【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)，又是葡萄球菌腸毒素家族9個(gè)血清型中最重要和研究較多的超抗原。SEB作為一種超抗原，無(wú)需經(jīng)過(guò)抗原遞呈細(xì)胞的識(shí)別和加工，就可以直接通過(guò)橋聯(lián)抗原遞呈細(xì)胞的MHCⅡ類分子結(jié)合槽外側(cè)區(qū)與T淋巴細(xì)胞TCR的Vβ區(qū)，刺激機(jī)體免疫系統(tǒng)大量的T細(xì)胞活化。通常SEB進(jìn)入機(jī)體后，初期可短暫激活Vβ8~+T細(xì)胞并誘發(fā)大量增殖，繼而過(guò)度增殖的細(xì)胞發(fā)生凋亡導(dǎo)致克隆清除，而出現(xiàn)中樞或外周耐受。盡管有許多關(guān)于SEB對(duì)成年動(dòng)物及新生鼠免疫器官、T淋巴細(xì)胞及細(xì)胞因子影響的研究，但妊娠期母鼠接觸SEB對(duì)子代新生鼠細(xì)胞免疫的影響，目前國(guó)內(nèi)外未見(jiàn)報(bào)道。因此，我們選用妊娠16天孕鼠尾靜脈注射15μg SEB，同時(shí)建立PBS對(duì)照，在子代新生鼠出生后第0-5天檢測(cè)胸腺、脾臟及外周血中CD4/CD8T細(xì)胞及TCR Vβ8~+T細(xì)胞亞群；通過(guò)新生鼠胸腺體外培養(yǎng)觀察ConA或SEB刺激后胸腺細(xì)胞的分化發(fā)育；通過(guò)脾臟淋巴細(xì)胞體外培養(yǎng)觀察其對(duì)ConA或SEB刺激的增殖應(yīng)答情況。 方法： 1.選用清潔級(jí)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(jī)分組，每組12只，實(shí)驗(yàn)組（SEB組）孕鼠通過(guò)尾靜脈注射15μg的SEB，同時(shí)設(shè)立對(duì)照組，給予等體積的PBS（磷酸鹽緩沖液）。 2.在妊娠期滿（21天）時(shí)，用4%水合氯醛麻醉孕鼠后打開腹腔和子宮，取出胎鼠并分離胸腺和脾臟，稱量胎體、胎盤、胸腺及脾臟的干濕重。 3.在新生鼠出生后的第0-5天，獲取每組新生鼠的胸腺、脾臟和外周血，用網(wǎng)搓法制備胸腺及脾臟的單細(xì)胞懸液，脾臟的單細(xì)胞懸液及外周血用紅細(xì)胞裂解液去除紅細(xì)胞。將獲得的細(xì)胞懸液分為兩份，一份用熒光抗體CD3-FITC、CD4-APC和CD8-PE進(jìn)行細(xì)胞染色，另一份用Vβ8.2-FITC、CD4-APC和CD8-PE進(jìn)行染色，染色后經(jīng)流式細(xì)胞儀檢測(cè)T細(xì)胞亞群的比例。 4.無(wú)菌分離出生后第0天的兩組新生鼠胸腺，采用胸腺器官培養(yǎng)法（thymus organculture，TOC）進(jìn)行體外培養(yǎng)，并加入ConA或SEB共培養(yǎng)三天。在培養(yǎng)結(jié)束時(shí)收集胸腺并制備單細(xì)胞懸液，染色后經(jīng)流式細(xì)胞儀檢測(cè)胸腺中T細(xì)胞亞群的比例。 5.無(wú)菌分離出生后第3天的兩組新生鼠脾臟，用淋巴細(xì)胞分離液分離脾臟淋巴細(xì)胞，體外與ConA或SEB連續(xù)共培養(yǎng)三天。在第1、2、3天培養(yǎng)結(jié)束時(shí)，收集脾臟淋巴細(xì)胞，染色后經(jīng)流式細(xì)胞儀檢測(cè)脾臟中T細(xì)胞亞群的比例。若檢測(cè)脾臟淋巴細(xì)胞增殖能力時(shí)，在每一時(shí)間點(diǎn)結(jié)束前5小時(shí)加入3H-TDR共培養(yǎng)至結(jié)束，然后通過(guò)γ計(jì)數(shù)儀檢測(cè)rpm. 結(jié)果： 1.妊娠期母鼠接觸SEB后對(duì)胎鼠體重等指標(biāo)的影響 妊娠期第16天母鼠靜脈注射SEB后，可明顯減少胎鼠胸腺及脾臟的濕重和干重(P0.01)；但與PBS組比較，SEB組的胎體及胎盤的干、濕重?zé)o統(tǒng)計(jì)學(xué)差異。2.