peripheral blood of the SEB group was significantly higher than that of the PBS group, and the level of PBS+SEB, SEB+SEB IFN- gamma and IL-4 was compared with the control group (PBS+PBS, PBS+PBS,) after the adult stage was given SEB stimulation again. SEB+PBS) obviously decreased.
6. the effects of exposure to SEB on DNA methylation of adult rats in pregnancy showed that the level of methylation of IFN- gamma and IL-4 genes in the spleen of group SEB was significantly lower than that of the PBS group.
Conclusion:
1. exposure to SEB during pregnancy changed the proportion of CD4/CD8T cells and V beta 8.2T cells in the central and peripheral parts of adult rats.
2. exposure to SEB during pregnancy altered the immune responses of CD4/CD8T cells and V beta 8.2T cells of offspring rats to re SEB stimulation.
3. Exposure to SEB during pregnancy decreases the proliferation of splenic lymphocytes in vitro, which may be related to the decrease of CD4+T cell subsets.
4. SEB exposure during pregnancy increases the levels of cytokines IL-4 and IFN-gamma in peripheral blood of offspring adult rats, which may be related to the decrease of DNA methylation.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.25
【参考文献】
相关期刊论文 前2条
1 管俊昌,夏佩莹,唐素兰;金黄色葡萄球菌L型产B型肠毒素及其基因的研究[J];蚌埠医学院学报;2004年04期
2 刘从森;刘婷婷;孔晓明;朱翔;刘勇;闵宏林;管俊昌;;妊娠母鼠静脉注射葡萄球菌肠毒素B对胎鼠胸腺发育的影响[J];蚌埠医学院学报;2011年06期
本文编号：2168375
本文链接：https://www.wllwen.com/yixuelunwen/fuchankeerkelunwen/2168375.html
最近更新
教材专著
