發(fā)布時(shí)間：2018-05-11 23:20

  本文選題：p + p’-DDE��； 參考：《華中科技大學(xué)》2008年碩士論文

【摘要】： 大量研究表明,許多環(huán)境污染物具有內(nèi)分泌干擾作用,特別是其對(duì)男性生殖功能的影響已引起了人們的高度重視。有機(jī)氯農(nóng)藥DDT (2,2-bis(4-chlorophenyl) -1,1, 1-trichloroethane)和六六六(benzene-hexachloride, BHC)等是污染范圍廣、危害大、在環(huán)境中存留時(shí)間長(zhǎng)并可通過(guò)食物鏈的生物放大作用而進(jìn)入機(jī)體的持久性有機(jī)污染物(persistent organic pollutants,POPs),可在機(jī)體脂肪組織和血脂中存留數(shù)十年,且主要以其代謝產(chǎn)物p,p’-DDE和β-BHC的形式存在。因此人們認(rèn)為,p,p’-DDE是DDT在環(huán)境和機(jī)體內(nèi)最持久濃度最高的代謝產(chǎn)物,而β-BHC是六六六在環(huán)境和機(jī)體內(nèi)難于降解的濃度最高的代謝產(chǎn)物。已有研究表明,p,p’-DDE和β-BHC屬于環(huán)境內(nèi)分泌干擾物(EDCs),被列為優(yōu)先消除的持久性有機(jī)污染物(POPs)。鑒于其遠(yuǎn)期的重大影響,環(huán)境抗雄激素以及相關(guān)的環(huán)境和生殖問(wèn)題的研究已經(jīng)成為目前研究的熱點(diǎn)。有關(guān)p,p’-DDE或β-BHC內(nèi)分泌干擾健康效應(yīng)的報(bào)道也開始逐漸增多,但其單獨(dú)作用以及兩者聯(lián)合作用對(duì)生殖系統(tǒng)機(jī)制的研究不是很多,尤其是p,p’-DDE或β-BHC誘導(dǎo)支持細(xì)胞凋亡的影響鮮有報(bào)道。而本研究的主要目的就是以支持細(xì)胞作為靶細(xì)胞,以p,p’-DDE與β-BHC為受試物來(lái)初步探討進(jìn)而揭示p,p’-DDE和β-BHC對(duì)支持細(xì)胞凋亡的可能作用機(jī)制。這也是本實(shí)驗(yàn)的創(chuàng)新點(diǎn)。 第一部分大鼠睪丸支持細(xì)胞的離體培養(yǎng)及p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞的活力影響 目的:探討p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞活力的影響來(lái)確定細(xì)胞的作用劑量。 方法:對(duì)離體培養(yǎng)的支持細(xì)胞分別用10μmol/L、30μmol/L、50μmol/L、70μmol/L p,p’-DDE、β-BHC及10μmol/L、30μmol/L、50μmol/L p,p’-DDE、β-BHC聯(lián)合染毒24 h后用MTT比色方法測(cè)其在490 nm的吸光度值。 結(jié)果: p,p’-DDE、β-BHC 50μmol/L、70μmol/L與溶劑對(duì)照組有顯著性差異(P 0.05);p,p’-DDE與β-BHC聯(lián)合染毒50μmol/L與溶劑對(duì)照組有顯著性差異(P 0.05);其他濃度組與溶劑對(duì)照組沒(méi)有顯著性差異。 結(jié)論:可以確定濃度高于50μmol/L的p,p’-DDE及β-BHC可以影響支持細(xì)胞的活力。p,p’-DDE、β-BHC聯(lián)合作用對(duì)支持細(xì)胞活性的影響大于兩者單獨(dú)作用。 第二部分p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞凋亡的影響 目的:檢測(cè)p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞凋亡的影響。方法:應(yīng)用吖啶橙/溴化乙錠(AO/EB)雙重?zé)晒馊旧珜?duì)經(jīng)過(guò)不同濃度(10、30和50μmol/L)的p,p’-DDE、β-BHC及其聯(lián)合處理24 h的支持細(xì)胞及先用Caspase-3抑制劑Ac-DEVD-CHO處理2 h再用50μmol/L p,p’-DDE與β-BHC聯(lián)合處理24 h的支持細(xì)胞進(jìn)行測(cè)定。 結(jié)果:支持細(xì)胞的凋亡率隨著p,p’-DDE、β-BHC及其聯(lián)合作用濃度的增大而提高(P0.05),p,p’-DDE、β-BHC及其聯(lián)合染毒各劑量組與Caspase-3抑制劑Ac-DEVD-CHO組比較,差異有統(tǒng)計(jì)學(xué)意義(P 0.05)。 第三部分p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞Caspase-3、Caspase-8、Caspase-9 mRNA轉(zhuǎn)錄水平的影響 目的:通過(guò)測(cè)定p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞作用后Caspase-3、Caspase-8及Caspase-9 mRNA的表達(dá)情況,探討p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞內(nèi)Caspase蛋白的影響,為進(jìn)一步探索其作用機(jī)制提供依據(jù)。 方法:用RT-PCR方法檢測(cè)支持細(xì)胞內(nèi)Caspase-3、Caspase-8及Caspase-9 mRNA的表達(dá)情況。 結(jié)果:支持細(xì)胞內(nèi)Caspase-3、Caspase-8及Caspase-9 mRNA隨著p,p’-DDE、β-BHC及其聯(lián)合濃度的增加而表達(dá)增加。 結(jié)論:p,p’-DDE、β-BHC及其聯(lián)合在mRNA水平上可以增加Caspase-3、Caspase-8和Caspase-9的表達(dá)。 