本文關(guān)鍵詞：代表性多溴聯(lián)苯醚的類固醇激素合成干擾作用及胚胎干細(xì)胞毒性研究　出處：《南京醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

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【摘要】：多溴聯(lián)苯醚(Polybrominated Diphenyl Ethers,PBDEs)是一類廣泛使用的溴代阻燃劑。由于其熱穩(wěn)定性好,阻燃效率高,目前已被廣泛應(yīng)用于紡織、家具、建材和電子等潛在可燃產(chǎn)品當(dāng)中。自20世紀(jì)70年代生產(chǎn)和使用多溴聯(lián)苯醚至今,已造成全球性環(huán)境污染。環(huán)境流行病學(xué)資料調(diào)查顯示,PBDEs廣泛存在于各種環(huán)境介質(zhì)中,并可以通過消化道、呼吸道和皮膚等途徑進(jìn)入機(jī)體。在人體血液、乳汁和脂肪等組織中均可以檢測出PBDEs的存在。PBDEs具有難降解性、環(huán)境穩(wěn)定性、高脂溶性和生物放大作用等特點(diǎn),能夠通過食物鏈的轉(zhuǎn)移,使生物受到毒害,最終導(dǎo)致對人體健康的危害。研究發(fā)現(xiàn),多溴聯(lián)苯醚對試驗(yàn)動物有致癌性、生殖毒性、神經(jīng)發(fā)育毒性和內(nèi)分泌干擾毒性,作用的靶器官主要包括神經(jīng)系統(tǒng)、脂肪組織、甲狀腺和生殖系統(tǒng)等。因此,在美國及歐洲等地,相繼限制生產(chǎn)和使用低溴類如BDE-47；99；153等,但高溴類BDE-209因其阻燃性能好,其生物毒性不確定而仍在廣泛使用。BDE209在環(huán)境中極易發(fā)生脫溴反應(yīng),生成低溴類化合物,其危害依然不容忽視。因此,本研究選取BDE-47和BDE209為代表性多溴聯(lián)苯醚類化合物,利用體外培養(yǎng)小鼠睪丸間質(zhì)瘤細(xì)胞株(MLTC-1)和小鼠胚胎干細(xì)胞系(D3)為染毒模型,側(cè)重于研究多溴聯(lián)苯醚類化合物對類固醇激素合成的影響,以及對胚胎干細(xì)胞的細(xì)胞毒性和全能性維持的影響,并初步探討了其相關(guān)分子機(jī)制。 第一部分 BDE-47/BDE-209對類固醇激素合成的影響及相關(guān)分子機(jī)制 目的 觀察BDE-47/BDE-209對類固醇激素合成的影響,初步探討cAMP-PKA信號通路在多溴聯(lián)苯醚抑制孕酮合成中的作用。 方法 1.采用體外培養(yǎng)的小鼠睪丸間質(zhì)瘤細(xì)胞株(MLTC-1)作為染毒模型。 2.MTT法測定BDE-47/BDE-209對MLTC-1細(xì)胞活力的影響,確定染毒劑量。 3.不同濃度BDE-47/BDE-209染毒MLTC-124h,在培養(yǎng)液中加入hCG(O.1U/L)繼續(xù)染毒4h,用放射免疫法(RIA)檢測培養(yǎng)液上清中的孕酮水平。 4.不同濃度BDE-47/BDE-209染毒MLTC-124h,吸棄培養(yǎng)液,繼續(xù)用不含BDE-47/BDE-209的培養(yǎng)液培養(yǎng)24h后,培養(yǎng)液中加入hCG繼續(xù)培養(yǎng)4h, RIA檢測培養(yǎng)液上清中的孕酮水平。 5.不同濃度BDE-47/BDE-209染毒MLTC-1細(xì)胞24h,培養(yǎng)液中分別加入CT (30ng/ml)、forskolin (10μmol/L)和hCG (0.1U/L)繼續(xù)染毒4h,RIA法檢測細(xì)胞培養(yǎng)上清中的孕酮和cAMP水平。 6.不同濃度BDE-47/BDE-209染毒MLTC-1細(xì)胞24h,培養(yǎng)液中分別加入8-Br-cAMP (500nmol/L)、22R-HC(25μmol/L)和孕烯醇酮(10μmol/L)繼續(xù)染毒4h,RIA法檢測細(xì)胞培養(yǎng)上清中的孕酮水平。 