妊娠期孕鼠接觸SEB對子代成年大鼠T細胞的印跡效應(yīng)及機制的研究
發(fā)布時間：2018-08-06 17:02
【摘要】：目的： 金黃色葡萄球菌腸毒素B（Staphylococcal enterotoxin B，SEB）既是一種毒素性物質(zhì)，又是一種超抗原。SEB作為一種腸毒素少量進入人體內(nèi)即可導(dǎo)致嚴重的疾病，作為一種超抗原可以刺激機體免疫系統(tǒng)中大量T細胞活化增殖。SEB進入機體后，初期可激活Vβ8+T細胞并誘發(fā)大量增殖，繼而過度增殖的細胞發(fā)生凋亡導(dǎo)致克隆清除，而出現(xiàn)中樞或外周耐受。本實驗室前期研究發(fā)現(xiàn)妊娠期孕鼠接觸SEB導(dǎo)致子代新生鼠T細胞亞群比例的改變，表明妊娠期孕鼠接觸SEB已在新生兒期對子代鼠細胞免疫產(chǎn)生影響，那么這種改變是否可保留至成年期形成印跡效應(yīng)，目前國內(nèi)外尚未見報道。因此，我們在前期研究基礎(chǔ)上，研究妊娠期孕鼠接觸SEB對子代成年大鼠胸腺、脾臟和血液中CD4/CD8T細胞及Vβ8.2T細胞亞群的影響；研究妊娠期孕鼠接觸SEB對子代成年大鼠體外培養(yǎng)的脾淋巴細胞增殖應(yīng)答的影響；研究子代成年大鼠細胞免疫對再次接受SEB刺激的免疫應(yīng)答反應(yīng)；研究子代成年大鼠外周血中IFN-γ及IL-4的水平及脾臟淋巴細胞中IFN-γ及IL-4基因的甲基化水平。其研究結(jié)果將為妊娠期接觸SEB對子代細胞免疫的影響提供實驗依據(jù)，也為探討一些胎源性疾病提供理論依據(jù)。 方法： 1．選用清潔級成年SD大鼠，交配及確定受孕后，孕鼠飼養(yǎng)至妊娠16天隨機分組，實驗組（SEB組）孕鼠通過尾靜脈注射15μg SEB，同時設(shè)立對照組，給予等體積的PBS（磷酸鹽緩沖液），繼續(xù)飼養(yǎng)孕鼠至自然分娩，讓子代新生鼠生長至成年時期，即獲得子代成年大鼠。 2．取子代成年大鼠胸腺、脾臟及外周血，制備單細胞懸液，熒光抗體（CD3FITC、CD8PE、CD4APC）染色后流式細胞儀檢測CD4/CD8T細胞。 3．取子代成年大鼠的胸腺及外周血，制備單細胞懸液，熒光抗體（Vβ8.2FITC、CD8PE、CD3APC）染色后流式細胞儀檢測Vβ8.2T細胞。 4．再次給予子代成年大鼠靜脈注射SEB，同時設(shè)立同窩子代大鼠注射SEB作為對照組，5天后獲取其胸腺、脾臟及外周血，分離其淋巴細胞，熒光抗體（CD3FITC、CD8PE、CD4APC）染色后流式細胞儀檢測CD4/CD8T細胞。 5．再次給予子代成年大鼠靜脈注射SEB，同時設(shè)立同窩的子代大鼠作為對照組，5天后取其胸腺及外周血，分離其淋巴細胞，染色后經(jīng)流式細胞儀檢測Vβ8.2T細胞。 6．無菌條件下取出子代成年大鼠的脾臟，分離淋巴細胞并進行體外培養(yǎng)，分別給予ConA及SEB連續(xù)刺激3天，每天取出培養(yǎng)的細胞檢測其增殖指數(shù)及其T細胞亞群。 7．獲取子代成年大鼠的血漿，ELISA檢測IFN-γ及IL-4的水平。 8．獲取子代成年大鼠的脾臟，通過MeDIP-qPCR檢測IL-4及IFN-γ基因的甲基化水平。 結(jié)果： 1．孕鼠接觸SEB對子代成年大鼠細胞免疫的影響（1）妊娠期接觸SEB對子代成年大鼠胸腺、脾臟及外周血中T細胞亞群的影響：SEB組子代成年雌雄性大鼠胸腺、脾臟及外周血中CD4+CD8T細胞的比例較PBS組明顯增加，而CD4CD8+T細胞的比例較PBS組明顯減少；（2）妊娠期接觸SEB對子代成年大鼠胸腺及外周血Vβ8.2T細胞的影響：妊娠期接觸SEB可明顯減少子代成年雌雄性大鼠胸腺及外周血中Vβ8.2T細胞的比例。 