三氯生对孕鼠胎盘发育的影响及其分子机制
发布时间：2019-03-16 19:56
【摘要】：三氯生(Triclosan,TCS)作为一种非离子型卤代酚类广谱抗菌剂广泛使用于护肤品、牙膏、临床护理清洁液等。181名母亲志愿者的尿液和脐带血液中TCS检出率分别为100%和51%。母乳中也能检测到TCS。我们研究室的前期研究已报道,孕妇尿液中TCS浓度增加与自然流产的发生显示正相关。用100mg/kg高剂量TCS处理孕第5-18天小鼠能引起胎鼠的体重降低和死亡率增加。我们进一步观察到TCS暴露能引起孕鼠血中四碘甲状腺原氨酸(Thyroxine,T4)和三碘甲状腺原氨酸(Triiodothyonine,T3)水平降低。临床资料显示,母亲的低甲状腺激素与胎儿宫内发育迟缓密切相关。TCS(10mg/kg)暴露引起孕鼠的T4和T3水平降低和胎鼠宫内发育迟缓。胎盘是母胎间交换物质的器官。胎儿的宫内发育依靠胎盘从母体摄得营养,而产生的废物经过胎盘排出。胎盘的营养物质转运与胎盘的发育和形态结构,氨基酸转运体和葡萄糖转运体的表达和功能活性有关。有研究证明,滋养细胞表达T3和T4受体,甲状腺激素通过激活PI3K-Akt-mTOR和ERK信号通路能刺激滋养细胞增殖和促进胎盘生长发育。此外,甲状腺激素也可以调节胎盘合体滋养细胞氨基酸转运体(SNAT1、SNAT2、SNAT4、TAUT)和葡萄糖转运体(GLUT1、GLUT3)的表达和功能活性。由此推测,孕鼠TCS暴露有可能通过降低T3和T4水平影响胎盘的发育和功能,从而导致胎鼠的宫内发育迟缓。研究目的:阐明TCS暴露对孕鼠胎盘发育的影响及其甲状腺激素介导的分子机制。实验方法:1.孕第6-18天ICR小鼠进行1,4,8mg/kg/day剂量的TCS灌胃。以下简称1-TCS孕鼠、4-TCS孕鼠和8-TCS孕鼠。2.胎盘用4%多聚甲醛固定和石蜡包埋。5μm切片进行苏木素-伊红(HE)染色,计算胎盘和迷路的体积。3.用5-溴-2-脱氧尿嘧啶核苷(BrdU)腹腔注射,3小时后取出胎盘固定,石蜡包埋,进行BrdU和增殖细胞核抗原(PCNA)免疫染色,进行BrdU阳性(BrdU+)和PCNA阳性(PCNA+)细胞计数。4.放射示踪法标记(14C-MeAIB,14C-Glucose)测量胎盘氨基酸和葡萄糖的转运功能。5.用酶联免疫法检测血清T4、T3、促甲状腺激素(TSH),雌激素(E2)和孕酮(P4),胎盘的白介素1β(IL-1β)和白介素6(IL-6)。6.用蛋白印迹法(Westernblot)检测Akt、mTOR、p70S6K、ERK1/2蛋白和磷酸、TNF-α蛋白。7.实时定量PCR检测胎盘PCNA、CyclinD3、SNAT1、SNAT2、SNAT4、TAUT、GLUT1、GLUT3。实验结果1.在妊娠第6-18天的8-TCS孕鼠,胎鼠的体重和存活率降低,而1-TCS孕鼠或4-TCS孕鼠没有发生胎鼠体重和存活率的改变。2.在妊娠第19天的8-TCS孕鼠,胎盘的重量减轻、总体积和迷路区体积减小,但是未出现胎盘血栓和组织坏死。同样,1-TCS孕鼠或4-TCS孕鼠也没有出现胎盘生长和发育的障碍。3.在妊娠第13天的8-TCS孕鼠,胎盘BrdU+细胞和PCNA+细胞数量明显低于对照孕鼠;PCNA和Cyclin D3 mRNA水平也明显降低,提示TCS抑制胎盘的细胞增殖。4.在妊娠第17天的8-TCS孕鼠,14C-MeAIB和14C-Glucose在单位重量胎盘的水平明显低于对照孕鼠,但是单位重量胎儿体内的相对水平没有明显改变,提示TCS能破坏胎盘的营养物质(葡萄糖、氨基酸)转运。5.在妊娠第17天的8-TCS孕鼠,无论胎盘氨基酸转运体SNAT1和SNAT4的表达水平或者葡萄糖转运体GLUT1都低于对照孕鼠,但是GLUT3和SNAT2表达无明显改变。6.在妊娠第17天的8-TCS孕鼠,T3和T4的血清水平都低于对照孕鼠,伴有TSH水平增加,但是E2和P4水平没有明显改变。7.在妊娠第17天的8-TCS孕鼠,胎盘Akt、mTOR、p70S6K的磷酸化水平明显降低,而ERK1/2磷酸化水平没有异常。对妊娠第15-17天8-TCS孕鼠进行L-T4(5mg/kg)处理能提高胎盘Akt、mTOR、p70S6K的磷酸化水平,提示TCS诱导的低甲状腺激素可以抑制Akt-mTOR-p70S6K信号通路。8.对妊娠第10-13天8-TCS孕鼠进行L-T4处理能增加胎盘Brdu+细胞和PCNA+细胞数量。对妊娠第15-17天TCS-孕鼠进行L-T4处理能改善14C-MeAIB和14C-Glucose通过胎盘的转运功能,增加存活胎鼠的体重。mTOR抑制剂雷帕霉素处理能阻断L-T4对胎盘细胞增殖,营养物质转运和胎鼠发育的保护作用。总结孕鼠进行TCS暴露能引起甲状腺激素水平降低,抑制Akt-mTOR-p70S6K信号通路,抑制胎盘的细胞增殖和葡萄糖转运体/氨基酸转运体表达,破坏胎盘的发育和营养物质的转运,导致胎鼠宫内发育不良。
[Abstract]:Triclosan (TCS) as a non-ionic halogenated phenolic broad-spectrum antibacterial agent is widely used in skin care products, toothpaste, clinical nursing cleaning liquid, etc. The detection rate of TCS in the urine and umbilical cord blood of 181 mother volunteers is 100% and 51%, respectively. TCS can also be detected in breast milk. The early studies of our laboratory have reported that the increase in TCS concentration in the urine of pregnant women is positively related to the occurrence of spontaneous abortion. The 5-18 day mice treated with a high dose of TCS at 100 mg/ kg can cause a decrease in body weight and an increase in mortality in the fetuses. We further observe that the exposure of TCS can cause the levels of tetraiodothyronine (T4) and triiodothyronine (T3) in the blood of pregnant rats to be reduced. The clinical data show that the mother's low thyroid hormone is closely related to the intrauterine growth retardation. The exposure of TCS (10 mg/ kg) to the T4 and T3 levels of the pregnant rats and the intrauterine growth retardation of the fetus. The placenta is the organ of the mother-to-child exchange substance. The intrauterine growth of the fetus depends on the placenta from the mother, and the resulting waste is discharged through the placenta. The transfer of nutrients from the placenta is related to the development and morphological structure of the placenta, the expression and functional activity of the amino acid transporter and the glucose transporter. It is shown that the TT3 and T4 receptors of the trophoblastic cells can stimulate the proliferation of the trophoblastic cells and promote the development of the placenta by activating the PI3K-Akt-mTOR and ERK signaling pathways. In addition, the thyroid hormone can also