LPA对牦牛卵丘细胞扩张因子HAS2、PTGS2和PTX3表达的影响

doi:10.11843/j.issn.0366-6964.2024.02.013

摘要/Abstract

摘要： 本研究以溶血磷脂酸(lysophosphatidic acid, LPA)在卵丘细胞扩张中的作用为切入点,旨在探讨不同浓度LPA对牦牛卵丘细胞(yak cumulus cells, YCCs)中卵丘扩张因子(透明质酸合成酶2(hyaluronate synthase 2, HAS2)、前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2, PTGS2)和正五聚蛋白3(pentraxin 3, PTX3))表达的影响。本研究以健康成年(3~4岁)雌牦牛的YCCs为研究对象,取对数生长期的YCCs,将不同浓度的LPA(空白对照、阴性对照、5 、15 、30和50 μmol·L^-1)作用于体外培养的YCCs,分别培养12、24、36、48 h后,CCK-8检测YCCs的细胞活性,采用RT-qPCR和Western-blot法检测YCCs中HAS2、PTGS2和PTX3 mRNA和蛋白相对表达量,细胞免疫荧光染色法检测卵丘扩张因子HAS2、PTGS2和PTX3在YCCs中的分布。试验中每个处理组3个重复。结果显示,LPA孵育12、24、36和48 h,对YCCs的活性有着明显的促进作用。当孵育时间为24 h时,LPA对YCCs活性的促进作用最明显。相比较于对照组而言,当LPA浓度为15 μmol·L^-1时,不同孵育时间中YCCs的活性提升最为显著(P<0.05)。当LPA浓度为15 μmol·L^-1时,与空白对照组相比,HAS2、PTGS2和PTX3的mRNA和蛋白相对表达量最高(P<0.05),且YCCs中HAS2、PTGS2和PTX3的荧光强度明显增强。当LPA浓度大于15 μmol·L^-1时,HAS2、PTGS2和PTX3的mRNA和蛋白相对表达量逐渐下降。本研究表明,LPA对YCCs活性具有促进作用。当孵育时间为24 h,并且LPA浓度为15 μmol·L^-1时,活性提升最为显著(P<0.05)。LPA可以增强YCCs中卵丘扩张因子HAS2、PTGS2、PTX3的表达,且其作用浓度具有剂量依赖性,最佳浓度为15 μmol·L^-1。研究结果为阐明LPA促进牦牛卵丘细胞扩张的分子机制提供了理论依据,为进一步提高牦牛卵母细胞的质量和体外受精(in vitro fertilization, IVF)成功率提供理论基础。

关键词: 牦牛, 溶血磷脂酸, 卵丘细胞, 卵丘扩张因子

Abstract: The purpose of this study was to investigate the effect of lysophosphatidic acid(LPA) with different concentrations on the expression of hyaluronate synthase 2(HAS2), prostaglandin-endoperoxide synthase 2(PTGS2) and pentraxin 3(PTX3) in Yak cumulus cells (YCCs). The YCCs of healthy adult (3-4 years old) female yaks were taken as the research object. YCCs in logarithmic growth phase were taken, and different concentrations of LPA (blank control, negative control, 5, 15, 30, and 50 μmol·L^-1) were applied to the cultured YCCs in vitro. After 12, 24, 36, and 48 hours of cultivation, CCK-8 was used to detect the cellular viability of YCCs. The relative expression levels of HAS2, PTGS2, and PTX3 mRNA and protein in YCCs were detected by RT-qPCR and Western-blot assays. Immunofluorescence staining was used to detect the distribution of cumulus dilatation factors HAS2, PTGS2, and PTX3 in YCCs. In this experiment, each treatment group had 3 replicates. The results showed that, LPA incubation for 12, 24, 36, and 48 h had a significant promoting effect on the viability of YCCs. When the incubation time was 24 h, LPA had the most significant promoting effect on the viability of YCCs. Compared to the control group, when the LPA concentration was 15 μmol·L^-1, the viability of YCCs increased most significantly at different incubation times(P<0.05). Compared with the blank control group, the highest relative expression levels of HAS2, PTGS2, and PTX3 mRNA and protein were observed(P<0.05) when the LPA concentration was 15 μmol·L^-1, and the fluorescence intensity of HAS2, PTGS2, and PTX3 in YCCs was significantly enhanced. However, when the LPA concentration exceeded 15 μmol·L^-1, the relative expression levels of HAS2, PTGS2, and PTX3 mRNA and protein gradually decreased. The study results suggests that LPA has a promoting effect on the viability of YCCs. When the incubation time is 24 hours and the LPA concentration is 15 μmol·L^-1, the viability improvement is the most significant(P<0.05). LPA can enhance the expression of the cumulus expansion factors HAS2, PTGS2, and PTX3 in YCCs, and its effect is dose-dependent, with the optimal concentration being 15 μmol·L^-1. The research results provide a theoretical basis for elucidating the molecular mechanism of LPA promoting the expansion of yak cumulus cells, and for further improving the quality of yak oocytes and the success rate of in vitro fertilization (IVF).

Key words: yak, lysophosphatidic acid, cumulus cells, cumulus expansion factors

中图分类号:

S823.85

刘斌, 王萌, 潘阳阳, 王靖雷, 徐庚全. LPA对牦牛卵丘细胞扩张因子HAS2、PTGS2和PTX3表达的影响[J]. 畜牧兽医学报, 2024, 55(2): 552-561.

LIU Bin, WANG Meng, PAN Yangyang, WANG Jinglei, XU Gengquan. Effect of LPA on the Expression of HAS2, PTGS2 and PTX3 in Cumulus Cells of Yak (Bos grunniens)[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 552-561.

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