摘要
为探讨玉米赤霉烯酮(ZEA)的雄性生殖毒性及其机制,本试验以TM3细胞为材料,加入不同浓度的ZEA,设对照组,5、10、20μg/L ZEA染毒组,培养72h后,应用噻唑蓝比色法(MTT)检测TM3细胞活力,免疫印迹法检测原癌基因c-myc、c-fos、c-jun及连接蛋白Cx43、P-Cx43蛋白的表达情况,应用划痕标记染料示踪(SLDT)技术观察GJIC功能的变化,同时通过免疫荧光观察Cx43在细胞内的分布情况。结果显示,各ZEA染毒组对TM3细胞生长有一定的促进作用;免疫印迹检测发现,与对照组相比,10、20μg/L染毒组c-fos的表达量明显升高(P<0.05),5、10、20μg/L染毒组c-myc、c-jun表达量极显著增加(P<0.01),而连接蛋白Cx43表达量在10μg/L染毒组中显著下降(P<0.05),在20μg/L染毒组中极显著下降(P<0.01),其磷酸化水平在20μg/L染毒组显著下降(P<0.05);荧光显微镜观察到10、20μg/L染毒组荧光黄(LY)在细胞间的扩散距离较对照组有极显著下降(P<0.01);ZEA染毒后,分布于细胞膜上的Cx43逐渐减少,出现在细胞质和细胞核上。结果表明,低浓度的ZEA即可诱发Cx43及其磷酸化水平的下调,Cx43在细胞内的异常分布,导致GJIC的功能受到抑制,同时引起原癌基因c-myc、cfos、c-jun异常的表达,低浓度的ZEA对TM3细胞的增殖产生了明显的促进作用。
In order to explain zearalenone-induced male reproductive toxicity and its mechanism,the effect of low concentration of zearalenone on proto-oncogene and GJIC were investigated in TM3cells(mice Leydig cell line).TM3 cells were used as materials,and treated with different concentrations of ZEA(0,5,10 and 20μg/L)for 72 h.The cell viability was determined by MTT assay,the function of GJIC and distribution of Cx43 were observed by fluorescence microscope.Then the expression of c-myc,c-fos,c-jun,Cx43,P-Cx43 protein was detected by western blot method.The results showed that,each ZEA group could promote the growth of TM3 cells.Compared with the control group,the expression of c-fos protein was significantly increased(P<0.05)with 10,20μg/L of ZEA and c-myc,c-jun protein were obviously increased(P<0.01)with5,10,20μg/L of ZEA.Meanwhile,the expression of Cx43 protein was significantly decreased(P<0.05)with 10μg/L of ZEA and significantly diminished(P<0.01)with 20μg/L of ZEA,its phosphorylation-level were obviously lessen(P<0.05)with 20μg/L of ZEA.SLDT assay showed that the diffused distance of LY between cells was obviously decreased(P<0.01)with10,20μg/L of ZEA.Through the immunofluorescence we could find that the distribution of Cx43 was mostly in the intracellular region rather than cell membrane after being exposed to ZEA.The results indicated that low concentration of ZEA could play a down-regulation effect of Cx43 and its phosphorylation level and induce an abnormal distribution of Cx43 in cells,then lead to the inhibition of GJIC.In addition,ZEA caused the abnormal expression of proto-oncogene:c-myc,cfos,c-jun.Low concentration of ZEA finally promoted the growth of TM3 cells.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2015年第3期467-471,共5页
Chinese Journal of Veterinary Science
基金
江苏省大学生实践创新训练计划资助项目(201411117028Z)
江苏高校优势学科建设工程资助项目(PAPD)
江苏省普通高校自然科学基金资助项目(08KJD230002)