2．再次注射SEB對妊娠期已接觸SEB孕鼠的子代成年大鼠CD4/CD8T細胞的影響妊娠期已接觸SEB的子代成年雌雄性大鼠，與同窩對照組比較，再次接觸SEB時可明顯減少胸腺中CD4+CD8T細胞的比例，而增加CD4CD8+T細胞的比例；在子代成年雌雄性大鼠脾臟及外周血中T細胞亞群表現(xiàn)出與胸腺中相類似的變化。 3．再次注射SEB對妊娠期已接觸SEB的子代成年大鼠Vβ8.2T細胞的影響妊娠期已接觸SEB的子代成年雌雄性大鼠，再次注射SEB時對胸腺中Vβ8.2T細胞的比例與對照組比較無差異，但外周血中Vβ8.2T細胞的比例卻較同窩對照組明顯增加。 4．SEB刺激對體外培養(yǎng)的子代成年大鼠脾細胞的影響實驗發(fā)現(xiàn)隨著刺激時間增加體外培養(yǎng)的子代成年大鼠脾細胞的增殖能力逐漸增強，而且SEB組的子代成年雌雄性大鼠脾細胞接受SEB的刺激能力較ConA組弱；檢測T細胞亞群發(fā)現(xiàn)，，給予SEB刺激時SEB組培養(yǎng)的脾細胞中CD4+CD8T細胞的比例較ConA組減弱，但對CD4CD8+T細胞的比例無明顯影響。 5．妊娠期孕鼠接觸SEB對子代成年大鼠外周血中細胞因子的影響ELISA檢測結(jié)果發(fā)現(xiàn)SEB組子代成年大鼠外周血中IFN-γ及IL-4的水平分別較PBS組明顯增加；在成年期再次給予SEB刺激后，其（PBS+SEB，SEB+SEB）IFN-γ及IL-4水平均較各自對照組（PBS+PBS，SEB+PBS）明顯減少。 6．妊娠期孕鼠接觸SEB對子代成年大鼠DNA甲基化的影響實驗發(fā)現(xiàn)SEB組子代成年大鼠脾臟中IFN-γ及IL-4基因的甲基化水平均較PBS組明顯減少。 結(jié)論： 1．妊娠期接觸SEB改變了子代成年大鼠中樞及外周中CD4/CD8T細胞及Vβ8.2T細胞的比例； 2．妊娠期接觸SEB改變子代成年大鼠CD4/CD8T細胞及Vβ8.2T細胞對再次SEB刺激的免疫應(yīng)答反應(yīng)； 3.妊娠期接觸SEB減弱子代成年大鼠脾淋巴細胞體外的增殖能力，增殖能力減低可能與CD4+T細胞亞群減少有關(guān)； 4.妊娠期接觸SEB增加子代成年大鼠外周血中的細胞因子IL-4及IFN-γ水平，可能與其DNA甲基化減少有關(guān)。
[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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R714.25
本文編號：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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R714.25
【參考文獻】
相關(guān)期刊論文 前2條
1 管俊昌,夏佩瑩,唐素蘭;金黃色葡萄球菌L型產(chǎn)B型腸毒素及其基因的研究[J];蚌埠醫(yī)學(xué)院學(xué)報;2004年04期
2 劉從森;劉婷婷;孔曉明;朱翔;劉勇;閔宏林;管俊昌;;妊娠母鼠靜脈注射葡萄球菌腸毒素B對胎鼠胸腺發(fā)育的影響[J];蚌埠醫(yī)學(xué)院學(xué)報;2011年06期
本文編號：2168375
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