三氯生對孕鼠胎盤發(fā)育的影響及其分子機制
發(fā)布時間：2019-03-16 19:56
【摘要】：三氯生(Triclosan,TCS)作為一種非離子型鹵代酚類廣譜抗菌劑廣泛使用于護膚品、牙膏、臨床護理清潔液等。181名母親志愿者的尿液和臍帶血液中TCS檢出率分別為100%和51%。母乳中也能檢測到TCS。我們研究室的前期研究已報道,孕婦尿液中TCS濃度增加與自然流產(chǎn)的發(fā)生顯示正相關。用100mg/kg高劑量TCS處理孕第5-18天小鼠能引起胎鼠的體重降低和死亡率增加。我們進一步觀察到TCS暴露能引起孕鼠血中四碘甲狀腺原氨酸(Thyroxine,T4)和三碘甲狀腺原氨酸(Triiodothyonine,T3)水平降低。臨床資料顯示,母親的低甲狀腺激素與胎兒宮內(nèi)發(fā)育遲緩密切相關。TCS(10mg/kg)暴露引起孕鼠的T4和T3水平降低和胎鼠宮內(nèi)發(fā)育遲緩。胎盤是母胎間交換物質(zhì)的器官。胎兒的宮內(nèi)發(fā)育依靠胎盤從母體攝得營養(yǎng),而產(chǎn)生的廢物經(jīng)過胎盤排出。胎盤的營養(yǎng)物質(zhì)轉(zhuǎn)運與胎盤的發(fā)育和形態(tài)結(jié)構,氨基酸轉(zhuǎn)運體和葡萄糖轉(zhuǎn)運體的表達和功能活性有關。有研究證明,滋養(yǎng)細胞表達T3和T4受體,甲狀腺激素通過激活PI3K-Akt-mTOR和ERK信號通路能刺激滋養(yǎng)細胞增殖和促進胎盤生長發(fā)育。此外,甲狀腺激素也可以調(diào)節(jié)胎盤合體滋養(yǎng)細胞氨基酸轉(zhuǎn)運體(SNAT1、SNAT2、SNAT4、TAUT)和葡萄糖轉(zhuǎn)運體(GLUT1、GLUT3)的表達和功能活性。由此推測,孕鼠TCS暴露有可能通過降低T3和T4水平影響胎盤的發(fā)育和功能,從而導致胎鼠的宮內(nèi)發(fā)育遲緩。研究目的:闡明TCS暴露對孕鼠胎盤發(fā)育的影響及其甲狀腺激素介導的分子機制。實驗方法:1.孕第6-18天ICR小鼠進行1,4,8mg/kg/day劑量的TCS灌胃。以下簡稱1-TCS孕鼠、4-TCS孕鼠和8-TCS孕鼠。2.胎盤用4%多聚甲醛固定和石蠟包埋。5μm切片進行蘇木素-伊紅(HE)染色,計算胎盤和迷路的體積。3.用5-溴-2-脫氧尿嘧啶核苷(BrdU)腹腔注射,3小時后取出胎盤固定,石蠟包埋,進行BrdU和增殖細胞核抗原(PCNA)免疫染色,進行BrdU陽性(BrdU+)和PCNA陽性(PCNA+)細胞計數(shù)。4.放射示蹤法標記(14C-MeAIB,14C-Glucose)測量胎盤氨基酸和葡萄糖的轉(zhuǎn)運功能。5.用酶聯(lián)免疫法檢測血清T4、T3、促甲狀腺激素(TSH),雌激素(E2)和孕酮(P4),胎盤的白介素1β(IL-1β)和白介素6(IL-6)。6.用蛋白印跡法(Westernblot)檢測Akt、mTOR、p70S6K、ERK1/2蛋白和磷酸、TNF-α蛋白。7.實時定量PCR檢測胎盤PCNA、CyclinD3、SNAT1、SNAT2、SNAT4、TAUT、GLUT1、GLUT3。實驗結(jié)果1.在妊娠第6-18天的8-TCS孕鼠,胎鼠的體重和存活率降低,而1-TCS孕鼠或4-TCS孕鼠沒有發(fā)生胎鼠體重和存活率的改變。2.在妊娠第19天的8-TCS孕鼠,胎盤的重量減輕、總體積和迷路區(qū)體積減小,但是未出現(xiàn)胎盤血栓和組織壞死。同樣,1-TCS孕鼠或4-TCS孕鼠也沒有出現(xiàn)胎盤生長和發(fā)育的障礙。3.在妊娠第13天的8-TCS孕鼠,胎盤BrdU+細胞和PCNA+細胞數(shù)量明顯低于對照孕鼠;PCNA和Cyclin D3 mRNA水平也明顯降低,提示TCS抑制胎盤的細胞增殖。4.在妊娠第17天的8-TCS孕鼠,14C-MeAIB和14C-Glucose在單位重量胎盤的水平明顯低于對照孕鼠,但是單位重量胎兒體內(nèi)的相對水平?jīng)]有明顯改變,提示TCS能破壞胎盤的營養(yǎng)物質(zhì)(葡萄糖、氨基酸)轉(zhuǎn)運。5.在妊娠第17天的8-TCS孕鼠,無論胎盤氨基酸轉(zhuǎn)運體SNAT1和SNAT4的表達水平或者葡萄糖轉(zhuǎn)運體GLUT1都低于對照孕鼠,但是GLUT3和SNAT2表達無明顯改變。6.在妊娠第17天的8-TCS孕鼠,T3和T4的血清水平都低于對照孕鼠,伴有TSH水平增加,但是E2和P4水平?jīng)]有明顯改變。7.在妊娠第17天的8-TCS孕鼠,胎盤Akt、mTOR、p70S6K的磷酸化水平明顯降低,而ERK1/2磷酸化水平?jīng)]有異常。對妊娠第15-17天8-TCS孕鼠進行L-T4(5mg/kg)處理能提高胎盤Akt、mTOR、p70S6K的磷酸化水平,提示TCS誘導的低甲狀腺激素可以抑制Akt-mTOR-p70S6K信號通路。8.對妊娠第10-13天8-TCS孕鼠進行L-T4處理能增加胎盤Brdu+細胞和PCNA+細胞數(shù)量。對妊娠第15-17天TCS-孕鼠進行L-T4處理能改善14C-MeAIB和14C-Glucose通過胎盤的轉(zhuǎn)運功能,增加存活胎鼠的體重。mTOR抑制劑雷帕霉素處理能阻斷L-T4對胎盤細胞增殖,營養(yǎng)物質(zhì)轉(zhuǎn)運和胎鼠發(fā)育的保護作用�？偨Y(jié)孕鼠進行TCS暴露能引起甲狀腺激素水平降低,抑制Akt-mTOR-p70S6K信號通路,抑制胎盤的細胞增殖和葡萄糖轉(zhuǎn)運體/氨基酸轉(zhuǎn)運體表達,破壞胎盤的發(fā)育和營養(yǎng)物質(zhì)的轉(zhuǎn)運,導致胎鼠宮內(nèi)發(fā)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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114
本文編號：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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114
【參考文獻】
相關期刊論文 前1條
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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