以线粒体为靶点研究重金属铅对睾丸间质细胞损伤的毒理作用机制

发布时间：2018-03-26 06:33

  本文选题：铅　切入点：睾丸间质细胞　出处：《暨南大学》2013年硕士论文

【摘要】：目的: 研究重金属铅对睾丸间质细胞的损伤作用，定位到线粒体，阐明其毒理作用机制，为临床男性性功能低下的治疗提供实验依据。 方法: 以100uM浓度的醋酸铅作用于睾丸间质细胞株R2C，加入25μM的羟基胆固醇作为孕酮合成底物，在1、3、6、12和24h分别收取正常和铅暴露细胞样品，应用放射免疫学技术检测培养上清中孕酮的含量；应用Real time RT-PCR和Western blot的方法验证孕酮合成相关酶的表达水平；应用流式细胞仪技术检测线粒体膜电位，并以透射电镜的技术观察线粒体形态学变化；应用ELISA的方法检测细胞内的cAMP水平，，并通过Western blot的方法检测cAMP-PKA/PKC-ERK1/2通路上相关蛋白的表达及其磷酸化水平，最后又通过免疫荧光技术检测了MEK/ERK与线粒体共定位的变化。 结果: 对比同一时间点的正常对照组，重金属铅（100μM）使R2C细胞培养上清中的孕酮含量明显减少，并呈现时效关系，在12h（P0.01）与24h（P0.001）具有统计学差异。检测孕酮合成相关酶的基因和蛋白表达水平结果显示，StAR基因表达从1h开始就出现明显的下调趋势，CYP11A1基因表达从6h开始出现明显的下调趋势，而3β-HSD基因表达从3h开始出现明显的下调趋势；StAR蛋白从1h开始就出现明显的下调趋势，CYP11A1蛋白从1h就开始下调，3β-HSD蛋白从12h开始出现明显的下调趋势。流式细胞仪技术检测结果显示，重金属铅作用1h就能够观察到线粒体膜电位降低9.37%，随后3和6h略有回升，接着在12h和24h保持下降10%的水平，与正常组比较均具有统计学差异。电镜结果发现醋酸铅作用1h开始就发生了损伤，直到24h呈现时间效应关系。cAMP-PKA/PKC-ERK1/2信号通路相关蛋白检测显示，铅暴露使细胞内cAMP水平自1h开始显著下降，PKA和PKC表达下降，ERK和MEK蛋白磷酸化水平也相应显著下调。免疫荧光结果发现重金属铅作用后，MEK/ERK与线粒体共定位减少。 结论: 重金属铅能够抑制睾丸间质细胞分泌孕酮的功能，能够在基因和蛋白水平上抑制孕酮合成关键酶StAR、CYP11A1和3β-HSD的表达。铅导致线粒体发生损伤，使线粒体膜电位下降。重金属铅对睾丸间质细胞的毒理作用与cAMP-PKA/PKC-ERK1/2信号通路的抑制密切相关。铅促使睾丸间质细胞内cAMP水平下降，cAMP的下降同时抑制PKA和PKC的蛋白表达，显示PKA和PKC都同时参与了该类固醇激素合成通路；PKA/PKC的下调抑制了MEK1/2和ERK1/2的磷酸化，进而影响StAR的活化，抑制孕酮的合成和分泌。这一机制的提出为部分因雄激素缺乏而导致的男性功能衰减的治疗和男性性腺功能衰退的治疗提供了实验依据。
[Abstract]:Objective:. To study the damage effect of heavy metal lead on Leydig cells of testis, locate mitochondria, elucidate its toxicological mechanism, and provide experimental basis for the treatment of male sexual dysfunction. Methods:. The Leydig cell line R2C was treated with 100uM concentration of lead acetate, and 25 渭 M hydroxyl cholesterol was added as the synthesis substrate of progesterone. Normal and lead exposed cell samples were collected at 1 ~ (3) O ~ (6) O _ (12) and 24 h, respectively. Radioimmunoassay was used to detect progesterone content in culture supernatant; Real time RT-PCR and Western blot were used to verify the expression of progesterone synthase; flow cytometry was used to detect mitochondrial membrane potential. The morphological changes of mitochondria were observed by transmission electron microscopy (TEM), the level of cAMP in cells was detected by ELISA, and the expression and phosphorylation of related proteins in cAMP-PKA/PKC-ERK1/2 pathway were detected by Western blot. Finally, the co-localization of MEK/ERK and mitochondria was detected by immunofluorescence technique. Results:. Compared with the normal control group at the same time point, the concentration of progesterone in the supernatant of R2C cells was significantly decreased by heavy metal lead (100 渭 M). The gene and protein expression level of progesterone synthase were detected. The results