妊娠期母鼠接觸SEB對(duì)新生鼠中樞及外周CD4/CD8T細(xì)胞亞群的影響 2.1妊娠期母鼠接觸SEB對(duì)新生鼠胸腺中CD4/CD8T細(xì)胞亞群的影響 妊娠期第16天母鼠接觸SEB后，在新生鼠出生后的第0-5天，SEB組新生鼠胸腺中CD4~+T細(xì)胞的比例明顯高于PBS組(P0.01或P0.05)，而SEB組CD8~+T細(xì)胞的比例明顯低于PBS組的(P0.01或P0.05)。 2.2妊娠期母鼠接觸SEB對(duì)新生鼠脾臟中CD4/CD8T細(xì)胞亞群的影響 在新生鼠出生后第0-5天，SEB組新生鼠脾臟中CD4~+T細(xì)胞的比例在各時(shí)間點(diǎn)均明顯高于PBS組(P0.01或P0.05)。但兩組在各時(shí)間點(diǎn)的CD8~+T細(xì)胞比例無(wú)統(tǒng)計(jì)學(xué)差異。 2.3妊娠期母鼠接觸SEB對(duì)新生鼠外周血中CD4/CD8T細(xì)胞亞群的影響 在新生鼠出生后第0-5天，PBS組和SEB組新生鼠外周血CD4~+T細(xì)胞與CD8~+T細(xì)胞亞群比例的總體變化趨勢(shì)與脾臟中的相類似。兩組外周血中CD4/CD8T細(xì)胞比值均呈上升趨勢(shì)，從第0-1天的比值小于1逐漸增加到之后的大于1，但SEB組在各時(shí)間點(diǎn)的CD4/CD8T細(xì)胞的比值均明顯高于PBS組(P0.01或P0.05)。 3.妊娠期母鼠接觸SEB對(duì)新生鼠中樞及外周TCR Vβ8.2~+T細(xì)胞的影響 妊娠期第16天母鼠接觸SEB后，在新生鼠出生后的第0-3天，SEB組新生鼠胸腺中的CD4~+TCR Vβ8.2~+T細(xì)胞比例較PBS組的明顯減少(P0.01或P0.05)，在第4-5天兩組的CD4~+TCR Vβ8.2~+T細(xì)胞比例趨于平穩(wěn)，且無(wú)統(tǒng)計(jì)學(xué)差異；而在出生后的第0-5天，SEB組新生鼠胸腺及外周血中CD8~+TCR Vβ8.2~+T細(xì)胞比例及外周血中的CD4~+TCR Vβ8.2~+T細(xì)胞比例均較PBS組的明顯減少(P0.01或P0.05)。 4.妊娠期母鼠接觸SEB對(duì)體外培養(yǎng)新生鼠胸腺分化發(fā)育的影響 4.1妊娠期母鼠接觸SEB對(duì)體外培養(yǎng)新生鼠胸腺中CD4/CD8T細(xì)胞亞群的影響 PBS組和SEB組新生鼠胸腺體外與ConA或SEB共培養(yǎng)三天后，SEB組新生鼠胸腺中CD4~+T細(xì)胞及CD8~+T細(xì)胞的比例在ConA（SEB~+ConA組）或SEB（SEB~+SEB組）刺激后均較PBS組（PBS~+ConA組；PBS~+SEB組）的明顯減少(P0.01或P0.05)；而SEB組CD4~+CD8~+T細(xì)胞的比例較PBS組明顯增加(P0.01或P0.05)，但胸腺中CD4-CD8-T細(xì)胞的比例在兩組間無(wú)統(tǒng)計(jì)學(xué)差異。 4.2妊娠期母鼠接觸SEB對(duì)體外培養(yǎng)新生鼠胸腺中TCR Vβ8.2~+T細(xì)胞的影響 PBS組和SEB組新生鼠胸腺體外與ConA或SEB共培養(yǎng)三天后，SEB組的CD4~+TCRVβ8.2~+T細(xì)胞和CD8~+TCR Vβ8.2~+T細(xì)胞比例均較PBS組明顯減少(P0.01或P0.05)。 5.妊娠期母鼠接觸SEB對(duì)新生鼠脾臟淋巴細(xì)胞體外增殖的影響 5.1妊娠期母鼠接觸SEB對(duì)新生鼠脾臟T淋巴細(xì)胞亞群增殖的影響 PBS組和SEB組新生鼠脾臟淋巴細(xì)胞體外與ConA或SEB共培養(yǎng)三天后，SEB組CD4~+T細(xì)胞比例在ConA或SEB刺激第一天分別較PBS組明顯增加，但在第2-3天卻明顯降低(P0.01或P0.05)，但兩組的CD8~+T細(xì)胞比例無(wú)統(tǒng)計(jì)學(xué)差異。而SEB組新生鼠脾臟淋巴細(xì)胞中CD4~+TCR Vβ8.2~+T細(xì)胞比例在ConA或SEB刺激的第1天及CD8~+TCR Vβ8.2~+T細(xì)胞比例在連續(xù)刺激三天分別較PBS組的明顯增加(P0.01或P0.05)，但兩組CD4~+TCR Vβ8.2~+T細(xì)胞比例在ConA或SEB刺激的第2-3天無(wú)差異性。 