代表性多溴联苯醚的类固醇激素合成干扰作用及胚胎干细胞毒性研究

发布时间：2018-01-18 03:20

本文关键词：代表性多溴联苯醚的类固醇激素合成干扰作用及胚胎干细胞毒性研究　出处：《南京医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：多溴联苯醚(Polybrominated Diphenyl Ethers,PBDEs)是一类广泛使用的溴代阻燃剂。由于其热稳定性好,阻燃效率高,目前已被广泛应用于纺织、家具、建材和电子等潜在可燃产品当中。自20世纪70年代生产和使用多溴联苯醚至今,已造成全球性环境污染。环境流行病学资料调查显示,PBDEs广泛存在于各种环境介质中,并可以通过消化道、呼吸道和皮肤等途径进入机体。在人体血液、乳汁和脂肪等组织中均可以检测出PBDEs的存在。PBDEs具有难降解性、环境稳定性、高脂溶性和生物放大作用等特点,能够通过食物链的转移,使生物受到毒害,最终导致对人体健康的危害。研究发现,多溴联苯醚对试验动物有致癌性、生殖毒性、神经发育毒性和内分泌干扰毒性,作用的靶器官主要包括神经系统、脂肪组织、甲状腺和生殖系统等。因此,在美国及欧洲等地,相继限制生产和使用低溴类如BDE-47；99；153等,但高溴类BDE-209因其阻燃性能好,其生物毒性不确定而仍在广泛使用。BDE209在环境中极易发生脱溴反应,生成低溴类化合物,其危害依然不容忽视。因此,本研究选取BDE-47和BDE209为代表性多溴联苯醚类化合物,利用体外培养小鼠睾丸间质瘤细胞株(MLTC-1)和小鼠胚胎干细胞系(D3)为染毒模型,侧重于研究多溴联苯醚类化合物对类固醇激素合成的影响,以及对胚胎干细胞的细胞毒性和全能性维持的影响,并初步探讨了其相关分子机制。第一部分 BDE-47/BDE-209对类固醇激素合成的影响及相关分子机制目的观察BDE-47/BDE-209对类固醇激素合成的影响,初步探讨cAMP-PKA信号通路在多溴联苯醚抑制孕酮合成中的作用。方法 1.采用体外培养的小鼠睾丸间质瘤细胞株(MLTC-1)作为染毒模型。 2.MTT法测定BDE-47/BDE-209对MLTC-1细胞活力的影响,确定染毒剂量。 3.不同浓度BDE-47/BDE-209染毒MLTC-124h,在培养液中加入hCG(O.1U/L)继续染毒4h,用放射免疫法(RIA)检测培养液上清中的孕酮水平。 4.不同浓度BDE-47/BDE-209染毒MLTC-124h,吸弃培养液,继续用不含BDE-47/BDE-209的培养液培养24h后,培养液中加入hCG继续培养4h, RIA检测培养液上清中的孕酮水平。 5.不同浓度BDE-47/BDE-209染毒MLTC-1细胞24h,培养液中分别加入CT (30ng/ml)、forskolin (10μmol/L)和hCG (0.1U/L)继续染毒4h,RIA法检测细胞培养上清中的孕酮和cAMP水平。 6.不同浓度BDE-47/BDE-209染毒MLTC-1细胞24h,培养液中分别加入8-Br-cAMP (500nmol/L)、22R-HC(25μmol/L)和孕烯醇酮(10μmol/L)继续染毒4h,RIA法检测细胞培养上清中的孕酮水平。 7. Real-time PCR法测定类固醇激素合成急性调节蛋白(StAR)、胆固醇侧链裂解酶(P450scc)、3β-HSD mRNA表达水平的变化。 8.蛋白印迹法(western blot)测定StAR、P450scc、3β-HSD蛋白表达水平的变化。结果 1.根据MTT结果确定BDE-47及BDE-209染毒剂量均分别为：0、0.04、0.2、1、5和25μmol/L 2.在hCG刺激下,BDE-47显著抑制孕酮的合成,且呈剂量一反应关系。在25μmol/L组,孕酮下降了87.6%。相较而言,BDE209对孕酮合成的抑制作用较弱,仅在25μmol/L组出现显著性差异。 3.去除化学物继续培养24h后,BDE-47/BDE-209各剂量组的孕酮水平均无显著性差异。 4.在forskolin刺激下,BDE-47和BDE-209均显著抑制孕酮的合成,但在CT刺激下,BDE-47和BDE-209各剂量组的孕酮水平均无显著差异。 5.在hCG和forskolin刺激下,BDE-47显著抑制MLTC-1细胞内cAMP水平。但在CT的刺激下,这种抑制作用消失。而BDE-209处理组,hCG、forskolin、CT刺激下,细胞内cAMP水平均无明显改变。 6.培养液中加入8-Br-cAMP后,BDE-47和BDE-209仍抑制孕酮的合成。 7.在培养液中加入22R-HC和孕烯醇酮后,BDE-47显著抑制二者刺激下的孕酮合成；但对于BDE-209处理组,未发现该抑制效应。 8. BDE-47/BDE-209染毒MLTC-1细胞24h后,StAR mRNA和蛋白水平均无显著变化。 9.BDE-47染毒MLTC-1细胞24h后,P450scc mRNA和蛋白水平均呈剂量-反应关系下降,而3β-HSD仅观察到mRNA水平下降,蛋白水平没有明显改变。BDE-209处理组,仅观察到P450scc和3β-HSD mRNA水平下降,未观察到其蛋白水平下降。