围生期双酚A暴露对仔鼠调节性T细胞的影响

发布时间：2018-07-06 17:21

本文选题：双酚A + 调节性T细胞　；参考：《安徽医科大学》2013年硕士论文

【摘要】：研究背景双酚A（bispheno1A，BPA）是生产碳酸聚酯、塑料制品等工业产品的原材料，用于奶瓶、可重复用水杯、牙密封剂、食品与饮料的包装材料、水管、热敏纸、卡板纸及其他产品的添加剂等制造。据报道，2008年BPA一年的产量高达80亿磅，而且以每年6%~10%的速度增长。BPA在高温、聚合不完全、长期使用等情况下通过酯键水解而释放，导致人群及动物的慢性低剂量暴露。 BPA具有拟雌激素作用，通过干扰雌激素活性等途径对人体多个系统如内分泌、生殖、发育等产生有害作用，而孕期及生命早期（包括婴幼儿期、青春期等）暴露BPA，可能与发育期免疫功能异常及相关疾病有关，这引起了广泛的关注。流行病学研究提示，生命早期暴露如BPA等内分泌干扰物可能是过去几十年发达国家儿童哮喘、过敏性皮炎、糖尿病、儿童肿瘤发病率上升的原因之一。动物实验对围生期或成年期BPA等内分泌干扰物暴露诱导的免疫毒性的研究越趋增多，但至今BPA暴露引起的免疫发育毒性机制尚未完全阐明。Treg细胞对机体发挥维持免疫稳态和免疫负性调控的关键作用，有报道称成年期BPA暴露能抑制Treg细胞数量，使T/B细胞及巨噬细胞等应答过度，导致免疫功能异常及免疫性疾病。本课题组假设母鼠孕期及哺乳期BPA暴露，诱导的仔鼠免疫功能的异常与失衡，可能与BPA抑制Treg细胞数量这一途径有关。研究目的通过检测孕期和哺乳期经水暴露不同剂量BPA母鼠所生仔鼠断乳期及青春期脾脏Treg细胞数量、特征性转录因子Foxp3mRNA表达水平及血清中IL-10、TGF-β含量，结合仔鼠生长发育状况，，探讨围生期BPA暴露对子代Treg细胞的影响。研究方法选用SPF级8周龄ICR雌鼠、9周龄ICR雄鼠；适应性饲养1周，按照雌：雄比例2:1合笼交配，雌鼠查到阴栓确认为妊娠，将孕鼠随机分为空白对照组、溶剂对照组（DMSO）、BPA低中高剂量组（2.2829、22.829、228.29μg/L）。母鼠自妊娠开始日（GD0）至仔鼠断乳（PND21）经饮水暴露BPA，PND21与PND42处死仔鼠，收集外周血并取脾脏；流式细胞仪检测仔鼠脾脏Treg细胞数量；荧光定量RT-PCR检测脾脏Treg细胞特征性转录因子Foxp3mRNA表达水平；ELISA检测血清中IL-10、TGF-β含量；使用SPSS15.0统计软件分析实验结果。结果 1一般情况母鼠孕期状况良好，未出现死亡、流产、死胎、死产等情况；组间饮水量差异无统计学意义，约为0.12ml/（g·d）；母鼠孕期体重差异无统计学意义。仔鼠出生性别比及平均窝仔数差异均无统计学意义；仅PND42时高剂量组仔鼠体重较空白对照组有显著增加（P0.05），其余各时点各组间仔鼠体重差异均无统计学意义。 2围生期BPA暴露对仔鼠Treg细胞的影响 2.1对仔鼠脾脏Treg细胞数量的影响围生期BPA暴露后仔鼠脾脏Treg细胞数量随母鼠BPA暴露剂量的增加而呈下降趋势。PND21时中、高剂量组Treg细胞数量与对照组相比明显降低，差异有统计学意义（P0.05，P0.01），而低剂量组则无明显差异；PND42时各组间Treg细胞数量均无明显差异，BPA暴露组仔鼠Treg细胞数量与对照组相比则表现为轻微下降，但与PND21相比，BPA暴露组Treg细胞数量均所有回升。 2.2对仔鼠脾脏Foxp3mRNA表达的影响暴露组仔鼠Foxp3mRNA表达水平随母鼠BPA暴露剂量的增加而呈下降趋势。PND21时中、高剂量组Foxp3mRNA表达水平与对照组相比有明显降低（P0.05，P0.01），而低剂量组则无明显差异；PND42时各组间Foxp3mRNA表达水平相比均无明显差异，与对照组相比，BPA暴露组表现为轻微下降，但与PND21时相比，BPA暴露组Foxp3mRNA表达水平均所有回升。 3对Treg细胞相关细胞因子的影响 3.1对仔鼠血清IL-10水平的影响PND21时高中低剂量组仔鼠血清IL-10含量随剂量增加而逐渐下降，高剂量组IL-10含量与对照组相比有显著降低（P0.05），低、中剂量组与对照组相比虽有降低但差异均无统计学意义；PND42时各组仔鼠血清IL-10含量均无显著差异，暴露组与对照组相比均有减少，与PND21相比则均有所回升。 3.2对血清TGF-β水平的影响PND21时高中低剂量组仔鼠血清TGF-β含量随剂量增加而逐渐上升，高、中剂量组TGF-β含量与对照组相比有显著增加（P0.05，P0.01），低剂量组与对照组相比差异无统计学意义；PND42时低、中、高剂量组与对照组相比呈逐渐增加，但差异无统计学意义，与PND21相比则5组均有下降，而暴露组下降幅度更为明显。 4仔鼠血清IL-10、TGF-β水平与Treg细胞数量的相关性相关性分析显示仔鼠血清IL-10含量与Treg细胞数量之间存在不显著正相关；而TGF-β含量与Treg细胞数量之间存在显著负相关。结论 1围生期BPA暴露对孕鼠、断乳前仔鼠体重无明显影响。 2围生期暴露BPA降低仔鼠断乳期Treg细胞数量及Foxp3mRNA表达水平。 3围生期暴露BPA对Treg细胞相关细胞因子有影响：降低血清中IL-10含量，增加血清中TGF-β含量。 4围生期暴露BPA，仔鼠Treg细胞数量的减少与相关细胞因子含量存在相关。
