DEHP对大鼠睾丸支持细胞间隙连接蛋白43表达和间隙连接通讯功能的影响
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摘要
目的通过对原代培养的大鼠睾丸支持细胞进行邻苯二甲酸二乙基己酯(di-2-ethylhexyl phthalate,DEHP)染毒,探讨DEHP体外对大鼠睾丸支持细胞间隙连接蛋白43(connexin 43,Cx43)表达及间隙连接通讯(gap junction intercellularcommunication,GJIC)功能的影响。方法体外分离和原代培养大鼠睾丸支持细胞,应用不同浓度的DEHP(10、50、100nmol/L)作用于支持细胞24 h,应用荧光定量PCR法检测Cx43基因mRNA表达,用Weastern blot方法检测Cx43蛋白表达,划痕标记荧光染料示踪方法检测GJIC功能。结果与对照组相比,DEHP各组支持细胞Cx43基因mRNA和蛋白表达均下降(P<0.05),呈浓度依赖关系,同时支持细胞GJIC功能降低。结论 DEHP在体外可通过抑制大鼠睾丸支持细胞Cx43基因表达而抑制GJIC功能,进而影响支持细胞功能。
[Objective]To investigate the effects of di-2-ethylhexyl phthalate(DEHP) on expressions of Connexin 43(Cx43) genes,and on the gap junctional intercellular communication(GJIC) in rat sertoli cells in vitro.[Methods]The rat sertoli cells were separated and primary cultured,and the sertoli cells were treated with different concentrations of DEHP(10,50,100 nmol/L) for 24h.The real-time fluorescence quantitative PCR was used to detect mRNA of Cx43 gene,and the western blot was used to detect the Cx43 protein.Furthermore,the GJIC was estimated by scrape-loading and dye transfer technique(SLDT).[Results] Compared with the control group,the mRNA and protein expressions of Cx43 gene in different DEHP groups were decreased(P<0.05)in a concentration dependent manner,and the GJIC in different DEHP groups was decreased.[Conclusion] DEHP can inhibit sertoli cell GJIC by down-regulating the Cx43 gene expression,which results in sertoli cell dysfunction.
[Objective]To investigate the effects of di-2-ethylhexyl phthalate(DEHP) on expressions of Connexin 43(Cx43) genes,and on the gap junctional intercellular communication(GJIC) in rat sertoli cells in vitro.[Methods]The rat sertoli cells were separated and primary cultured,and the sertoli cells were treated with different concentrations of DEHP(10,50,100 nmol/L) for 24h.The real-time fluorescence quantitative PCR was used to detect mRNA of Cx43 gene,and the western blot was used to detect the Cx43 protein.Furthermore,the GJIC was estimated by scrape-loading and dye transfer technique(SLDT).[Results] Compared with the control group,the mRNA and protein expressions of Cx43 gene in different DEHP groups were decreased(P<0.05)in a concentration dependent manner,and the GJIC in different DEHP groups was decreased.[Conclusion] DEHP can inhibit sertoli cell GJIC by down-regulating the Cx43 gene expression,which results in sertoli cell dysfunction.
引文
[1]Heudorf U,Mersch-Sundermann V,Angerer J.Phthalates:toxicologyand exposure[J].Int J Hyg Enviro Health,2007,210:623-634.
[2]Guo Y,Wu Q,Kannan K.Phthalate metabolites in urine from China,and implications for human exposures[J].Environ Int,2011,37:893-898.
[3]Silva MJ,Reidy JA,Herbert AR,et al.Detection of phthalate metabo-lites in human amniotic fluid[J].Bull Environ Contam Toxicol,2004,72:1226-1231.
[4]Andrade AJ,Grande SW,Talsness CE,et al.A dose response studyfollowing in utero and lactational exposure to di-(2-ethylhexyl)phthalate(DEHP):reproductive effects on adult male offspring rats[J].Toxicol-ogy,2006,228:85-97.
[5]Borch J,Metzdorff SB,Vinggaard AM,et al.Mechanisms underlyingthe anti-androgenic effect of diethylhexyl phthalate in fetal rat testis[J].Toxicology,2006,223:144-155.
[6]Supornsilchai V,Soder O,Svechnikov K.Stimulation of the pituitary-adrenal axis and of adrenocortical steroidogenesis ex vivo by administra-tion of di-2-ethylhexyl phthalate to prepubertal male rats[J].J Endocri-nol,2007,192:33-39.
[7]Singh S,Li SS.Phthalates:toxicogenomics and inferred human diseases[J].Genomics,2011,97:148-157.
[8]余晓晖,胡云飞.睾丸支持细胞的生物学特性及研究进展[J].中国组织工程研究与临床康复杂志,2010,14(44):8311-8314.
[9]Sohl G,Willecke K.Gap junctions and the connexin protein family[J].Cardiovasc Res,2004,62:228-232.
[10]贾喜菲,宁静慧.间隙连接蛋白43与妇科疾病[J].医学综述杂志,2011,17(3):345-347.
[11]Duffy HS,Delmar M,Spray DC.Formation of the gap junction nexus:blinding partners for connexins[J].J Physiol Paris,2002,96:243-249.
[2]Guo Y,Wu Q,Kannan K.Phthalate metabolites in urine from China,and implications for human exposures[J].Environ Int,2011,37:893-898.
[3]Silva MJ,Reidy JA,Herbert AR,et al.Detection of phthalate metabo-lites in human amniotic fluid[J].Bull Environ Contam Toxicol,2004,72:1226-1231.
[4]Andrade AJ,Grande SW,Talsness CE,et al.A dose response studyfollowing in utero and lactational exposure to di-(2-ethylhexyl)phthalate(DEHP):reproductive effects on adult male offspring rats[J].Toxicol-ogy,2006,228:85-97.
[5]Borch J,Metzdorff SB,Vinggaard AM,et al.Mechanisms underlyingthe anti-androgenic effect of diethylhexyl phthalate in fetal rat testis[J].Toxicology,2006,223:144-155.
[6]Supornsilchai V,Soder O,Svechnikov K.Stimulation of the pituitary-adrenal axis and of adrenocortical steroidogenesis ex vivo by administra-tion of di-2-ethylhexyl phthalate to prepubertal male rats[J].J Endocri-nol,2007,192:33-39.
[7]Singh S,Li SS.Phthalates:toxicogenomics and inferred human diseases[J].Genomics,2011,97:148-157.
[8]余晓晖,胡云飞.睾丸支持细胞的生物学特性及研究进展[J].中国组织工程研究与临床康复杂志,2010,14(44):8311-8314.
[9]Sohl G,Willecke K.Gap junctions and the connexin protein family[J].Cardiovasc Res,2004,62:228-232.
[10]贾喜菲,宁静慧.间隙连接蛋白43与妇科疾病[J].医学综述杂志,2011,17(3):345-347.
[11]Duffy HS,Delmar M,Spray DC.Formation of the gap junction nexus:blinding partners for connexins[J].J Physiol Paris,2002,96:243-249.