showed that the expression of star gene was down-regulated from 1h to 6h, and CYP11A1 gene expression was down-regulated from 6h to 6h. However, the expression of 3 尾 -HSD gene showed a down-regulation trend from 3 h to 1 h. The CYP11A1 protein began to down-regulate from 1 h to 12 h. Flow cytometry showed that the expression of 3 尾 -HSD protein was down-regulated from 1 h to 12 h, and the results of flow cytometry showed that the expression of CYP11A1 protein was down-regulated from 1 h to 12 h. After exposure to heavy metal lead for 1 hour, the mitochondrial membrane potential decreased by 9.377.The mitochondrial membrane potential increased slightly at 3 and 6 h, and then decreased by 10% at 12 h and 24 h, respectively. The results of electron microscope showed that the damage occurred at 1 h after exposure to lead acetate, until 24 hours after exposure to lead acetate. The results showed that the signal pathway related proteins of cAMP-PKA / PKC-ERK1 / 2 showed a time-dependent relationship. The level of cAMP and PKC expression decreased significantly from 1 h after lead exposure, and the phosphorylation of ERK and MEK protein were also significantly down-regulated. The results of immunofluorescence showed that the co-localization of MEK / ERK and mitochondria was decreased after heavy metal lead exposure. Conclusion:. Heavy metal lead can inhibit the secretion of progesterone in the interstitial cells of testis, and inhibit the expression of the key enzymes of progesterone synthesis, StARP11A1 and 3 尾 -HSD, at the gene and protein levels. The toxic effect of heavy metal lead on Leydig cells was closely related to the inhibition of cAMP-PKA/PKC-ERK1/2 signaling pathway. Lead induced the decrease of cAMP level in Leydig cells and inhibited the expression of PKA and PKC proteins. The results showed that both PKA and PKC were involved in the downregulation of PKA / PKC in the steroid hormone synthesis pathway, which inhibited the phosphorylation of MEK1/2 and ERK1/2, thus affecting the activation of StAR. Inhibition of progesterone synthesis and secretion. This mechanism provides experimental evidence for the treatment of male functional decline caused partly by androgen deficiency and the treatment of male gonadal dysfunction.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【参考文献】

中国期刊全文数据库 前5条

1 汪美贞;贾秀英;;铅对雄性生殖毒性的研究进展[J];动物学杂志;2006年01期

2 金海丽;铅毒性的研究进展[J];广东微量元素科学;2004年10期

3 张秋玲;周桂侠;;国内铅对雌性生殖毒性的研究进展[J];工业卫生与职业病;2008年02期

4 张秋玲;戴雪松;李刚;;铅暴露对雄性生殖系统毒性的研究进展[J];中国工业医学杂志;2009年05期

5 任军慧,朱伟杰;铅离子对雄(男)性生殖系统的毒性影响[J];生殖与避孕;2005年02期

中国重要会议论文全文数据库 前1条

1 周丹;沈勤;;ERK信号转导通路在LPS诱导的炎症过程中的表达[A];华东六省一市生物化学与分子生物学会2008年学术交流会论文摘要汇编[C];2008年

本文编号：1666751