结论 1、BDE-47显著抑制MLTC-1类固醇激素的合成,去除化学物后MLTC-1细胞合成类固醇激素的能力基本恢复。BDE-47显著抑制MLTC-1细胞孕酮的合成,其机制可能涉及cAMP-依赖的蛋白激酶信号通路。此外,BDE-47还抑制P450scc和3β-HSD酶的活性及表达水平。 2、BDE-209抑制MLTC-1类固醇激素的合成,去除化学物后MLTC-1细胞合成类固醇激素的能力基本恢复。BDE-209对MLTC-1细胞孕酮合成的影响,并不涉及cAMP-依赖的蛋白激酶信号通路,但是可以抑制P450scc的mRNA转录。 3、BDE-47与BDE-209相比,对类固醇激素合成的影响更显著。第二部分 BDE一47对小鼠胚胎干细胞的毒性研究及相关分子机制目的观察BDE-47对小鼠胚胎干细胞的毒性作用及对干细胞全能性维持的影响,并初步探讨相关分子机制。方法 1.采用体外培养的D3mESCs作为染毒模型。 2.不同浓度BDE-47染毒D3mESCs24h和48h,观察细胞形态改变。 3.不同浓度BDE-47染毒D3mESCs24h和48h, MTT法测定BDE-47对D3mESCs细胞活力的影响,确定后续试验染毒剂量。 4.不同浓度BDE-47染毒D3mESCs24h,利用流式细胞仪分析细胞周期改变。 5.不同浓度BDE-47染毒D3mESCs24h,利用流式细胞仪和DeadEndTM荧光测定TUNEL系统检测细胞凋亡情况。Real-time RT-PCR测定促凋亡基因P53的表达水平变化。 6.不同浓度BDE-47染毒D3mESCs24h,用碱性磷酸酶染色试剂盒检测染毒后的D3mESCs的染色状况；用碱性磷酸酶活性分析试剂盒检测染毒后的D3mESCs碱性磷酸酶活性改变。 7.不同浓度BDE-47染毒D3mESCs24h,固定细胞后,用细胞免疫荧光法检测染毒后的D3mESCs的转录因子Oct-4/Sox-2/Nanog表达情况。 8. Real-time RT-PCR测定不同浓度BDE-47染毒后转录因子Oct-4/Sox-2/Nanog的表达水平变化以及mmu-miR-145和mmu-miR-34a的表达情况。 9.蛋白印迹法(western blot)测定Oct-4/Sox-2/Nanog蛋白表达水平的变化。结果 1.不同剂量BDE47染毒24h和48h后,D3mESCs的细胞形态与对照组比较,均在25μM剂量组及以上出现改变,镜下观察细胞密度明显减少,少数细胞碎裂,细胞出现明显萎缩,部分克隆边缘塌陷。而且染毒48h, D3mESCs细胞形态改变更加明显。 2.MTT结果显示,不同剂量BDE47染毒24h后,D3mESCs各剂量组细胞活力无明显差异。不同剂量BDE47染毒48h后,100gM剂量组出现细胞活力改变,细胞存活率降低。确定后续试验染毒剂量为0.04μM,1μM,25μM，100μM；染毒时间为24h。 3.流式细胞周期结果显示,不同剂量BDE47染毒24h后,D3mESCs细胞周期各期水平无显著性差异。 4.流式细胞凋亡结果显示,不同剂量BDE47染毒24h后,在25μM及100μM剂量组,D3mESCs细胞凋亡增加,统计具有显著差异(P0.05).DeadEndTM荧光测定TUNEL系统原位分析细胞凋亡结果显示,随着染毒剂量增加,细胞内凋亡信号增加,凋亡细胞显著增加。 5.AP染色结果显示,不同剂量BDE47染毒24h后,D3mESCs在1μM及以上剂量组出现细胞着色减淡,部分细胞未着色。AP活性检测结果显示,D3mESCs在1μM剂量组即出现AP活性的显著下降。 6.细胞免疫荧光结果显示,不同剂量BDE47染毒24h后,D3mESCs维持其干细胞多能性的转录因子Oct-4、Sox-2,Nanog表达降低。 7.不同剂量BDE47染毒24h后,D3mESCs维持其干细胞多能性的转录因子Oct-4、Sox-2,Nanog mRNA和蛋白水平均呈剂量-效应关系下降；而MircoRNA mmu-miR-145和mmu-miR-34a水平呈剂量-效应关系上升。结论 1、BDE-47能引起D3mESCs细胞形态改变。染毒48h后,在100gM剂量组引起细胞活力显著降低。BDE47能导致细胞凋亡增加,促凋亡基因P53表达升高。 2、BDE-47能影响D3mESCs全能性的维持,显著抑制D3mESCs维持全能性的转录因子Oct-4.Sox-2,Nanog的表达,其机制可能涉及促进相关MicroRNA mmu-miR-145和mmu-miR-34a的高表达。
[Abstract]:Polybrominated diphenyl ethers (Polybrominated Diphenyl, Ethers, PBDEs) is a kind of widely used brominated flame retardant. Due to its good thermal stability, flame retardant and high efficiency, has been widely used in textile, furniture, building materials and other electronic products potentially combustible. Since 1970s the production