經(jīng)水?dāng)z入三氯乙烯對小鼠調(diào)節(jié)性T細(xì)胞影響的研究
發(fā)布時間：2019-07-08 18:47
【摘要】：研究背景有機(jī)溶劑三氯乙烯（TCE）在工業(yè)上被廣泛用作清洗劑和去脂劑。隨著電子產(chǎn)業(yè)的迅猛發(fā)展，TCE的使用量逐年上升，已成為重要的職業(yè)毒物和廣泛存在的環(huán)境污染物。除了引起明顯的靶器官毒性外，研究表明TCE暴露與包括自身免疫在內(nèi)的免疫相關(guān)效應(yīng)明顯相關(guān)，而受損的免疫系統(tǒng)有助于組織損傷的發(fā)生和發(fā)展。但TCE暴露引起的免疫性損傷機(jī)制目前尚未完全闡明。 研究目的通過對飲水?dāng)z入TCE后小鼠脾臟Treg細(xì)胞的數(shù)量、特異性轉(zhuǎn)錄因子Foxp3mRNA和分泌的細(xì)胞因子IL-10檢測，探討TCE暴露對小鼠Treg細(xì)胞的影響；結(jié)合小鼠肝損傷和肝臟促炎細(xì)胞因子的檢測，分析經(jīng)水?dāng)z入TCE對小鼠調(diào)節(jié)性T細(xì)胞的影響與TCE暴露所致肝損傷的關(guān)系。 研究方法選用6周齡雌性BALB/c小鼠飼養(yǎng)于清潔級動物房，染毒前適應(yīng)性飼養(yǎng)1周，按隨機(jī)數(shù)字表法分為空白對照組、溶劑對照組以及2.5mg/ml TCE組和5mg/ml TCE組，TCE經(jīng)飲水染毒，分別在2、4、8、12周處死動物，采集外周血并無菌取肝臟和脾臟。采用流式細(xì)胞儀檢測脾臟Treg細(xì)胞數(shù)量，熒光定量RT-PCR檢測Treg細(xì)胞特異轉(zhuǎn)錄因子Foxp3mRNA表達(dá)水平，ELISA檢測血清中Treg細(xì)胞分泌的IL-10含量；用生化分析法測定肝功能指標(biāo)ALT和AST，取肝臟組織制成切片進(jìn)行組織病理學(xué)觀察，ELISA法檢測肝臟組織勻漿液中促炎細(xì)胞因子IL-1、IL-6和TNF-α含量；用SPSS13.0統(tǒng)計軟件包對實驗結(jié)果進(jìn)行統(tǒng)計分析。 結(jié)果 1一般情況在整個實驗期間各組小鼠狀態(tài)良好，未出現(xiàn)個體因染毒而死亡；各組小鼠飲水量無明顯差異(F=0.89)，平均飲水量為0.13ml/(g·d)，2.5mg/ml和5.0mg/ml TCE組小鼠平均暴露量分別為450mg/(kg·d)和730mg/(kg·d)。各時間點(diǎn)各組小鼠體重增長、肝臟和脾臟臟器系數(shù)差異均無統(tǒng)計學(xué)意義。 2飲水?dāng)z入TCE對小鼠Treg細(xì)胞的影響 2.1對脾臟Treg細(xì)胞數(shù)量的影響在2、4、8、12周，溶劑對照組Treg細(xì)胞數(shù)與空白對照組之間差異無統(tǒng)計學(xué)意義；在2、4周，TCE暴露組Treg細(xì)胞數(shù)量均低于空白對照組及溶劑對照組（P0.05）,且高劑量組比低劑量組下降趨勢明顯；在8、12周，TCE暴露組Treg細(xì)胞數(shù)量的下降有所恢復(fù)，只有8周時5.0mg/ml TCE組顯著低于空白對照組（P0.05），其余組與對照組之間差異均無統(tǒng)計學(xué)意義。 2.2對脾臟Foxp3mRNA表達(dá)的影響在2、4、8、12周，溶劑對照組Foxp3mRNA表達(dá)與空白對照組之間差異無統(tǒng)計學(xué)意義；TCE暴露組Foxp3mRNA表達(dá)低于空白對照組及溶劑對照組（P0.05），且高劑量組比低劑量組下降趨勢明顯；TCE組Foxp3mRNA表達(dá)下調(diào)在4周時達(dá)最低，在8、12周有所回升，在12周時4組之間差異均無統(tǒng)計學(xué)意義。 2.3對血清中Treg細(xì)胞分泌的IL-10水平的影響在2、4、8、12周，溶劑對照組血清IL-10水平與空白對照組間差異均無統(tǒng)計學(xué)意義；在2、4周，TCE暴露組血清中IL-10水平均顯著低于空白對照組及溶劑對照組（P0.05）；在8、12周，，TCE組IL-10水平與空白對照組及溶劑對照組相比差異均無統(tǒng)計學(xué)意義。 