7. Real-time PCR法測定類固醇激素合成急性調(diào)節(jié)蛋白(StAR)、膽固醇側(cè)鏈裂解酶(P450scc)、3β-HSD mRNA表達(dá)水平的變化。 8.蛋白印跡法(western blot)測定StAR、P450scc、3β-HSD蛋白表達(dá)水平的變化。 結(jié)果 1.根據(jù)MTT結(jié)果確定BDE-47及BDE-209染毒劑量均分別為：0、0.04、0.2、1、5和25μmol/L 2.在hCG刺激下,BDE-47顯著抑制孕酮的合成,且呈劑量一反應(yīng)關(guān)系。在25μmol/L組,孕酮下降了87.6%。相較而言,BDE209對孕酮合成的抑制作用較弱,僅在25μmol/L組出現(xiàn)顯著性差異。 3.去除化學(xué)物繼續(xù)培養(yǎng)24h后,BDE-47/BDE-209各劑量組的孕酮水平均無顯著性差異。 4.在forskolin刺激下,BDE-47和BDE-209均顯著抑制孕酮的合成,但在CT刺激下,BDE-47和BDE-209各劑量組的孕酮水平均無顯著差異。 5.在hCG和forskolin刺激下,BDE-47顯著抑制MLTC-1細(xì)胞內(nèi)cAMP水平。但在CT的刺激下,這種抑制作用消失。而BDE-209處理組,hCG、forskolin、CT刺激下,細(xì)胞內(nèi)cAMP水平均無明顯改變。 6.培養(yǎng)液中加入8-Br-cAMP后,BDE-47和BDE-209仍抑制孕酮的合成。 7.在培養(yǎng)液中加入22R-HC和孕烯醇酮后,BDE-47顯著抑制二者刺激下的孕酮合成；但對于BDE-209處理組,未發(fā)現(xiàn)該抑制效應(yīng)。 8. BDE-47/BDE-209染毒MLTC-1細(xì)胞24h后,StAR mRNA和蛋白水平均無顯著變化。 9.BDE-47染毒MLTC-1細(xì)胞24h后,P450scc mRNA和蛋白水平均呈劑量-反應(yīng)關(guān)系下降,而3β-HSD僅觀察到mRNA水平下降,蛋白水平?jīng)]有明顯改變。BDE-209處理組,僅觀察到P450scc和3β-HSD mRNA水平下降,未觀察到其蛋白水平下降。 結(jié)論 1、BDE-47顯著抑制MLTC-1類固醇激素的合成,去除化學(xué)物后MLTC-1細(xì)胞合成類固醇激素的能力基本恢復(fù)。BDE-47顯著抑制MLTC-1細(xì)胞孕酮的合成,其機(jī)制可能涉及cAMP-依賴的蛋白激酶信號通路。此外,BDE-47還抑制P450scc和3β-HSD酶的活性及表達(dá)水平。 2、BDE-209抑制MLTC-1類固醇激素的合成,去除化學(xué)物后MLTC-1細(xì)胞合成類固醇激素的能力基本恢復(fù)。BDE-209對MLTC-1細(xì)胞孕酮合成的影響,并不涉及cAMP-依賴的蛋白激酶信號通路,但是可以抑制P450scc的mRNA轉(zhuǎn)錄。 3、BDE-47與BDE-209相比,對類固醇激素合成的影響更顯著。 第二部分 BDE一47對小鼠胚胎干細(xì)胞的毒性研究及相關(guān)分子機(jī)制 目的 觀察BDE-47對小鼠胚胎干細(xì)胞的毒性作用及對干細(xì)胞全能性維持的影響,并初步探討相關(guān)分子機(jī)制。 方法 1.采用體外培養(yǎng)的D3mESCs作為染毒模型。 2.不同濃度BDE-47染毒D3mESCs24h和48h,觀察細(xì)胞形態(tài)改變。 3.不同濃度BDE-47染毒D3mESCs24h和48h, MTT法測定BDE-47對D3mESCs細(xì)胞活力的影響,確定后續(xù)試驗(yàn)染毒劑量。 4.不同濃度BDE-47染毒D3mESCs24h,利用流式細(xì)胞儀分析細(xì)胞周期改變。 5.不同濃度BDE-47染毒D3mESCs24h,利用流式細(xì)胞儀和DeadEndTM熒光測定TUNEL系統(tǒng)檢測細(xì)胞凋亡情況。Real-time RT-PCR測定促凋亡基因P53的表達(dá)水平變化。 6.