经水摄入三氯乙烯对小鼠调节性T细胞影响的研究

发布时间：2019-07-08 18:47

【摘要】：研究背景有机溶剂三氯乙烯（TCE）在工业上被广泛用作清洗剂和去脂剂。随着电子产业的迅猛发展，TCE的使用量逐年上升，已成为重要的职业毒物和广泛存在的环境污染物。除了引起明显的靶器官毒性外，研究表明TCE暴露与包括自身免疫在内的免疫相关效应明显相关，而受损的免疫系统有助于组织损伤的发生和发展。但TCE暴露引起的免疫性损伤机制目前尚未完全阐明。研究目的通过对饮水摄入TCE后小鼠脾脏Treg细胞的数量、特异性转录因子Foxp3mRNA和分泌的细胞因子IL-10检测，探讨TCE暴露对小鼠Treg细胞的影响；结合小鼠肝损伤和肝脏促炎细胞因子的检测，分析经水摄入TCE对小鼠调节性T细胞的影响与TCE暴露所致肝损伤的关系。研究方法选用6周龄雌性BALB/c小鼠饲养于清洁级动物房，染毒前适应性饲养1周，按随机数字表法分为空白对照组、溶剂对照组以及2.5mg/ml TCE组和5mg/ml TCE组，TCE经饮水染毒，分别在2、4、8、12周处死动物，采集外周血并无菌取肝脏和脾脏。采用流式细胞仪检测脾脏Treg细胞数量，荧光定量RT-PCR检测Treg细胞特异转录因子Foxp3mRNA表达水平，ELISA检测血清中Treg细胞分泌的IL-10含量；用生化分析法测定肝功能指标ALT和AST，取肝脏组织制成切片进行组织病理学观察，ELISA法检测肝脏组织匀浆液中促炎细胞因子IL-1、IL-6和TNF-α含量；用SPSS13.0统计软件包对实验结果进行统计分析。结果 1一般情况在整个实验期间各组小鼠状态良好，未出现个体因染毒而死亡；各组小鼠饮水量无明显差异(F=0.89)，平均饮水量为0.13ml/(g·d)，2.5mg/ml和5.0mg/ml TCE组小鼠平均暴露量分别为450mg/(kg·d)和730mg/(kg·d)。各时间点各组小鼠体重增长、肝脏和脾脏脏器系数差异均无统计学意义。 2饮水摄入TCE对小鼠Treg细胞的影响 2.1对脾脏Treg细胞数量的影响在2、4、8、12周，溶剂对照组Treg细胞数与空白对照组之间差异无统计学意义；在2、4周，TCE暴露组Treg细胞数量均低于空白对照组及溶剂对照组（P0.05）,且高剂量组比低剂量组下降趋势明显；在8、12周，TCE暴露组Treg细胞数量的下降有所恢复，只有8周时5.0mg/ml TCE组显著低于空白对照组（P0.05），其余组与对照组之间差异均无统计学意义。 2.2对脾脏Foxp3mRNA表达的影响在2、4、8、12周，溶剂对照组Foxp3mRNA表达与空白对照组之间差异无统计学意义；TCE暴露组Foxp3mRNA表达低于空白对照组及溶剂对照组（P0.05），且高剂量组比低剂量组下降趋势明显；TCE组Foxp3mRNA表达下调在4周时达最低，在8、12周有所回升，在12周时4组之间差异均无统计学意义。 2.3对血清中Treg细胞分泌的IL-10水平的影响在2、4、8、12周，溶剂对照组血清IL-10水平与空白对照组间差异均无统计学意义；在2、4周，TCE暴露组血清中IL-10水平均显著低于空白对照组及溶剂对照组（P0.05）；在8、12周，，TCE组IL-10水平与空白对照组及溶剂对照组相比差异均无统计学意义。 2.4饮水摄人TCE小鼠脾脏Treg细胞数量与Foxp3mRNA表达水平和血清中IL-10含量相关性相关分析显示小鼠脾脏Treg细胞数量与Foxp3mRNA表达水平和血清中IL-10含量均存在显著正相关（r=0.630，0.593，P0.01）。 3饮水摄入TCE对小鼠肝脏的损伤 3.1对肝功能的影响在2、4、8、12周，溶剂对照组小鼠ALT和AST水平与空白对照组比较差异均无统计学意义；在2、4周，TCE暴露组小鼠ALT和AST与空白和溶剂对照组比较明显升高（P0.05），在4周时达到最高，8和12周时有所下降，但均高于空白对照组和溶剂对照组。 3.2对肝脏组织病理学的影响空白对照组、溶剂对照组小鼠肝细胞排列整齐，胞质均匀，胞核大而圆，核仁明显，核膜清晰；TCE染毒组小鼠肝组织有明显的炎细胞浸润，在4周时最明显，8和12周时炎细胞浸润有所减少。 3.3对肝组织中促炎细胞因子的影响 3.3.1对肝脏IL-1含量的影响在2、4、8、12周，溶剂对照组小鼠肝脏IL-1水平与空白对照组比较差异均无统计学意义；在2、4周，5mg/ml TCE暴露组小鼠肝脏IL-1较空白对照组明显升高，4周时达到最高（P 0.05），8和12周时有所下降，但均高于空白对照和溶剂对照组。 3.3.2对肝脏IL-6含量的影响在2、4、8和12周，溶剂对照组小鼠肝脏IL-6水平与空白对照组比较差异均无统计学意义；与空白对照和溶剂对照组比较，5mg/ml TCE组在4和8周时明显升高，4周时最高（P 0.05）；2.5mg/ml TCE组在8周时明显升高（P 0.05）。 3.3.3对肝脏TNF-α含量的影响在2、4、8和12周，溶剂对照组与空白对照组之间差异均无统计学意义；在2、4、8周，TCE暴露组小鼠肝脏TNF-α含量均显著高于空白对照组及溶剂对照组，4周时达到最高（P0.05）。 3.3.4肝功能与肝脏IL-1、IL-6和TNF-α含量的相关性相关性分析显示肝功能ALT和AST，与肝脏中IL-1、IL-6和TNF-α含量之间存在显著正相关(r=0.526,0.510,0.614,0.518,0.525,0.630, P 0.01)。 4肝功能、肝脏IL-1、IL-6和TNF-α含量与Treg细胞数量的相关性相关性分析显示肝功能ALT和AST，肝脏IL-1、IL-6、TNF-α含量与Treg细胞数量之间存在显著负相关(r=0.723,0.632,0.525,0.419,0.567, P 0.01)。结论 1经水摄入TCE对小鼠调节性T细胞有显著影响，导致Treg细胞数量、特异性转录因子Foxp3mRNA表达和分泌的细胞因子IL-10表达水平显著降低。 2经水摄入TCE可导致小鼠肝脏损伤，ALT、AST以及肝脏组织中促炎细胞因子水平升高，并观察到炎细胞浸润。 3经水摄入TCE对小鼠调节性T细胞影响与其导致的肝脏损伤存在显著相关。
文内图片：

图片说明：不同时间不同组别小鼠脾脏Treg数量（%）（x±s,n=3）
[Abstract]:The research background organic solvent trichloroethylene (TCE) is widely used as a cleaning agent and a degreasing agent in the industry. With the rapid development of the electronic industry, the usage of TCE is increasing year by year, which has become an important occupational toxicant and a wide range of environmental pollutants. In addition to causing significant target organ toxicity, studies have shown that TCE exposure is significantly associated with immune-related effects, including autoimmune, and the compromised immune system contributes to the occurrence and development of tissue damage. However, the mechanism of immune injury caused by TCE