[Abstract]:Research background bisphenol A (bispheno1A, BPA) is the raw material for the production of carbonated polyesters, plastic products and other industrial products, used in bottles, reproducible cups, dental sealants, food and beverage packaging materials, water pipes, thermosensitive paper, cardboard and other products additives. It is reported that in 2008, the output of BPA was as high as 8 billion pounds, and per year. The rate of 6%~10% increased in.BPA at high temperature, incomplete polymerization, and was released by hydrolysis of ester bonds in the case of long term use, resulting in chronic low dose exposure to people and animals.
BPA has the effect of estrogenic activity by interfering with the activity of estrogen, such as endocrine, reproduction, and development, and so on. The exposure to BPA in pregnancy and early life (including infancy, puberty, etc.) may be associated with abnormal immune function and related diseases in the development period, which has aroused widespread concern. Studies have suggested that early exposure to endocrine disruptors, such as BPA, may be one of the reasons for the increase in the incidence of childhood asthma, allergic dermatitis, diabetes, and children's tumours in developed countries for the past few decades. Animal experiments have increased the immunological toxicity induced by endocrine disruptors such as perinatal or adult BPA, but up to now, BPA The mechanism of Immunogenicity Induced by exposure has not yet fully elucidated the key role of.Treg cells in maintaining immune homeostasis and negative immunological regulation. It is reported that adult BPA exposure can inhibit the number of Treg cells and excessively respond to T/B cells and macrophages and lead to immune dysfunction and immune diseases. The abnormal and unbalanced immune function induced by BPA exposure during pregnancy and lactation may be related to the inhibition of the number of Treg cells by BPA.
Objective to investigate the effect of perinatal BPA exposure on the progeny Treg cells by detecting the weaning period and the number of Treg cells in puberty spleens, the expression level of the characteristic transcription factor Foxp3mRNA and the content of IL-10, TGF- beta in the serum, and the growth and development of the offspring of BPA mice.