5.2妊娠期母鼠接觸SEB對(duì)新生鼠脾臟淋巴細(xì)胞增殖指數(shù)的影響 PBS組和SEB組新生鼠脾臟淋巴細(xì)胞體外與ConA或SEB共培養(yǎng)三天后，，3H摻入法檢測(cè)結(jié)果發(fā)現(xiàn)SEB組新生鼠脾臟淋巴細(xì)胞在ConA或SEB刺激后第1天增殖能力明顯強(qiáng)于PBS組，但在第2-3天的增殖能力卻又明顯低于PBS組(P0.01或P0.05)。 結(jié)論： 1.妊娠期母鼠接觸SEB可明顯影響胎鼠胸腺及脾臟的發(fā)育； 2.妊娠期母鼠接觸SEB可通過(guò)中樞和外周兩種克隆清除方式導(dǎo)致新生鼠TCRVβ8.2~+T細(xì)胞比例減少，但SEB并非僅僅作用于TCR Vβ8.2~+T細(xì)胞，可能對(duì)其它的T淋巴細(xì)胞也產(chǎn)生影響； 3.在受到抗原刺激時(shí)，妊娠期母鼠接觸SEB并不影響新生鼠胸腺中DN細(xì)胞向DP細(xì)胞的發(fā)育過(guò)程，但明顯減少DP細(xì)胞向SP細(xì)胞的成熟過(guò)程； 4. CD4~+T細(xì)胞可能是妊娠期接觸SEB母鼠的新生鼠脾臟淋巴細(xì)胞增殖的主要細(xì)胞群體； 5.妊娠期母鼠接觸SEB已在新生兒期對(duì)新生鼠CD4~+T細(xì)胞、CD8~+T細(xì)胞以及TCRVβ8.2~+T細(xì)胞形成印跡效應(yīng)，可能成為胎源性疾病的起源。
[Abstract]:Objective:
Staphylococcal enterotoxin B (SEB) is not only a toxic substance, but also the most important and widely studied superantigen in the nine serotypes of the staphylococcal enterotoxin family. As a superantigen, SEB can directly present cells by bridging antigen without recognising and processing antigen presenting cells. MHC class II molecules bind to the V beta region of the T lymphocyte TCR to stimulate the activation of a large number of T cells in the body's immune system. Usually SEB enters the body, initially, it can activate V beta 8~+ T cells and induce a large number of proliferation, and then apoptosis of overproliferated cells leads to clonal clearance, resulting in central or peripheral tolerance. The effect of SEB on immune organs, T lymphocytes and cytokines of adult and newborn mice has not been reported at home and abroad, but the effect of maternal exposure to SEB during pregnancy on cellular immunity of offspring newborn mice has not been reported at present. CD4/CD8T cells and TCR V-beta 8~+ T cell subsets in thymus, spleen and peripheral blood were detected at day 0-5, and the differentiation and development of thymocytes stimulated by ConA or SEB were observed in vitro in neonatal rats.
