p，p’-DDE和β-BHC诱导大鼠睾丸支持细胞凋亡Caspase途径的研究

发布时间：2018-05-11 23:20

本文选题：p + p’-DDE　；参考：《华中科技大学》2008年硕士论文

【摘要】： 大量研究表明,许多环境污染物具有内分泌干扰作用,特别是其对男性生殖功能的影响已引起了人们的高度重视。有机氯农药DDT (2,2-bis(4-chlorophenyl) -1,1, 1-trichloroethane)和六六六(benzene-hexachloride, BHC)等是污染范围广、危害大、在环境中存留时间长并可通过食物链的生物放大作用而进入机体的持久性有机污染物(persistent organic pollutants,POPs),可在机体脂肪组织和血脂中存留数十年,且主要以其代谢产物p,p’-DDE和β-BHC的形式存在。因此人们认为,p,p’-DDE是DDT在环境和机体内最持久浓度最高的代谢产物,而β-BHC是六六六在环境和机体内难于降解的浓度最高的代谢产物。已有研究表明,p,p’-DDE和β-BHC属于环境内分泌干扰物(EDCs),被列为优先消除的持久性有机污染物(POPs)。鉴于其远期的重大影响,环境抗雄激素以及相关的环境和生殖问题的研究已经成为目前研究的热点。有关p,p’-DDE或β-BHC内分泌干扰健康效应的报道也开始逐渐增多,但其单独作用以及两者联合作用对生殖系统机制的研究不是很多,尤其是p,p’-DDE或β-BHC诱导支持细胞凋亡的影响鲜有报道。而本研究的主要目的就是以支持细胞作为靶细胞,以p,p’-DDE与β-BHC为受试物来初步探讨进而揭示p,p’-DDE和β-BHC对支持细胞凋亡的可能作用机制。这也是本实验的创新点。第一部分大鼠睾丸支持细胞的离体培养及p,p’-DDE、β-BHC及其联合作用对支持细胞的活力影响目的:探讨p,p’-DDE、β-BHC及其联合作用对支持细胞活力的影响来确定细胞的作用剂量。方法:对离体培养的支持细胞分别用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联合染毒24 h后用MTT比色方法测其在490 nm的吸光度值。结果: p,p’-DDE、β-BHC 50μmol/L、70μmol/L与溶剂对照组有显著性差异(P 0.05);p,p’-DDE与β-BHC联合染毒50μmol/L与溶剂对照组有显著性差异(P 0.05);其他浓度组与溶剂对照组没有显著性差异。结论:可以确定浓度高于50μmol/L的p,p’-DDE及β-BHC可以影响支持细胞的活力。p,p’-DDE、β-BHC联合作用对支持细胞活性的影响大于两者单独作用。第二部分p,p’-DDE、β-BHC及其联合作用对支持细胞凋亡的影响目的:检测p,p’-DDE、β-BHC及其联合作用对支持细胞凋亡的影响。方法:应用吖啶橙/溴化乙锭(AO/EB)双重荧光染色对经过不同浓度(10、30和50μmol/L)的p,p’-DDE、β-BHC及其联合处理24 h的支持细胞及先用Caspase-3抑制剂Ac-DEVD-CHO处理2 h再用50μmol/L p,p’-DDE与β-BHC联合处理24 h的支持细胞进行测定。结果:支持细胞的凋亡率随着p,p’-DDE、β-BHC及其联合作用浓度的增大而提高(P0.05),p,p’-DDE、β-BHC及其联合染毒各剂量组与Caspase-3抑制剂Ac-DEVD-CHO组比较,差异有统计学意义(P 0.05)。第三部分p,p’-DDE、β-BHC及其联合作用对支持细胞Caspase-3、Caspase-8、Caspase-9 mRNA转录水平的影响目的:通过测定p,p’-DDE、β-BHC及其联合作用对支持细胞作用后Caspase-3、Caspase-8及Caspase-9 mRNA的表达情况,探讨p,p’-DDE、β-BHC及其联合作用对支持细胞内Caspase蛋白的影响,为进一步探索其作用机制提供依据。方法:用RT-PCR方法检测支持细胞内Caspase-3、Caspase-8及Caspase-9 mRNA的表达情况。