本文選題：褪黑素 + Leydig細(xì)胞　； 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：目的：觀察褪黑素對(duì)Leydig細(xì)胞睪酮合成的抑制作用，初步探討褪黑素影響睪酮分泌的作用機(jī)制。 方法：（1）采用Leydig細(xì)胞原代培養(yǎng)模型，通過I型膠原酶消化法分離C57BL/6J小鼠睪丸間質(zhì)細(xì)胞，以400（30μm）目不銹鋼網(wǎng)對(duì)小鼠睪丸間質(zhì)細(xì)胞進(jìn)行過濾、純化，對(duì)細(xì)胞進(jìn)行臺(tái)盼藍(lán)染色，用3β-羥基固醇脫氫酶（3β-HSD）特異性染色法鑒定細(xì)胞，觀察細(xì)胞形態(tài)學(xué)改變。（2）在培養(yǎng)的Leydig細(xì)胞中加入不同濃度(0，10-6，10-8，10-10和10-12mol/L)的褪黑素或/和褪黑素受體拮抗劑Luzindole（1μmol/L），作用30min后再加褪黑素，CCK-8法檢測細(xì)胞活性和增殖，流式細(xì)胞儀檢測細(xì)胞周期、線粒體膜電位和細(xì)胞活性氧（reactive oxidative species，ROS）水平，ELISA法檢測睪酮水平。（3）將Leydig細(xì)胞分為對(duì)照組、褪黑素處理組與褪黑素/Luzindole聯(lián)合作用組，，免疫熒光法測定褪黑激素受體和睪酮合成相關(guān)基因Mel1a，GATA-4，StAR和3β-HSD的蛋白分布和表達(dá)。 結(jié)果：（1）體外培養(yǎng)的細(xì)胞形態(tài)呈梭形、增殖速度快、貼壁生長狀態(tài)良好。臺(tái)盼藍(lán)染色法顯示，原代培養(yǎng)的Leydig細(xì)胞存活率在90%以上。3β-HSD特異性染色后，可見多數(shù)細(xì)胞胞質(zhì)呈藍(lán)黑色。成功建立了小鼠Leydig細(xì)胞的體外原代培養(yǎng)模型。（2）與對(duì)照組相比，加入褪黑素后，Leydig細(xì)胞增殖受到抑制，細(xì)胞培養(yǎng)上清液中睪酮含量降低，細(xì)胞線粒體膜電位下降，活性氧水平增高。褪黑素對(duì)Leydig細(xì)胞睪酮合成的抑制作用可被褪黑素受體拮抗劑Luzindole所阻斷。（3）加入褪黑素后，睪酮合成關(guān)鍵調(diào)節(jié)因子Mel1a、 GATA-4、StAR和3β-HSD的分布與表達(dá)降低，該效應(yīng)可被褪黑素受體拮抗劑Luzindole阻斷。 結(jié)論： 1、建立了Leydig細(xì)胞原代培養(yǎng)模型，發(fā)現(xiàn)褪黑素可以抑制Leydig細(xì)胞的增殖，和睪酮的合成。 2、褪黑素對(duì)睪酮合成的抑制作用可能是通過下調(diào)睪酮合成關(guān)鍵調(diào)節(jié)因子Mel1a、 GATA-4、StAR和3β-HSD等實(shí)現(xiàn)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R339.2

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