Method:
1. Choose clean adult SD rats, after mating and definite conception, the pregnant rats were fed to 16 days of gestation and divided into 12 groups randomly. The pregnant rats in the experimental group (SEB group) were injected with 15 UG SEB through the caudal vein, and the control group was set up to give the same volume of PBS (phosphate buffer).
2. At the end of pregnancy (21 days), the pregnant rats were anesthetized with 4% chloral hydrate, then the abdominal cavity and uterus were opened, the fetal rats were removed and the thymus and spleen were separated. The dry and wet weight of the fetus, placenta, thymus and spleen were weighed.
3. The thymus, spleen and peripheral blood of each group of newborn rats were obtained on the 0th to 5th day after birth. The single cell suspension of thymus and spleen was prepared by mesh rubbing method. The single cell suspension of spleen and peripheral blood were removed by erythrocyte lysate. The cell suspension was divided into two parts, one with fluorescent antibody CD3-FITC, CD4-APC and CD8-PE. Cell staining was performed, and the other was stained with V-beta 8.2-FITC, CD4-APC and CD8-PE. The ratio of T cell subsets was detected by flow cytometry.
4. The thymus of two groups of newborn mice were cultured in vitro by thymus organ culture (TOC) and added ConA or SEB for three days. At the end of culture, the thymus was collected and the single cell suspension was prepared. The ratio of T cell subsets in the thymus was detected by flow cytometry after staining.
5. Splenic lymphocytes were isolated from the spleens of two groups of newborn mice on the third day after birth and co-cultured with ConA or SEB for three consecutive days in vitro. At the end of the first, second and third days of culture, splenic lymphocytes were collected and stained, and the proportion of T cell subsets in the spleen was detected by flow cytometry. At the end of each time point, 3H-TDR was added 5 hours before the end of each time point, and then rpm was detected by gamma counter.
Result:
1. the influence of SEB on weight of fetal rats during pregnancy.
The wet weight and dry weight of thymus and spleen were significantly reduced after intravenous injection of SEB on the 16th day of gestation (P 0.01), but there was no significant difference between SEB group and PBS group.
2.1 the effect of SEB exposure on CD4/CD8T cell subsets in thymus of neonatal rats during gestation period
The proportion of CD4~+ T cells in the thymus of SEB group was significantly higher than that of PBS group (P 0.01 or P 0.05) on the day 0-5 after birth, and that of CD8~+ T cells in SEB group was significantly lower than that of PBS group (P 0.01 or P 0.05).
2.2 the effect of SEB exposure on CD4/CD8T cell subsets in spleen of neonatal rats during gestation period
The ratio of CD4~+T cells in spleen of SEB group was significantly higher than that of PBS group at each time point (P 0.01 or P 0.05) at 0-5 days after birth, but there was no significant difference between the two groups at each time point.
2.3 the effect of SEB exposure on CD4/CD8T cell subsets in peripheral blood of neonatal rats during gestation period
The percentage of CD4~+ T cells in peripheral blood and CD8~+ T cells in the PBS and SEB groups was similar to that in the spleen at day 0-5 after birth. The ratio of 8T cells was significantly higher than that of group PBS (P0.01 or P0.05).
