弓形虫感染对小鼠睾丸间质细胞睾酮分泌的影响及机制研究

Effect and Mechanism of Toxoplasma gondii Infection on Testosterone Secretion in Mouse Leydig Cells

为探究弓形虫RH株速殖子体外感染对小鼠睾丸间质细胞睾酮分泌的影响,构建了弓形虫与小鼠睾丸间质细胞共培养试验模型。将纯化后的弓形虫虫体与细胞按照0∶1、1∶5、1∶1、5∶1、10∶1、20∶1的比例共培养6 h,筛选最佳的弓形虫和细胞共培养比例。在弓形虫与细胞比例为20∶1条件下,共培养3 h、6 h、9 h和12 h,筛选最佳共培养时间。同时,使用瑞氏-吉姆萨染色液对细胞进行染色,观察共培养情况下细胞状态。培养结束,ELISA检测细胞上清的睾酮浓度,Western blot检测细胞内睾酮分泌相关蛋白StAR、CYP11A1、3β-HSD和CYP17A1的表达水平。结果表明,弓形虫与细胞共培养6 h,当弓形虫与细胞比例高于5∶1时,睾酮分泌量显著增加(P<0.05)。弓形虫与细胞比例为20∶1时,睾酮分泌量随培养时间的延长而增加,且感染组明显高于对照组(P<0.05)。染色结果发现,弓形虫1 h内即可进入MLTC-1细胞,12 h细胞内出现大量空泡,60 h左右细胞大量破裂。Western blot结果显示,共培养12 h时,感染组CYP11A1、3β-HSD和CYP17A1的蛋白表达量较对照组显著降低(P<0.05)。说明弓形虫可在短时间内进入MLTC-1细胞并通过影响类固醇激素合成酶的表达来刺激睾酮的分泌,但随着时间的延长,弓形虫会抑制相关酶的表达,抑制细胞的正常功能。

To investigate the effect of Toxoplasma gondii RH strain tachyzoite infection on testosterone secretion of mouse Leydig cells in vitro,a co-culture model of Toxoplasma gondii and mouse Leydig cells was established.The purified Toxoplasma gondii were co-cultured with cells at the ratio of 0∶1,1∶5,1∶1,5∶1,10∶1 and 20∶1 for 6 h,and the best co-culture ratio of Toxoplasma gondii and cells was selected.When the ratio of Toxoplasma gondii to cells was 20:1,the best co-culture time was screened by co-culture for 3 h,6 h,9 h and 12 h.At the same time,Swiss-Giemsa staining was used to observe the state of cells in co-culture.At the end of culture,the concentration of testosterone in the supernatant was detected by ELISA,and the expressions of testosterone secretion related proteins StAR,CYP11A1,3β-HSD and CYP17A1 were detected by Western blot.The results showed that when the ratio of Toxoplasma gondii to cells was higher than 5∶1,the secretion of testosterone increased significantly when Toxoplasma gondii was co-cultured with cells for 6 hours(P<0.05).When the ratio of Toxoplasma gondii to cells was 20∶1,the secretion of testosterone increased with the prolongation of culture time,and the testosterone secretion in the infection group was significantly higher than that in the control group(P<0.05).The staining results showed that Toxoplasma gondii could enter MLTC-1 cells within 1 hour,a large number of vacuoles appeared in the cells at 12 hours,and a large number of cells ruptured at about 60 hours.The results of Western blot showed that the protein expressions of CYP11A1,3β-HSD and CYP17A1 in the infection group were significantly lower than those in the control group at 12 h of co-culture(P<0.05).It is suggested that Toxoplasma gondii can enter MLTC-1 cells in a short time and stimulate the secretion of testosterone by affecting the expression of steroid synthase,but with the passage of time,Toxoplasma gondii can inhibit the expression of related enzymes and inhibit the normal function of cells.