regulate the expression and functional activity of the amino acid transporter (SNAT1, SNAT2, SNAT4, TAUT) and the glucose transporter (GLUT1, GLUT3) of the placenta. It is thus speculates that the exposure of the pregnant rat TCS can affect the development and function of the placenta by reducing the levels of T3 and T4, resulting in a slow intrauterine growth of the fetal rat. Objective: To study the effects of TCS exposure on the development of placenta in pregnant rats and the molecular mechanism mediated by thyroid hormone. Experimental method:1. The 6-18 day ICR mice were given a 1,4,8 mg/ kg/ day dose of TCS. Hereinafter referred to as 1-TCS pregnant rat,4-TCS pregnant rat and 8-TCS pregnant rat.2. The placenta was fixed with 4% paraformaldehyde and paraffin-embedded. The 5. m u.m section was stained with hematoxylin-eosin (HE) to calculate the volume of the placenta and the labyrinth. BrdU positive (BrdU +) and PCNA-positive (PCNA +) cell counts were performed with 5-bromo-2-deoxy-1-deoxy-1-(BrdU) intraperitoneal injection. Radiotracer method (14C-MeAIB, 14C-Gluctose) was used to measure the transport function of placenta amino acid and glucose. Serum T4, T3, thyroid stimulating hormone (TSH), estrogen (E2) and progesterone (P4), interleukin-1 (IL-1) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay. Akt, mTOR, p70S6K, ERK1/2 protein and phosphate and TNF-linked protein were detected by Western blot. The PCNA, CyclinD3, SNAT1, SNAT2, SNAT4, TAUT, GLUT1 and GLUT3 were detected by real-time quantitative PCR. Experimental results 1. No change in body weight and survival was observed in the 1-TCS pregnant rat or 4-TCS pregnant rat in the 8-TCS pregnant rat at 6-18 days of gestation, while the 1-TCS pregnant rat or the 4-TCS pregnant rat did not have a change in the body weight and the survival rate of the fetus. In the 8-TCS pregnant rat on Day 19 of gestation, the weight of the placenta was reduced, the total volume and the volume of the lost area were reduced, but no placental thrombus and tissue necrosis were present. Similarly,1-TCS pregnant or 4-TCS pregnant rats have no barriers to placental growth and development. The number of placental BrdU + cells and PCNA + cells in the 8-TCS pregnant rats on the 13th day of pregnancy was significantly lower than that of control pregnant rats, and the level of PCNA and Cyclin D3 mRNA was also significantly reduced, suggesting that TCS inhibited the proliferation of cells in the placenta. In the 8-TCS pregnant rat on day 17 of gestation, the level of 14C-MeAIB and 14C-Glucose in the unit weight of the placenta was significantly lower than that of the control pregnant rat, but the relative levels in the unit weight of the fetus did not change significantly, suggesting that TCS can disrupt the transfer of nutrients (glucose, amino acids) in the placenta. The expression levels of SNAT1 and SNAT4 in the placenta amino acid transporter, or GLUT1 of the glucose transporter, were lower in the 8-TCS pregnant rat on Day 17 of gestation, but the expression of GLUT3 and SNAT2 did not change significantly. The serum levels of the 8-TCS pregnant rats, T3 and T4 on Day 17 of gestation were lower than that of the control pregnant rats with an increase in TSH, but the E2 and P4 levels did not change significantly. The level of phosphorylation of the placenta Akt, mTOR, p70S6K was significantly lower during the day 17 of gestation, while the level of ERK1/2 phosphorylation was not abnormal. The treatment of L-T4 (5 mg/ kg) of 8-TCS pregnant rats on the 15th to 17th day of pregnancy can improve the phosphorylation of Akt, mTOR, p70S6K in the placenta. It is suggested that the low thyroid hormone induced by TCS can inhibit the signal pathway of Akt-mTOR-p70S6K. The L-T4 treatment of the 8-TCS pregnant rats on the 10th to 13th day of pregnancy can increase the number of the placental