3. the effect of SEB exposure on TCR and V 8.2~+T cells in neonatal rats during pregnancy
The proportion of CD4~+TCR V beta 8.2~+ T cells in the thymus of SEB group was significantly lower than that of PBS group (P 0.01 or P 0.05) on day 16 of gestation, and the proportion of CD4~+TCR V beta 8.2~+ T cells in the thymus of SEB group tended to be stable on day 4-5 of gestation, and there was no statistical difference between the two groups. The proportion of CD8~+TCR V beta 8.2~+ T cells in thymus and peripheral blood and the proportion of CD4~+TCR V beta 8.2~+ T cells in peripheral blood were significantly lower than those in PBS group (P 0.01 or P 0.05).
4. the effect of SEB exposure on the differentiation and development of thymus in neonatal rats in gestation period
4.1 the effect of SEB exposure on CD4/CD8T cell subsets in neonatal rat thymus during pregnancy
Three days after co-culture with ConA or SEB in vitro, the proportion of CD4~+ T cells and CD8~+ T cells in the thymus of PBS group and SEB group was significantly lower than that of PBS group (PBS + ConA group; PBS + SEB group) after stimulation by ConA (SEB + ConA group) or SEB (SEB + SEB group). Significantly increased (P0.01 or P0.05), but the proportion of CD4-CD8-T cells in thymus was not significantly different between the two groups.
4.2 the effect of SEB exposure on TCR V beta 8.2~+T cells in the thymus of neonatal rats in gestation period
Three days after co-culture with ConA or SEB in vitro, the proportion of CD4~+TCRV beta 8.2~+ T cells and CD8~+TCR V beta 8.2~+ T cells in SEB group were significantly lower than that in PBS group (P 0.01 or P 0.05).
5. the effect of SEB exposure on the proliferation of splenic lymphocytes in neonatal rats in gestation period
5.1 the effect of SEB exposure on the proliferation of T lymphocyte subsets in spleen of neonatal rats during gestation period
Three days after co-culture with ConA or SEB in vitro, the ratio of CD4~+ T cells in the spleen lymphocytes of PBS and SEB groups increased significantly on the first day of ConA or SEB stimulation, but decreased significantly on the second and third days (P 0.01 or P 0.05), but there was no significant difference in the ratio of CD8~+ T cells between the two groups. The ratio of TCR V-beta 8.2~+ T cells on the first day of ConA or SEB stimulation and the ratio of CD8~+TCR V-beta 8.2~+ T cells on the third day of continuous stimulation were significantly higher than that of PBS group (P 0.01 or P 0.05), but the ratio of CD4~+TCR V-beta 8.2~+ T cells on the second and third days of ConA or SEB stimulation had no difference.
5.2 the effect of SEB exposure on the proliferation index of spleen lymphocytes in neonatal rats during gestation period
Three days after co-culture with ConA or SEB in vitro, 3H incorporation assay showed that the proliferation of splenic lymphocytes in SEB group was significantly stronger than that in PBS group on the first day after ConA or SEB stimulation, but significantly lower than that in PBS group on the second and third days (P 0.01 or P 0.05).
Conclusion:
1. exposure to SEB in pregnant rats can significantly affect the development of thymus and spleen in fetal rats.
2. Exposure to SEB during pregnancy can reduce the proportion of TCRV-beta 8.2~+ T cells in neonatal rats through central and peripheral clonal clearance. However, SEB does not only affect TCR-V-beta 8.2~+ T cells, but may also affect other T lymphocytes.
3. Exposure to SEB during pregnancy did not affect the development of DN cells into DP cells, but significantly reduced the maturation of DP cells into SP cells.
4. CD4~+T cells may be the main cell population of splenic lymphocyte proliferation in neonatal rats exposed to SEB during pregnancy.
5. The imprinting effect of SEB exposure on neonatal rat CD4~+ T cells, CD8~+ T cells and TCRV beta 8.2~+ T cells may be the origin of fetal diseases.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R378.11

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相關(guān)期刊論文 前2條

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2 管俊昌,劉勇,徐飛,夏佩瑩;金葡菌L型對(duì)小鼠體外胚胎發(fā)育的影響[J];中國(guó)人獸共患病雜志;2004年11期

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