双（对氯苯基）二氯乙烯与β-六氯化苯对大鼠睾丸支持细胞凋亡的影响

Effects of p,p＇-DDE and β-BHC on the apoptosis of Sertoli cells in vitro

目的探讨双（对氯苯基）二氯乙烯[1，1-dichloro-2，2-bis（p-chlorophenyl）ethylene，p，p＇-DDE]、β-六氯化苯（β-benzenehexachloride，β—BHC）及其联合作用对大鼠睾丸支持细胞凋亡半胱氨酸天冬氨酸蛋白酶（Caspase）途径的影响及其作用机制。方法以不同浓度的p，p’-DDE、β-BHC（分别为10、30、50μmol／L）及其联合（10μmol／LP，p＇-DDE＋10μmol／LB—BHC，30μmol／LP，p’-DDE＋30μmol／L β-BHC，50μmol／LP，β-DDE＋50μmol／Lβ-BHC）处理大鼠睾丸支持细胞24h，设Caspase-3抑制剂组（先用100μmol／LCaspase-3抑制剂Ac—DEVD—CHO处理支持细胞2h，再用50tunoL／LP，p’-DDE＋50μmol／LB—BHC联合处理），采用四甲基偶氮唑蓝比色法测定支持细胞的活性，吖啶橙／溴化乙锭（AO／EB）双重荧光染色测定支持细胞的凋亡率，RT—PCR法检测Caspase-3、Caspase-8及Caspase-9mRNA表达量的变化。结果10、30、50、70μmol／LP，p＇-DDE组吸光度（A值）分别为0．498±0．039、0．481±0．065、0．397±0．032和0．286±0．049，相同浓度3-BHC组A值分别为0．518±0．103、0．490±0．060、0．454±0．054和0．302±0．030，10、30、50μmol／LP，p＇-DDE与B—BHC联合组A值分别为0．483±0．048、0．473±0．058和0．337±0．052，50μmol／L浓度组与对照组比较，A值降低，差异有统计学意义（t值分别为4．599、2．716、6．537，P〈0．05）。AO／EB双重荧光染色分析结果显示，10、30、50μmol／L各染毒组的支持细胞凋亡率明显升高，与溶剂对照组（9．83±0．95）％、Caspase-3抑制剂Ac—DEVD—CHO组（15．60±2．74）％比较，P，p＇-DDE处理组分别为（23．73±2．24）％、（30．03±2．84）％和（38．04±4．98）％，B—BHC处理组分别为（24．42±3．53）％、（30．64±1．06）％和（39．88±0．17）％，联合处理组分别为（37．08±1．71）％、（42．18±3．32）％和（41．25±3．36）％。逆转录（RT）．PCR结果表明，Caspase-3、Caspase-8及Caspase-9mRNA表达量随着P，P’-DDE、β-BHC及其联合浓度的增加而增加。结论P，P’-DDE、β-BHC及其联合作用可通过激活启动Caspase-8和Caspase-9，进而激活下游效应Caspase-3引起支持细胞凋亡。

Objective To explore the effects of p,p＇-DDE and β-BHC on the apoptosis of Sertoli cells in vitro via activation of Caspase. Methods Sertoli cells were treated in vitro for 24 hours with a serial concentrations of p,p＇-DDE （ 10, 30 and 50 μmol/L） , β-BHC（ 10, 30 and 50 μmol/L） and p,p＇-DDE ±β-BHC（ 10, 30 and 50 μmol/L）. The inhibitory group was first treated with 100 μmol/L Caspase-3 inhibitor Ac-DEVD-CHO treating for 2 hours before 50 μmol/L p, p＇-DDE ± 50 μmol/L β-BHC 24 hours- treatment. The vitality of Sertoli cells was determined by MTr and the apoptosis rate was measured by AO/EB double fluorescence staining. The expressions of Caspase-3, Caspase-8 and Caspase-9 were determined by RT-PCR. Results Average optical density （A） values were 0.498 ±0.039, 0.481 ±0.065, 0.397 ± 0. 032 and 0. 286 ±0. 049 in p,p＇-DDE groups （ 10, 30, 50 and 70 μmol/L）, and 0. 518 ±0. 103, 0. 490 ±0. 060, 0.454 ± 0. 054 and 0. 302 ± 0. 030 in β-BHC groups （ 10, 30, 50 and 70 μmol/L）. In the mixture-treated groups （ 10, 30 and 50 μmol/L） , the average A values were 0. 483 ±0. 048, 0. 473 ±0. 058 and 0. 337 ±0. 052. Compared with the solvent control group （0. 527 ±0.022）, 50 μmol/L group of p,p＇- DDE,β-BHC or their mixture caused a significant decrease of Sertoli cell viability （ t values were 4. 599, 2. 716,6. 537 respectively,P 〈 0. 05）. AO/EB double fluorescence staining analysis showed that apoptosis rates of Sertoli cells were significantly increased with all treated groups. The expressions of Caspase-3, Caspase-8 and Caspase-9 were upregulated as the concentrations of p,p＇-DDE, β-BHC and their mixture were increased. Conclusion p,p＇-DDE, β-BHC and their mixture could induce the apoptosis of Sertoli cells in vitro which was associated with activation of Caspase-3 mediated by cleavage of Caspase-8 and Caspase-9.