褪黑素对小鼠Leydig细胞睾酮合成的抑制作用及机制

发布时间：2018-04-20 23:21

本文选题：褪黑素 + Leydig细胞　；参考：《苏州大学》2014年硕士论文

【摘要】：目的：观察褪黑素对Leydig细胞睾酮合成的抑制作用，初步探讨褪黑素影响睾酮分泌的作用机制。方法：（1）采用Leydig细胞原代培养模型，通过I型胶原酶消化法分离C57BL/6J小鼠睾丸间质细胞，以400（30μm）目不锈钢网对小鼠睾丸间质细胞进行过滤、纯化，对细胞进行台盼蓝染色，用3β-羟基固醇脱氢酶（3β-HSD）特异性染色法鉴定细胞，观察细胞形态学改变。（2）在培养的Leydig细胞中加入不同浓度(0，10-6，10-8，10-10和10-12mol/L)的褪黑素或/和褪黑素受体拮抗剂Luzindole（1μmol/L），作用30min后再加褪黑素，CCK-8法检测细胞活性和增殖，流式细胞仪检测细胞周期、线粒体膜电位和细胞活性氧（reactive oxidative species，ROS）水平，ELISA法检测睾酮水平。（3）将Leydig细胞分为对照组、褪黑素处理组与褪黑素/Luzindole联合作用组，，免疫荧光法测定褪黑激素受体和睾酮合成相关基因Mel1a，GATA-4，StAR和3β-HSD的蛋白分布和表达。结果：（1）体外培养的细胞形态呈梭形、增殖速度快、贴壁生长状态良好。台盼蓝染色法显示，原代培养的Leydig细胞存活率在90%以上。3β-HSD特异性染色后，可见多数细胞胞质呈蓝黑色。成功建立了小鼠Leydig细胞的体外原代培养模型。（2）与对照组相比，加入褪黑素后，Leydig细胞增殖受到抑制，细胞培养上清液中睾酮含量降低，细胞线粒体膜电位下降，活性氧水平增高。褪黑素对Leydig细胞睾酮合成的抑制作用可被褪黑素受体拮抗剂Luzindole所阻断。（3）加入褪黑素后，睾酮合成关键调节因子Mel1a、 GATA-4、StAR和3β-HSD的分布与表达降低，该效应可被褪黑素受体拮抗剂Luzindole阻断。结论： 1、建立了Leydig细胞原代培养模型，发现褪黑素可以抑制Leydig细胞的增殖，和睾酮的合成。 2、褪黑素对睾酮合成的抑制作用可能是通过下调睾酮合成关键调节因子Mel1a、 GATA-4、StAR和3β-HSD等实现的。
[Abstract]:Aim: to observe the inhibitory effect of melatonin on testosterone synthesis in Leydig cells and to explore the mechanism of the effect of melatonin on testosterone secretion. Methods the primary culture model of Leydig cells was used to isolate the interstitial cells from testis of C57BL/6J mice by type I collagenase digestion. Leydig cells of mice testis were filtered and purified with 400 ~ 30 渭 m stainless steel mesh, and the cells were stained with trypan blue. Cells were identified by 3 尾 -hydroxysteroid dehydrogenase 3 尾 -HSD-specific staining. Observe the morphological changes of Leydig cells cultured with different concentrations of melatonin or / and melatonin receptor antagonist Luzindole(1 渭 mol / L, add different concentrations of melatonin or / and melatonin receptor antagonist Luzindole(1 渭 mol / L to Leydig cells cultured in different concentrations, and then add melatonin CCK-8 to detect cell activity and proliferation, and flow cytometry to detect cell cycle. Mitochondrial membrane potential (MMP) and reactive oxygen species (Ros) levels of Leydig cells were determined by Elisa. The Leydig cells were divided into control group, melatonin treated group and melatonin / luzindole group. The protein distribution and expression of melatonin receptor and testosterone biosynthesis related gene Mel1aHSD, GATA-4, star and 3 尾 -HSD were detected by immunofluorescence assay. Results the cells cultured in vitro were fusiform, rapid proliferation and good adherent growth. Trypan blue staining showed that the survival rate of primary cultured Leydig cells was more than 90%. 3 尾 -HSD specific staining showed that the cytoplasm of most of the cells was blue black. The primary culture model of mouse Leydig cells in vitro was successfully established. Compared with the control group, the proliferation of mouse Leydig cells was inhibited after melatonin was added, the testosterone content in the supernatant of cell culture decreased, the mitochondrial membrane potential decreased and the level of reactive oxygen species increased. The inhibitory effect of melatonin on testosterone synthesis in Leydig cells could be blocked by melatonin receptor antagonist Luzindole). After melatonin was added to melatonin, the distribution and expression of Mel1a, GATA-4, star and 3 尾 -HSD, the key regulatory factors for testosterone synthesis, were decreased. This effect can be blocked by melatonin receptor antagonist Luzindole. Conclusion: 1. The primary culture model of Leydig cells was established, and it was found that melatonin could inhibit the proliferation of Leydig cells and the synthesis of testosterone. 2. The inhibitory effect of melatonin on testosterone synthesis may be achieved by down-regulating the key regulatory factors of testosterone synthesis, such as Mel1a, GATA-4StAR and 3 尾 -HSD.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R339.2

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