exposure is not yet fully set forth. Objective To study the effect of TCE exposure on the mouse Treg cells by detecting the number of Tregs, the specific transcription factor Foxp3mRNA and the secreted cytokine IL-10 in the mouse spleen after drinking water. The effect of water intake TCE on regulatory T cells in mice and the effect of TCE on liver injury caused by TCE exposure The results showed that 6-week-old female BALB/ c mice were fed to clean-grade animal house. The first week after exposure, the control group was divided into the blank control group, the solvent control group and the 2.5 mg/ ml TCE group and the 5 mg/ ml TCE group, and the TCE was treated with drinking water, at 2,4,8 and 12 weeks, respectively. Dead animals, collect peripheral blood and aseptically take the liver And the expression level of the specific transcription factor Foxp3mRNA of the Treg cell was detected by the fluorescence quantitative RT-PCR, and the content of the IL-10 secreted by the Treg cells in the serum was detected by ELISA, and the ALT and the ALT of the liver function indexes were determined by the biochemical analysis method. The levels of IL-1, IL-6 and TNF-1 in the tissue homogenate of the liver were detected by the method of AST and liver tissue, and the results of the experiment were analyzed by using the SPSS13.0 statistical software package. score The average exposure of mice in each group was 450 mg/ (kg 路 d) and 730mg, respectively, and the mean water consumption was 0.13 ml/ (g 路 d), 2.5 mg/ ml and 5.0 mg/ ml TCE group. / (kg 路 d). Body weight gain, liver and spleen organ coefficient difference in each group of time points no statistical significance. Water intake TCE was small The effect of the effect of murine Treg cells on the number of Treg cells in the spleen was not statistically significant between 2,4,8,12 weeks, and the number of Treg cells in the solvent control group and the blank control group was not statistically significant; in 2,4 weeks, the number of Treg cells in the TCE exposed group was lower than that of the blank control group and the solution In the control group (P0.05), the descending trend of the high-dose group was more obvious than that of the low-dose group, and the decrease of the number of Treg cells in the TCE-exposed group was recovered at 8 and 12 weeks, and the TCE group was significantly lower than that in the control group at 8 weeks (P0.05), and the rest group and the control group There was no significant difference between the expression of Foxp3mRNA in the spleen and the control group, and the expression of Foxp3mRNA in the control group was not statistically significant. The expression of Foxp3mRNA in TCE exposed group was lower than that of the blank control group and the solvent control group (P0.05). The decrease of Foxp3mRNA in TCE group was lower than that of low-dose group, and the decrease of Foxp3mRNA in TCE group reached the lowest at 4 weeks, and then recovered at 8 and 12 weeks, at 12 weeks. The levels of IL-10 in the serum of the TCE exposed group were significantly lower than that of the blank control group and the solvent control group at 2,4,8 and 12 weeks. Group (P0.05); in 8,12 weeks, the level of IL-10 in TCE group was compared with the blank control group and the solution There was no statistical significance in