2.4飲水?dāng)z人TCE小鼠脾臟Treg細(xì)胞數(shù)量與Foxp3mRNA表達(dá)水平和血清中IL-10含量相關(guān)性相關(guān)分析顯示小鼠脾臟Treg細(xì)胞數(shù)量與Foxp3mRNA表達(dá)水平和血清中IL-10含量均存在顯著正相關(guān)（r=0.630，0.593，P0.01）。 3飲水?dāng)z入TCE對小鼠肝臟的損傷 3.1對肝功能的影響在2、4、8、12周，溶劑對照組小鼠ALT和AST水平與空白對照組比較差異均無統(tǒng)計學(xué)意義；在2、4周，TCE暴露組小鼠ALT和AST與空白和溶劑對照組比較明顯升高（P0.05），在4周時達(dá)到最高，8和12周時有所下降，但均高于空白對照組和溶劑對照組。 3.2對肝臟組織病理學(xué)的影響空白對照組、溶劑對照組小鼠肝細(xì)胞排列整齊，胞質(zhì)均勻，胞核大而圓，核仁明顯，核膜清晰；TCE染毒組小鼠肝組織有明顯的炎細(xì)胞浸潤，在4周時最明顯，8和12周時炎細(xì)胞浸潤有所減少。 3.3對肝組織中促炎細(xì)胞因子的影響 3.3.1對肝臟IL-1含量的影響在2、4、8、12周，溶劑對照組小鼠肝臟IL-1水平與空白對照組比較差異均無統(tǒng)計學(xué)意義；在2、4周，5mg/ml TCE暴露組小鼠肝臟IL-1較空白對照組明顯升高，4周時達(dá)到最高（P 0.05），8和12周時有所下降，但均高于空白對照和溶劑對照組。 3.3.2對肝臟IL-6含量的影響在2、4、8和12周，溶劑對照組小鼠肝臟IL-6水平與空白對照組比較差異均無統(tǒng)計學(xué)意義；與空白對照和溶劑對照組比較，5mg/ml TCE組在4和8周時明顯升高，4周時最高（P 0.05）；2.5mg/ml TCE組在8周時明顯升高（P 0.05）。 3.3.3對肝臟TNF-α含量的影響在2、4、8和12周，溶劑對照組與空白對照組之間差異均無統(tǒng)計學(xué)意義；在2、4、8周，TCE暴露組小鼠肝臟TNF-α含量均顯著高于空白對照組及溶劑對照組，4周時達(dá)到最高（P0.05）。 3.3.4肝功能與肝臟IL-1、IL-6和TNF-α含量的相關(guān)性相關(guān)性分析顯示肝功能ALT和AST，與肝臟中IL-1、IL-6和TNF-α含量之間存在顯著正相關(guān)(r=0.526,0.510,0.614,0.518,0.525,0.630, P 0.01)。 4肝功能、肝臟IL-1、IL-6和TNF-α含量與Treg細(xì)胞數(shù)量的相關(guān)性相關(guān)性分析顯示肝功能ALT和AST，肝臟IL-1、IL-6、TNF-α含量與Treg細(xì)胞數(shù)量之間存在顯著負(fù)相關(guān)(r=0.723,0.632,0.525,0.419,0.567, P 0.01)。 結(jié)論 1經(jīng)水?dāng)z入TCE對小鼠調(diào)節(jié)性T細(xì)胞有顯著影響，導(dǎo)致Treg細(xì)胞數(shù)量、特異性轉(zhuǎn)錄因子Foxp3mRNA表達(dá)和分泌的細(xì)胞因子IL-10表達(dá)水平顯著降低。 2經(jīng)水?dāng)z入TCE可導(dǎo)致小鼠肝臟損傷，ALT、AST以及肝臟組織中促炎細(xì)胞因子水平升高，并觀察到炎細(xì)胞浸潤。 3經(jīng)水?dāng)z入TCE對小鼠調(diào)節(jié)性T細(xì)胞影響與其導(dǎo)致的肝臟損傷存在顯著相關(guān)。
文內(nèi)圖片：
圖片說明：不同時間不同組別小鼠脾臟Treg數(shù)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114
本文編號：2511796
文內(nèi)圖片：
圖片說明：不同時間不同組別小鼠脾臟Treg數(shù)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114
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