and use of PBDEs has so far, global environment pollution. Environmental epidemiology data show, PBDEs exist in various environmental media, and through the digestive tract, respiratory tract and skin into the body. In the way of human blood, breast milk and adipose tissue can detect the presence of.PBDEs resistant to degradation, environmental stability of PBDEs, the characteristics of high liposolubility and biomagnification, can be transferred through the food chain, the biological contamination, resulting in harm to human health. The study found that PBDEs are caused by the test animal Cancer, reproductive toxicity, toxicity and endocrine disruption of neural development toxicity, the target organ including nervous system, adipose tissue, thyroid and reproductive system. Therefore, in the United States and Europe, have restricted the production and use of PBDEs such as BDE-47; 99; 153, but because of its high bromine BDE-209 good flame retardant performance, the biological toxicity of uncertainty is still widely used in.BDE209 prone debromination reaction in the environment, generate low bromine compounds, its harm still cannot be ignored. Therefore, this study selected BDE-47 and BDE209 as the representative of polybrominated diphenyl ether compounds, the use of mouse Leydig tumor cell line culture in vitro (MLTC-1) and mouse embryonic stem cell line (D3) as the exposure model, focuses on the research of diphenyl ether compounds polybrominated effect on the synthesis of steroid hormones and cytotoxic effects on embryonic stem cells and pluripotency, and The related molecular mechanisms are preliminarily discussed.
Part one
The effect of BDE-47/BDE-209 on the synthesis of steroid hormone and its molecular mechanism
objective
The effect of BDE-47/BDE-209 on the synthesis of steroid hormones was observed and the effect of cAMP-PKA signaling pathway on the inhibition of progesterone synthesis by polybrominated diphenyl ethers was preliminarily investigated.
Method
1. the mouse testicular stromal tumor cell line (MLTC-1) cultured in vitro was used as a poisoned model.
The effect of BDE-47/BDE-209 on the activity of MLTC-1 cells was determined by 2.MTT method, and the dose was determined.