the difference between the control group and the control group. The correlation between the number of the Tregs and the expression of Foxp3mRNA and the level of IL-10 in the serum of the TCE mouse in the drinking water showed significant positive correlation with the level of the expression of Foxp3mRNA and the level of IL-10 in the serum (r = 0). .630,0.593,P0.01 ). The effect of TCE on the liver of the mice was less than that of the control group in 2,4,8 and 12 weeks, and the levels of ALT and AST in the control group and the blank control group were not statistically significant; in the 2,4 and TCE groups, the ALT and AST of the mice were compared with those of the control group. The blank and solvent control group was significantly higher (P0.05) and reached the highest,8 and 12 weeks at 4 weeks. In the control group, the liver cells of the control group were orderly, the cytoplasm was uniform, the nucleus was large and the nucleolus was large and the nucleolus was obvious, and the nuclear membrane clear; the mice liver tissue of the TCE exposure group has obvious inflammatory cell infiltration, The most obvious,8 and 12-week inflammatory cells at 4 weeks The effect of 3.3.3 on the pro-inflammatory cytokines in the liver tissue was 2,4,8,12 weeks, and the liver IL of the mouse liver in the solvent control group. There was no statistical difference between the level of -1 and the blank control group; in the 2,4 and 5 mg/ ml TCE exposed group, the liver IL-1 was significantly higher than that in the blank control group and reached the highest at 4 weeks (P 0.05). The effect of 3.3.2 on the content of IL-6 in the liver was higher than that of the blank control group and the solvent control group. The effect of 3.3.2 on the content of IL-6 in the liver was not statistically significant between the control group and the blank control group at 2,4,8 and 12 weeks. The TCE group increased significantly at 4 and 8 weeks and the highest at 4 weeks (P 0.05 The effect of 3.3.3 on the content of TNF-1 in the liver was not statistically significant at 2,4,8 and 12 weeks, and the content of TNF-1 in the liver of the mice at 2,4,8 and TCE groups was not significant. The correlation between the liver function and the content of IL-1, IL-6 and TNF-1 in the liver was significantly higher than that in the blank control group and the solvent control group (P0.05). The correlation between the liver function and the content of IL-1, IL-6 and TNF-1 in the liver showed a significant positive correlation with the levels of IL-1, IL-6 and TNF-1 in the liver (r = 0.526, 0.5). The correlation between the contents of liver function, liver IL-1, IL-6 and TNF-1 and the number of Treg cells showed a significant negative correlation with the number of Treg cells (r = 0). .7 (23, 0.632, 0.525, 0.419, 0.567, P 0.01). Conclusion 1 Water intake TCE has a significant effect on the regulatory T cells of mice, resulting in the number of Treg cells. The expression of the specific transcription factor Foxp3mRNA and the expression of the secreted cytokines IL-10 decreased significantly. The levels of pro-inflammatory cytokines in the liver injury, ALT, AST, and liver tissues of the mice were elevated and observed.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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