褪黑素对牦牛黄体细胞生长及功能影响的研究

doi:10.11843/j.issn.0366-6964.2022.02.013

摘要/Abstract

摘要： 本试验旨在探究褪黑素（melatonin，MT）对牦牛黄体细胞生长、抗氧化性及功能的影响。本试验以健康牦牛二代黄体细胞为研究对象，比较了不同浓度MT（0（Control组）、25、125、250、500 pg·mL^-1）对牦牛黄体细胞生长和功能的影响。通过CCK-8法检测黄体细胞的生长情况，并采用qRT-PCR检测增殖相关基因PCNA，凋亡相关基因BCL-2、BAX、FAS，抗氧化相关基因SOD1、SOD2、GPX1和CAT以及孕酮合成相关基因HSD3β、STAR和CYP11A1 mRNA的表达水平，应用ELISA检测黄体细胞ROS和孕酮水平。结果显示，添加不同浓度的MT培养能促进细胞增殖，加速黄体细胞进入平台期。MT能促进黄体细胞增殖基因PCNA和抑凋亡基因BCL-2 mRNA的表达并抑制促凋亡基因BAX、FAS的表达。同时，MT能显著降低黄体细胞中的ROS水平并促进黄体细胞分泌孕酮，且浓度在250 pg·mL^-1时上述作用最明显。在此浓度作用下，黄体细胞中抗氧化相关基因SOD2、GPX1及CAT和孕酮合成相关基因STAR、CYP11A1 mRNA 的表达量明显上调。与仅添加MT相比，应用MT及MT受体抑制剂Luzindole联合处理黄体细胞可显著升高黄体细胞的ROS水平并抑制SOD2、GPX1及CAT的表达，孕酮水平及STAR和CYP11A1表达量均显著下调。综上表明，MT可能通过上调PCNA、BCL-2，下调BAX、FAS mRNA表达量进而促进黄体细胞增殖。MT可能通过受体介导调控了黄体细胞的ROS和孕酮水平。本研究为MT治疗黄体异常引发的生殖疾病以及其在提高牦牛繁殖性能上的应用提供了一定的理论基础。

关键词: 褪黑素, 牦牛, 黄体细胞, 抗氧化性, 孕酮

Abstract: This experiment was intended to research the effect of melatonin(MT)on the growth, antioxidant activity and function in yak luteal cells. The 2nd-passage luteal cells from healthy yak were used as research object in this experiment, and the effect of different concentrations (0 (Control), 25, 125, 250, and 500 pg·mL^-1) of melatonin on the growth and function in yak luteal cells were compared. Cell proliferation and viability were evaluated by CCK-8 assay. qRT-PCR was conducted to analyse the mRNA expression levels of several genes, including proliferation-related gene PCNA, apoptosis-related genes BCL-2, BAX, FAS, antioxidant-related genes SOD1, SOD2, GPX1 and CAT, as well as progesterone synthesis-related genes HSD3β, STAR and CYP11A1. The levels of ROS and progesterone were determined by ELISA. The results showed that the supplementation of different concentrations of MT could promote luteal cells proliferation and accelerate them into the plateau phase. MT could promote the mRNA expression of cell-proliferated gene PCNA and anti-apoptosis-related gene BCL-2, and repress the expression of apoptosis-related genes BAX, FAS. MT could reduce the ROS level and promote progesterone secretion significantly in luteal cells, moreover, the above effects were most obvious when the MT concentration was 250 pg·mL^-1. At this concentration, the mRNA expression levels of antioxidant-related genes SOD2, GPX1, CAT, and progesterone synthesis-related genes STAR and CYP11A1 significantly up-regulated. Compared with MT treatment alone, the co-treatment with MT and MT receptor inhibitor Luzindole could significantly increase the ROS level and decrease the expression level of SOD2, GPX1 and CAT. The level of progesterone and the expression of STAR and CYP11A1 decreased significantly. In conclusion, MT promotes yak luteal cells proliferation by up-regulating the mRNA expression level of PCNA and BCL-2, and repressing the expression level of BAX and FAS. MT may regulate the ROS level and progesterone level through its receptor. This study provides theoretical basis for the application of MT in the treatment of reproductive diseases caused by corpus luteum abnormality and the improvement in the reproductive performance of yak.

Key words: melatonin, yak, luteal cells, antioxidant activity, progesterone

中图分类号:

S823.85.3

程华琴, 袁钰洁, 罗文, 高绍帅, 叶林霞, 李键, 殷实. 褪黑素对牦牛黄体细胞生长及功能影响的研究[J]. 畜牧兽医学报, 2022, 53(2): 459-469.

CHENG Huaqin, YUAN Yujie, LUO Wen, GAO Shaoshuai, YE Linxia, LI Jian, YIN Shi. Research on the Effect of Melatonin on the Growth and Function of Yak Luteal Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 459-469.

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