第四部分p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞Caspase-3、Caspase-8及Caspase-9蛋白表達(dá)的影響 目的:探討p,p’-DDE、β-BHC及其聯(lián)合作用對(duì)支持細(xì)胞的Caspase凋亡途徑是否有影響。 方法:用Western blotting方法來(lái)半定量在p,p’-DDE、β-BHC及其聯(lián)合作用不同濃度及不同作用時(shí)間內(nèi)的Caspase-3、Caspase-8及Caspase-9蛋白的表達(dá)情況。 結(jié)果:不同濃度的p,p’-DDE、β-BHC及其聯(lián)合處理支持細(xì)胞細(xì)胞24 h后,Caspase-3、Caspase-8及Caspase-9酶原蛋白帶均變細(xì),隨染毒濃度增加,酶原與β-actin的灰度值之比逐漸減小,即Caspase-3、Caspase-8及Caspase-9酶原裂解增加。相同濃度高劑量組p,p’-DDE、β-BHC及其聯(lián)合處理支持細(xì)胞細(xì)胞不同時(shí)間后,Caspase-3、Caspase-8及Caspase-9酶原蛋白帶也變細(xì),隨著染毒時(shí)間延長(zhǎng),酶原與β-actin的灰度值之比也逐漸減小。Caspase-3、Caspase-8及Caspase-9酶原蛋白的裂解,隨著毒物作用濃度的升高,毒物作用的時(shí)間延長(zhǎng)而增強(qiáng)。 結(jié)論:p,p’-DDE與β-BHC對(duì)大鼠睪丸支持細(xì)胞凋亡可能起著協(xié)同作用。 從上述結(jié)果可以看出:1. p,p’-DDE、β-BHC及其聯(lián)合引起支持細(xì)胞的凋亡的發(fā)生。支持細(xì)胞是曲細(xì)精管中唯一的體細(xì)胞,對(duì)于精子的發(fā)生具有不可替代的作用。p,p’-DDE是一種環(huán)境抗雄激素,β-BHC是環(huán)境擬雌激素,兩者單獨(dú)或聯(lián)合作用引起支持細(xì)胞的凋亡,這必然影響到生殖細(xì)胞功能從而導(dǎo)致生精功能的紊亂。2. p,p’-DDE、β-BHC及其聯(lián)合可以明顯提高Caspase-3、Caspase-8和Caspase-9 mRNA表達(dá)量。3. p,p’-DDE、β-BHC及其聯(lián)合可以活化酶原形式的Caspase-3、Caspase-8和Caspase-9,減少其表達(dá);50μmol/L p,p’-DDE、β-BHC及其聯(lián)合呈時(shí)間依賴性促進(jìn)酶原形式的Caspase-3、Caspase-8及Caspase-9的活化。綜合第二點(diǎn)、第三點(diǎn)可以看出p,p’-DDE與β-BHC對(duì)支持細(xì)胞的作用機(jī)制除了公認(rèn)的激素受體途徑之外還可以通過(guò)細(xì)胞凋亡通路。凋亡細(xì)胞的形態(tài)變化大多由胱天蛋白酶系(Caspases)引發(fā),一旦胞內(nèi)的這些酶被特異性激活,細(xì)胞即進(jìn)入凋亡。Caspase一經(jīng)活化導(dǎo)致底物裂解細(xì)胞凋亡將不可逆地進(jìn)行下去,因而又成為凋亡的重要標(biāo)志。在Caspase級(jí)聯(lián)反應(yīng)中,Caspsse-3是下游一個(gè)重要的公共凋亡效應(yīng)因子,在各種因素引發(fā)的凋亡程序中起最后的樞紐作用。支持細(xì)胞在給予p,p’-DDE、β-BHC及其聯(lián)合染毒24 h,細(xì)胞凋亡率隨著毒物濃度的增加而增加,且凋亡可以被Caspase-3抑制劑Ac-DEVD-CHO所抑制,表明p,p’-DDE、β-BHC及其聯(lián)合所致支持細(xì)胞的凋亡是通過(guò)Caspase-3途徑產(chǎn)生作用。Caspase-3、Caspase-8和Caspase-9 mRNA表達(dá)隨著p,p’-DDE、β-BHC及其聯(lián)合濃度的增加而增加,且其酶原呈劑量及時(shí)間依賴性減少,表明凋亡是通過(guò)激活啟動(dòng)Caspase-8和Caspase-9,進(jìn)而激活下游效應(yīng)Caspase-3的級(jí)聯(lián)反應(yīng)來(lái)實(shí)現(xiàn)。本研究為進(jìn)一步研究其具體的作用途徑提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:A large number of studies have shown that many environmental pollutants have endocrine disrupting effects, especially their effects on male reproductive function have aroused great attention. Organochlorine pesticides DDT (2,2-bis (4-chlorophenyl) -1,1, 1-trichloroethane) and 666 (benzene-hexachloride, BHC) are widely polluted and endanger the environment. Persistent organic pollutants (POPs), which is retained for a long time and through the biological amplification of the food chain, can be stored for decades in body fat tissue and blood lipids, and mainly in the form of its metabolites P, p '-DDE and beta -BHC. Therefore, P, p' -DDE is the environment and machine of DDT. The most persistent metabolite in the body, while beta -BHC is the highest metabolite of 666 in the environment and in the body, is the most