3., MLTC-124h was exposed to BDE-47/BDE-209 at different concentrations, and hCG (O.1U/L) was added to the culture medium, and 4H was continuously exposed. The progesterone level in the supernatant of the culture solution was detected by radioimmunoassay (RIA).
4. after exposure to different concentrations of BDE-47/BDE-209, MLTC-124h and BDE-47/BDE-209 were added to the culture medium, 24h was added to the culture medium. HCG was added to the culture medium, and then 4H was continued. RIA was used to detect progesterone level in the supernatant of the culture medium.
5., MLTC-1 cells 24h were exposed to BDE-47/BDE-209 with different concentrations of 24h. CT (30ng/ml), forskolin (10 mol/L mol/L) and hCG (0.1U/L) were added to the culture medium, 4H and RIA method were used to detect progesterone and level of the cells in the culture supernatant.
6., MLTC-1 cells 24h were exposed to BDE-47/BDE-209 with different concentrations of 24h. 8-Br-cAMP (500nmol/L), 22R-HC (25 mol/L mol/L) and progesterone (10 mol/L) were added to the culture medium, and 4H was added. The progesterone level in cell culture supernatant was detected by RIA method.
7. Real-time PCR method was used to determine the changes in the expression of acute regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3 beta -HSD mRNA in steroid hormones.
8. protein blotting (Western blot) was used to determine the changes in the expression level of StAR, P450scc, and 3 beta -HSD protein.
Result
1. according to the MTT results, the doses of BDE-47 and BDE-209 were determined respectively: 0,0.04,0.2,1,5 and 25 mu mol/L, respectively.
2., under the stimulation of hCG, BDE-47 significantly inhibited progesterone synthesis, and showed a dose response relationship. In 25 mol/L group, progesterone decreased 87.6%., compared with BDE209, the inhibition effect of BDE209 on progesterone synthesis was weaker, and there was significant difference only in 25 mol/L mol/L group.
3. there was no significant difference in progesterone level in each dose group of BDE-47/BDE-209 after 24h was removed.
4. under the stimulation of forskolin, BDE-47 and BDE-209 significantly inhibited progesterone synthesis. However, under CT stimulation, there was no significant difference in progesterone levels between BDE-47 and BDE-209 groups.
5., under the stimulation of hCG and forskolin, BDE-47 significantly inhibited the cAMP level in MLTC-1 cells. However, the inhibitory effect disappeared under the stimulation of CT. However, in BDE-209 treatment group, there was no significant change in intracellular cAMP level under hCG, forskolin and CT stimulation.
After adding 8-Br-cAMP to 6. medium, BDE-47 and BDE-209 still inhibit the synthesis of progesterone.
7. after adding 22R-HC and progesterone in culture medium, BDE-47 significantly inhibited progesterone synthesis stimulated by two, but no effect was found in BDE-209 treatment group.
There was no significant change in the level of StAR mRNA and protein after 8. BDE-47/BDE-209 infected MLTC-1 cells 24h.
9.BDE-47 toxicity in MLTC-1 cells after 24h, P450scc mRNA and protein levels showed a dose-response relationship and decreased 3 beta -HSD were observed only in mRNA decreased, protein levels did not change significantly in.BDE-209 treatment group were observed only in P450scc and 3 beta -HSD reduced the level of mRNA was not observed in the protein level decreased.
conclusion
1, BDE-47 significantly inhibited MLTC-1 synthesis of steroid hormone synthesis, ability to remove chemicals after steroid hormone synthesis in MLTC-1 cells recovered.BDE-47 significantly inhibited progesterone MLTC-1 cells, its mechanism may involve cAMP- dependent protein kinase pathway. In addition, the level of expression of BDE-47 and P450scc and beta 3 also inhibited the activity of -HSD.
2, the synthesis of BDE-209 MLTC-1 inhibition of steroid hormones, ability to remove chemicals after steroid hormone synthesis in MLTC-1 cells of the basic recovery effect of.BDE-209 on the synthesis of progesterone MLTC-1 cells does not involve protein kinase signal pathway of cAMP- dependent transcription of mRNA, but can inhibit P450scc.
3, the effect of BDE-47 on the synthesis of steroid hormone is more significant than that of BDE-209.