The study methods were SPF 8 weeks old ICR female rats and 9 weeks old ICR male rats. The female rats were bred for 1 weeks. The female rats were copulated with the male proportion of 2:1 cage. The female rats were found to be pregnant, and the pregnant rats were randomly divided into the blank control group, the solvent control group (DMSO), the low and high dose group of BPA (2.2829,22.829228.29 mu g/L). The female rats were from the beginning of pregnancy (GD0) to the offspring. PND21 was exposed to BPA by drinking water, PND21 and PND42 were sacrificed to kill the offspring, collect the peripheral blood and take the spleen; flow cytometry was used to detect the number of Treg cells in the spleen of the offspring of the offspring; the fluorescence quantitative RT-PCR was used to detect the Foxp3mRNA expression level of the characteristic transcription factors of the spleen Treg cells; ELISA detected IL-10, TGF- beta content in serum; and the analysis of the SPSS15.0 statistical software was used. Experimental results.
Result
1 normal female mice were in good condition during pregnancy. There was no death, abortion, stillbirth, and stillbirth. There was no statistical difference between the group and 0.12ml/ (G. D). There was no statistical significance in the weight difference between the female rats during pregnancy and the difference of the sex ratio and the average litter size of the offspring. The weight of the high dose group at PND42 was higher than that of the high dose group. There was a significant increase in the blank control group (P0.05).
The effect of 2 perinatal exposure to BPA exposure on Treg cells in offspring rats
2.1 effect on the number of Treg cells in spleens of the offspring rats, the number of Treg cells in the spleen of the offspring was decreased with the increase of BPA exposure dose after perinatal exposure, and the number of Treg cells in the high dose group was significantly lower than that of the control group (P0.05, P0.01), but there was no significant difference between the low dose group and the low dose group, while PND42 in the low dose group was not significantly different. There was no significant difference in the number of Treg cells in each group. The number of Treg cells in the BPA exposure group was slightly lower than that in the control group, but the number of Treg cells in the BPA exposed group all increased all compared with the PND21.
The effect of 2.2 on the expression of Foxp3mRNA in the spleens of the offspring rats, the expression level of Foxp3mRNA in the exposed group was decreased with the increase of BPA exposure dose in.PND21, and the Foxp3mRNA expression level in the high dose group was significantly lower than that in the control group (P0.05, P0.01), but there was no significant difference between the low dose group and the low dose group, and the Foxp3mRNA expression level between each group at PND42. There was no significant difference between the two groups. Compared with the control group, the BPA exposure group showed a slight decrease, but compared with the PND21 group, the expression level of Foxp3mRNA in the BPA exposure group all recovered.
The effect of 3 on Treg cell related cytokines
The effect of 3.1 on the serum IL-10 level of young mice PND21 decreased with the increase of dose in the low dose group of high and low dose group, and the content of IL-10 in high dose group was significantly lower than that of the control group (P0.05). The low dose group was lower than the control group, but the difference was not statistically significant compared with the control group, and the serum IL-10 content of each group in each group at PND42. There was no significant difference between the exposed group and the control group, and all of them rose again compared with PND21.
3.2 when the serum TGF- beta level was affected by PND21, the serum level of TGF- beta in the low dose group of high and low dose group increased with the increase of dose, and the content of TGF- beta in the middle dose group was significantly higher than that of the control group (P0.05, P0.01). The low dose group had no significant difference compared with the control group; the low dose group was lower than the control group, and the high dose group was compared with the control group. Gradually increased, but the difference was not statistically significant, compared with PND21, the 5 groups decreased, while the exposure group decreased more significantly.
The correlation analysis of the serum IL-10, TGF- beta level and the number of Treg cells in the sera of 4 offspring showed that there was no significant positive correlation between the serum IL-10 content and the number of Treg cells in the offspring, but there was a significant negative correlation between the content of TGF- beta and the number of Treg cells.
conclusion
1 the BPA exposure during perinatal period had no significant effect on the weight of pregnant rats before weaning.
2 exposure to BPA during perinatal period reduced the number of Treg cells and Foxp3mRNA expression in weaned rats.
3 exposure to BPA during perinatal period has an effect on Treg cell related cytokines: decreasing IL-10 content in serum and increasing TGF- beta content in serum.
4 during perinatal exposure to BPA, the reduction of Treg cell number in offspring rats was related to the content of related cytokines.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【共引文献】