结果:支持细胞内Caspase-3、Caspase-8及Caspase-9 mRNA随着p,p’-DDE、β-BHC及其联合浓度的增加而表达增加。结论:p,p’-DDE、β-BHC及其联合在mRNA水平上可以增加Caspase-3、Caspase-8和Caspase-9的表达。第四部分p,p’-DDE、β-BHC及其联合作用对支持细胞Caspase-3、Caspase-8及Caspase-9蛋白表达的影响目的:探讨p,p’-DDE、β-BHC及其联合作用对支持细胞的Caspase凋亡途径是否有影响。方法:用Western blotting方法来半定量在p,p’-DDE、β-BHC及其联合作用不同浓度及不同作用时间内的Caspase-3、Caspase-8及Caspase-9蛋白的表达情况。结果:不同浓度的p,p’-DDE、β-BHC及其联合处理支持细胞细胞24 h后,Caspase-3、Caspase-8及Caspase-9酶原蛋白带均变细,随染毒浓度增加,酶原与β-actin的灰度值之比逐渐减小,即Caspase-3、Caspase-8及Caspase-9酶原裂解增加。相同浓度高剂量组p,p’-DDE、β-BHC及其联合处理支持细胞细胞不同时间后,Caspase-3、Caspase-8及Caspase-9酶原蛋白带也变细,随着染毒时间延长,酶原与β-actin的灰度值之比也逐渐减小。Caspase-3、Caspase-8及Caspase-9酶原蛋白的裂解,随着毒物作用浓度的升高,毒物作用的时间延长而增强。结论:p,p’-DDE与β-BHC对大鼠睾丸支持细胞凋亡可能起着协同作用。从上述结果可以看出:1. p,p’-DDE、β-BHC及其联合引起支持细胞的凋亡的发生。支持细胞是曲细精管中唯一的体细胞,对于精子的发生具有不可替代的作用。p,p’-DDE是一种环境抗雄激素,β-BHC是环境拟雌激素,两者单独或联合作用引起支持细胞的凋亡,这必然影响到生殖细胞功能从而导致生精功能的紊乱。2. p,p’-DDE、β-BHC及其联合可以明显提高Caspase-3、Caspase-8和Caspase-9 mRNA表达量。3. p,p’-DDE、β-BHC及其联合可以活化酶原形式的Caspase-3、Caspase-8和Caspase-9,减少其表达;50μmol/L p,p’-DDE、β-BHC及其联合呈时间依赖性促进酶原形式的Caspase-3、Caspase-8及Caspase-9的活化。综合第二点、第三点可以看出p,p’-DDE与β-BHC对支持细胞的作用机制除了公认的激素受体途径之外还可以通过细胞凋亡通路。凋亡细胞的形态变化大多由胱天蛋白酶系(Caspases)引发,一旦胞内的这些酶被特异性激活,细胞即进入凋亡。Caspase一经活化导致底物裂解细胞凋亡将不可逆地进行下去,因而又成为凋亡的重要标志。在Caspase级联反应中,Caspsse-3是下游一个重要的公共凋亡效应因子,在各种因素引发的凋亡程序中起最后的枢纽作用。支持细胞在给予p,p’-DDE、β-BHC及其联合染毒24 h,细胞凋亡率随着毒物浓度的增加而增加,且凋亡可以被Caspase-3抑制剂Ac-DEVD-CHO所抑制,表明p,p’-DDE、β-BHC及其联合所致支持细胞的凋亡是通过Caspase-3途径产生作用。Caspase-3、Caspase-8和Caspase-9 mRNA表达随着p,p’-DDE、β-BHC及其联合浓度的增加而增加,且其酶原呈剂量及时间依赖性减少,表明凋亡是通过激活启动Caspase-8和Caspase-9,进而激活下游效应Caspase-3的级联反应来实现。本研究为进一步研究其具体的作用途径提供了实验依据。
[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.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

【引证文献】