Brdu + cells and the PCNA + cells. The L-T4 treatment of the 15-17-day TCS-pregnant rat was able to improve the transfer function of 14C-MeAIB and 14C-Gluctose through the placenta and increase the weight of the surviving fetuses. The mTOR inhibitor rapamycin treatment can block the protective effects of L-T4 on the proliferation of the placenta, the transport of nutrients and the development of the fetal rat. Conclusion The exposure of the TCS to the pregnant rats can cause the decrease of the thyroid hormone level, inhibit the expression of the Akt-mTOR-p70S6K signal, inhibit the cell proliferation of the placenta and the expression of the glucose transporter/ amino acid transporter, destroy the development of the placenta and transport of the nutrient substances, and lead to the intrauterine growth of the fetus.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2441915
[Abstract]:Triclosan (TCS) as a non-ionic halogenated phenolic broad-spectrum antibacterial agent is widely used in skin care products, toothpaste, clinical nursing cleaning liquid, etc. The detection rate of TCS in the urine and umbilical cord blood of 181 mother volunteers is 100% and 51%, respectively. TCS can also be detected in breast milk. The early studies of our laboratory have reported that the increase in TCS concentration in the urine of pregnant women is positively related to the occurrence of spontaneous abortion. The 5-18 day mice treated with a high dose of TCS at 100 mg/ kg can cause a decrease in body weight and an increase in mortality in the fetuses. We further observe that the exposure of TCS can cause the levels of tetraiodothyronine (T4) and triiodothyronine (T3) in the blood of pregnant rats to be reduced. The clinical data show that the mother's low thyroid hormone is closely related to the intrauterine growth retardation. The exposure of TCS (10 mg/ kg) to the T4 and T3 levels of the pregnant rats and the intrauterine growth retardation of the fetus. The placenta is the organ of the mother-to-child exchange substance. The intrauterine growth of the fetus depends on the placenta from the mother, and the resulting waste is discharged through the placenta. The transfer of nutrients from the placenta is related to the development and morphological structure of the placenta, the expression and functional activity of the amino acid transporter and the glucose transporter. It is shown that the TT3 and T4 receptors of the trophoblastic cells can stimulate the proliferation of the trophoblastic cells and promote the development of the placenta by activating the PI3K-Akt-mTOR and ERK signaling pathways. In addition, the thyroid hormone can also regulate the expression and functional activity of the amino acid transporter (SNAT1, SNAT2, SNAT4, TAUT) and the glucose transporter (GLUT1, GLUT3) of the placenta. It is thus speculates that the exposure of the pregnant rat TCS can affect the development and function of the placenta by reducing the levels of T3 and T4, resulting in a slow intrauterine growth of the fetal rat. Objective: To study the effects of TCS exposure on the development of placenta in pregnant rats and the molecular mechanism mediated by thyroid hormone. Experimental method:1. The 6-18 day ICR mice were given a 1,4,8 mg/ kg/ day dose of TCS. Hereinafter referred to as 1-TCS pregnant rat,4-TCS pregnant rat and 8-TCS pregnant rat.2. The placenta was fixed with 4% paraformaldehyde and paraffin-embedded. The 5. m u.m section was stained with hematoxylin-eosin (HE) to calculate the volume of the placenta and the labyrinth. BrdU positive (BrdU +) and PCNA-positive (PCNA +) cell counts were performed with 5-bromo-2-deoxy-1-deoxy-1-(BrdU) intraperitoneal injection. Radiotracer method (14C-MeAIB, 14C-Gluctose) was used to measure the transport function of placenta amino acid and glucose. Serum T4, T3, thyroid stimulating hormone (TSH), estrogen (E2) and progesterone (P4), interleukin-1 (IL-1) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay. Akt, mTOR, p70S6K, ERK1/2 protein and phosphate and TNF-linked protein were detected by Western blot. The PCNA, CyclinD3, SNAT1, SNAT2, SNAT4, TAUT, GLUT1 and GLUT3 were detected by real-time quantitative PCR. Experimental results 1. No change in body weight and survival was observed in the 1-TCS pregnant rat or 4-TCS pregnant rat in the 8-TCS pregnant rat at 6-18 days of gestation, while the 1-TCS pregnant rat or the 4-TCS pregnant rat did not have a change in the body weight and the survival rate of the fetus. In the 8-TCS pregnant rat on Day 19 of gestation, the weight of the placenta was reduced, the total volume and the volume of the lost area were reduced, but no placental thrombus and tissue necrosis were present. Similarly,1-TCS pregnant or 4-TCS pregnant rats have no barriers to placental growth and development. The number of placental BrdU + cells and PCNA + cells in the 8-TCS pregnant rats on the 13th day of pregnancy was significantly lower than that of control pregnant rats, and the level of PCNA and Cyclin D3 mRNA was also significantly reduced, suggesting that TCS inhibited the proliferation of cells in the placenta. In the 8-TCS pregnant rat on day 17 of gestation, the level of 14C-MeAIB and 14C-Glucose in the unit weight of the placenta was significantly lower than that of the control pregnant rat, but the relative levels in the unit weight of the fetus did not change significantly, suggesting that TCS can disrupt the transfer of nutrients (glucose, amino acids) in the placenta. The expression levels of SNAT1 and SNAT4 in the placenta amino acid transporter, or GLUT1 of the glucose transporter, were lower in the 8-TCS pregnant rat on Day 17 of gestation, but the expression of GLUT3 and SNAT2 did not change significantly. The serum levels of the 8-TCS pregnant rats, T3 and T4 on Day 17 of gestation were lower than that of the control pregnant rats with an increase in TSH, but the E2 and P4 levels did not change significantly. The level of phosphorylation of the placenta Akt, mTOR, p70S6K was significantly lower during the day 17 of gestation, while the level of ERK1/2 phosphorylation was not abnormal. The treatment of L-T4 (5 mg/ kg) of 8-TCS pregnant rats on the 15th to 17th day of pregnancy can improve the phosphorylation of Akt, mTOR, p70S6K in the placenta. It is suggested that the low thyroid hormone induced by TCS can inhibit the signal pathway of Akt-mTOR-p70S6K. The L-T4 treatment of the 8-TCS pregnant rats on the 10th to 13th day of pregnancy can increase the number of the placental Brdu + cells and the PCNA + cells. The L-T4 treatment of the 15-17-day TCS-pregnant rat was able to improve the transfer function of 14C-MeAIB and 14C-Gluctose through the placenta and increase the weight of the surviving fetuses. The mTOR inhibitor rapamycin treatment can block the protective effects of L-T4 on the proliferation of the placenta, the transport of nutrients and the development of the fetal rat. Conclusion The exposure of the TCS to the pregnant rats can cause the decrease of the thyroid hormone level, inhibit the expression of the Akt-mTOR-p70S6K signal, inhibit the cell proliferation of the placenta and the expression of the glucose transporter/ amino acid transporter, destroy the development of the placenta and transport of the nutrient substances, and lead to the intrauterine growth of the fetus.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
【参考文献】
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1 Prachi Gupta;Nisha Bhatia;Mohinder Pal Bansal;Ashwani Koul;;Lycopene modulates cellular proliferation, glycolysis and hepatic ultrastructure during hepatocellular carcinoma[J];World Journal of Hepatology;2016年29期
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