不同濃度BDE-47染毒D3mESCs24h,用堿性磷酸酶染色試劑盒檢測染毒后的D3mESCs的染色狀況；用堿性磷酸酶活性分析試劑盒檢測染毒后的D3mESCs堿性磷酸酶活性改變。 7.不同濃度BDE-47染毒D3mESCs24h,固定細(xì)胞后,用細(xì)胞免疫熒光法檢測染毒后的D3mESCs的轉(zhuǎn)錄因子Oct-4/Sox-2/Nanog表達(dá)情況。 8. Real-time RT-PCR測定不同濃度BDE-47染毒后轉(zhuǎn)錄因子Oct-4/Sox-2/Nanog的表達(dá)水平變化以及mmu-miR-145和mmu-miR-34a的表達(dá)情況。 9.蛋白印跡法(western blot)測定Oct-4/Sox-2/Nanog蛋白表達(dá)水平的變化。 結(jié)果 1.不同劑量BDE47染毒24h和48h后,D3mESCs的細(xì)胞形態(tài)與對照組比較,均在25μM劑量組及以上出現(xiàn)改變,鏡下觀察細(xì)胞密度明顯減少,少數(shù)細(xì)胞碎裂,細(xì)胞出現(xiàn)明顯萎縮,部分克隆邊緣塌陷。而且染毒48h, D3mESCs細(xì)胞形態(tài)改變更加明顯。 2.MTT結(jié)果顯示,不同劑量BDE47染毒24h后,D3mESCs各劑量組細(xì)胞活力無明顯差異。不同劑量BDE47染毒48h后,100gM劑量組出現(xiàn)細(xì)胞活力改變,細(xì)胞存活率降低。確定后續(xù)試驗(yàn)染毒劑量為0.04μM,1μM,25μM，100μM；染毒時(shí)間為24h。 3.流式細(xì)胞周期結(jié)果顯示,不同劑量BDE47染毒24h后,D3mESCs細(xì)胞周期各期水平無顯著性差異。 4.流式細(xì)胞凋亡結(jié)果顯示,不同劑量BDE47染毒24h后,在25μM及100μM劑量組,D3mESCs細(xì)胞凋亡增加,統(tǒng)計(jì)具有顯著差異(P0.05).DeadEndTM熒光測定TUNEL系統(tǒng)原位分析細(xì)胞凋亡結(jié)果顯示,隨著染毒劑量增加,細(xì)胞內(nèi)凋亡信號增加,凋亡細(xì)胞顯著增加。 5.AP染色結(jié)果顯示,不同劑量BDE47染毒24h后,D3mESCs在1μM及以上劑量組出現(xiàn)細(xì)胞著色減淡,部分細(xì)胞未著色。AP活性檢測結(jié)果顯示,D3mESCs在1μM劑量組即出現(xiàn)AP活性的顯著下降。 6.細(xì)胞免疫熒光結(jié)果顯示,不同劑量BDE47染毒24h后,D3mESCs維持其干細(xì)胞多能性的轉(zhuǎn)錄因子Oct-4、Sox-2,Nanog表達(dá)降低。 7.不同劑量BDE47染毒24h后,D3mESCs維持其干細(xì)胞多能性的轉(zhuǎn)錄因子Oct-4、Sox-2,Nanog mRNA和蛋白水平均呈劑量-效應(yīng)關(guān)系下降；而MircoRNA mmu-miR-145和mmu-miR-34a水平呈劑量-效應(yīng)關(guān)系上升。 結(jié)論 1、BDE-47能引起D3mESCs細(xì)胞形態(tài)改變。染毒48h后,在100gM劑量組引起細(xì)胞活力顯著降低。BDE47能導(dǎo)致細(xì)胞凋亡增加,促凋亡基因P53表達(dá)升高。 2、BDE-47能影響D3mESCs全能性的維持,顯著抑制D3mESCs維持全能性的轉(zhuǎn)錄因子Oct-4.Sox-2,Nanog的表達(dá),其機(jī)制可能涉及促進(jìn)相關(guān)MicroRNA mmu-miR-145和mmu-miR-34a的高表達(dá)。
[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.

【學(xué)位授予單位】：南京醫(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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