The second part
The toxicity of BDE 47 to mouse embryonic stem cells and its molecular mechanism
objective
The toxic effects of BDE-47 on mouse embryonic stem cells and the effect on the maintenance of stem cells were observed, and the related molecular mechanisms were preliminarily discussed.
Method
1. the D3mESCs cultured in vitro was used as a poisoned model.
2. D3mESCs24h and 48h were infected with different concentrations of BDE-47, and the morphological changes of cells were observed.
3. D3mESCs24h and 48h were poisoned by different concentrations of BDE-47, and the effect of BDE-47 on the activity of D3mESCs cells was determined by MTT method, and the dosage of the subsequent test was determined.
4. BDE-47 was infected with different concentrations of D3mESCs24h, and the cell cycle changes were analyzed by flow cytometry.
5. D3mESCs24h was exposed to BDE-47 at different concentrations. The apoptosis was detected by flow cytometry and DeadEndTM fluorescence. TUNEL system was used to detect apoptosis..Real-time RT-PCR was used to detect the expression level of Pro apoptotic gene P53.
6. D3mESCs24h was exposed to BDE-47 at different concentrations. The staining condition of D3mESCs was detected by alkaline phosphatase staining kit. Alkaline phosphatase activity assay kit was used to detect the change of D3mESCs alkaline phosphatase activity after exposure.
7. the expression of D3mESCs transcription factor Oct-4/Sox-2/Nanog was detected by cell immunofluorescence after immobilized cells with different concentrations of BDE-47 and immobilized cells for D3mESCs24h.
8. Real-time RT-PCR was used to determine the expression level of transcription factor Oct-4/Sox-2/Nanog and the expression of mmu-miR-145 and mmu-miR-34a after different concentrations of BDE-47.
9. protein blotting (Western blot) was used to determine the changes in the expression level of Oct-4/Sox-2/Nanog protein.
Result
1. different doses of BDE47 were 24h and 48h, group D3mESCs and control cells, there were changes in the 25 M dose group and above, the cell density decreased significantly under microscope, a few cells fragmentation, cells became atrophy and edge collapse. But some clones at 48h, change the morphology of D3mESCs cells is more obvious.
2.MTT results showed that different doses of BDE47 after 24h exposure, no significant difference in each D3mESCs group. Cell viability of different doses of BDE47 after 48h exposure, 100gM dose group showed cell viability, cell survival rate decreased. Determine the follow-up test at the dose of 0.04 M, 1 M, 25 M, 100 M exposure; time is 24h.
The results of 3. flow cytometry showed that there was no significant difference in the level of D3mESCs cell cycle at different doses of BDE47 after 24h.
4. apoptosis by flow cytometry showed that different doses of BDE47 after 24h exposure, in 25 M and 100 M dose group, the apoptosis of D3mESCs cells increased, with statistical significant difference (P0.05).DeadEndTM fluorescence determination of TUNEL system in situ analysis results of apoptosis showed that as the dose increased, the increase of intracellular apoptotic signal, apoptosis significantly increased.
5.AP staining showed that when BDE47 was exposed to 24h at different doses, cell stain was weakened in the dose of D3mESCs above 1 M and above, and the unstained.AP activity of some cells showed that D3mESCs showed a significant decrease in AP activity in 1 M M dose group.
The results of 6. cell immunofluorescence showed that D3mESCs maintained the pluripotent transcription factor Oct-4 of stem cells and decreased the expression of Sox-2 and Nanog after different doses of BDE47 for 24h.
7., after different doses of BDE47 were poisoned with 24h, the transcription factors Oct-4, Sox-2, Nanog mRNA and protein level of D3mESCs maintained their stem cell pluripotent, all showed a dose-response relationship, while MircoRNA mmu-miR-145 and mmu-miR-34a levels increased in dose effect relationship.
conclusion
1, BDE-47 can induce morphological changes of D3mESCs cells. After exposure to 48h, cell viability was significantly reduced in 100gM dose group,.BDE47 could induce apoptosis and increase the expression of P53.
2, BDE-47 can affect the maintenance of D3mESCs totipotency, and significantly inhibit the expression of transcription factor Oct-4.Sox-2 and Nanog, which maintain D3mESCs totipotency. Its mechanism may involve the promotion of high expression of MicroRNA mmu-miR-145 and mmu-miR-34a.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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