difficult to degrade. Studies have shown that P, p '-DDE and beta -BHC belong to environmental endocrine disruptors (EDCs) and are listed as the priority elimination of persistent organic pollutants (POPs). In view of their long-term significant effects, environmental resistance Research on androgens and related environmental and reproductive problems has become a hot spot. Reports on the health effects of P, p '-DDE or beta -BHC endocrine disrupting health are beginning to increase, but their separate effects and the combination of the two effects on reproductive system mechanisms are not much, especially P, p' -DDE or beta -BHC induces fine support. The main purpose of this study is to explore the possible mechanisms of P, p '-DDE and beta -BHC to support cell apoptosis by supporting cells as target cells and using P, p' -DDE and beta -BHC as subjects. This is also an innovation in this experiment.
Part one in vitro culture of rat testis sertoli cells and the effect of P, p '-DDE, beta -BHC and their combined action on the viability of Sertoli cells
Objective: To investigate the effects of P, p '-DDE, beta -BHC and their combined action on the viability of Sertoli cells in order to determine the dose of cells.
Methods: the support cells in the isolated culture were 10 mu mol/L, 30 mol/L, 50 mu mol/L, 70 mol/L P, p '-DDE, beta -BHC and 10 micron mol/L, 30 mu mol/L, 50 mu mol/L P.
Results: P, p '-DDE, beta -BHC 50 mu mol/L, 70 mol/L and solvent control group were significantly different (P 0.05); P, p' -DDE and beta -BHC combined 50 micron mol/L and solvent control group had significant difference (0.05), and there was no significant difference between the other concentration group and the solvent control group.
Conclusion: P, p '-DDE and beta -BHC can affect the activity of.P, p' -DDE, and the effect of the combination of beta -BHC on the activity of support cells is greater than that of the 50 u mol/L.
The second part is the effect of P, p '-DDE, beta -BHC and their combined action on the apoptosis of Sertoli cells.
Objective: to detect the effect of P, p '-DDE, beta -BHC and their combined action on the support of cell apoptosis. Methods: using acridine orange / ethidium bromide (AO/EB) double fluorescence staining for P, p' -DDE, beta -BHC and their combined treatment of 24 h cells with different concentrations (10,30 and 50 mol/L) and 50 micron P, p '-DDE and beta -BHC co processed 24 h support cells.
Results: the apoptosis rate of the supporting cells increased with the increase of P, p '-DDE, beta -BHC and the concentration of combined action (P0.05). The difference was statistically significant (P 0.05) compared with Ac-DEVD-CHO group of Caspase-3 inhibitor, P, p' -DDE, beta -BHC and its combined dosage groups.
The third part is the effect of P, p '-DDE, beta -BHC and their combined action on the transcriptional level of Caspase-3, Caspase-8 and Caspase-9 mRNA in supporting cells.
Objective: To explore the effect of P, p '-DDE, beta -BHC and its combined action on supporting intracellular proteins by measuring the expression of P, p' -DDE, beta -BHC and their combined action on Caspase-3, Caspase-8 and Caspase-9 mRNA after the action of supporting cells, and to provide a basis for further exploring its mechanism.
Methods: the expression of Caspase-3, Caspase-8 and Caspase-9 mRNA in Sertoli cells was detected by RT-PCR.
Results: the expression of Caspase-3, Caspase-8 and Caspase-9 mRNA in Sertoli cells increased with the increase of P, P -DDE, beta -BHC and their combined concentrations.
Conclusion: P, p '-DDE, beta -BHC and their combination can increase Caspase-3, Caspase-8 and Caspase-9 expression at mRNA level.
The fourth part is the effect of P, p '-DDE, beta -BHC and their combined action on the expression of Caspase-3, Caspase-8 and Caspase-9 proteins in supporting cells.
Objective: To investigate whether P, p '-DDE, beta -BHC and their combined effects affect the Caspase apoptotic pathway of Sertoli cells.
Methods: the expression of Caspase-3, Caspase-8 and Caspase-9 protein in P, p '-DDE, beta -BHC and their combined effects in different concentrations and different periods of action were semi quantified by Western blotting method.
Results: after different concentrations of P, p '-DDE, beta -BHC and the combined treatment of 24 h of cell cells, the bands of Caspase-3, Caspase-8 and Caspase-9 enzyme protein were all finer. As the concentration increased, the ratio of the gray value of the enzyme between the enzyme and the beta -actin decreased gradually, that is, Caspase-3, Caspase-8 and the fragmentation of the Caspase-9 enzyme. After -BHC and its combined treatment of cell cells at different time, the white band of Caspase-3, Caspase-8 and Caspase-9 zymogen also became thinner. With the prolonged exposure time, the ratio of the gray value of the enzyme between the enzyme and the beta -actin gradually decreased.Caspase-3, the lysis of the Caspase-8 and the Caspase-9 enzyme protein, and the time prolongation of the toxic action with the increase of the concentration of the toxicant. Long and enhanced.
Conclusion: P, p '-DDE and beta -BHC may play a synergistic role in the apoptosis of rat Sertoli cells.
From the above results, we can see that 1. P, p '-DDE, beta -BHC and their combination cause the apoptosis of supporting cells. Support cells are the only body cells in the seminiferous tubules, which have an irreplaceable role in spermatogenesis,.P, p' -DDE is an environmental androgen, and beta -BHC is an environmental estrogens, both alone or combined to cause branches. .2. P, p '-DDE, beta -BHC and its combination can significantly improve Caspase-3, Caspase-8 and Caspase-9 mRNA expression.3. P, p', and its combined activator, reducing its expression; reducing its expression; 50 micron. /L P, p '-DDE, beta -BHC and their combination are time dependent to promote the activation of Caspase-3, Caspase-8 and Caspase-9 in the proenzyme form. A comprehensive second point, third points can be seen P, p' -DDE and beta -BHC to support cells in addition to the recognized hormone receptor pathway in addition to the apoptosis pathway through cell apoptosis. Most of these are triggered by cystine (Caspases). Once the intracellular enzymes are activated specifically, the cells enter apoptotic.Caspase as soon as they are activated, resulting in the irreversible apoptosis of the substrate cracking cells, and thus become an important marker of apoptosis. In the cascade reaction of Caspase, Caspsse-3 is an important public apoptosis effect downstream. P, p '-DDE, beta -BHC and its combined exposure to 24 h, the apoptosis rate increases with the increase of poison concentration, and apoptosis can be suppressed by Caspase-3 inhibitor Ac-DEVD-CHO, indicating P, p' -DDE, beta -BHC and associated support cells. The apoptosis is induced by Caspase-3 pathway, and the expression of.Caspase-3, Caspase-8 and Caspase-9 mRNA increases with the increase of P, p '-DDE, beta -BHC and its combined concentration, and the dose and time dependence of the enzyme decreases, indicating that apoptosis is activated by activating Caspase-8 and Caspase-9, and then activating downstream effect Caspase-3 cascade. This study provides an experimental basis for further study of its specific role.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 吳慧明;螺蟲乙酯對(duì)雄性大鼠毒性及其機(jī)制研究[D];浙江大學(xué);2013年

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本文編號(hào)：1876090