接种密度对牛颗粒细胞形态及相关基因表达的影响  

Effects of Seeding Density on Morphology and Gene Expression of Bovine Granulosa Cells

作　　者：王栋梁[1] 成俊丽 李鹏飞[3] WANG Dongliang;CHENG Junli;LI Pengfei(ShuoZhou Vocational Technology College,Shuozhou 036002,China;College of Animal Science,Shanxi AgriculturalUniversity,Taigu 030801,China;College of Life Science,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]朔州职业技术学院,山西朔州036002 [2]山西农业大学动物科学学院,山西太谷030801 [3]山西农业大学生命科学学院,山西太谷030801

出　　处：《山西农业科学》2024年第3期147-152,共6页Journal of Shanxi Agricultural Sciences

基　　金：国家自然科学基金面上项目(31873002);山西省应用基础研究计划面上项目(20210302123380);山西农业大学横向科技项目(2022HX010,2021HX23,2020HX06,2019HX03)。

摘　　要：牛卵巢颗粒细胞是雌激素(Estrogen,E_(2))合成的主要来源,具有E_(2)活性的牛颗粒细胞体外模型的建立是研究E_(2)合成与分泌过程中潜在调控机制的重要工具,该细胞模型的建立可为E_(2)合成分子调控机制及牛卵巢卵泡发育机制的研究提供理论与技术支持。细胞接种密度是颗粒细胞体外培养模型的关键因素,高密度可引起细胞生理及分子的显著变化。研究通过不同接种密度下的形态变化和基因表达筛选牛颗粒细胞体外培养最优接种密度,采用长期无血清法培养牛原代颗粒细胞,培养液中添加FSH及IGF-1以诱导E_(2)的合成。培养7 d后,采集6孔板中高(3.0×10^(6)个细胞/孔)、中(2.0×10^(6)个细胞/孔)、低(1.0×10^(6)个细胞/孔)3种不同接种密度的细胞图像进行观测,并利用qRT-PCR技术检测3种不同接种密度中相关基因的表达情况。结果表明,低密度(1.0×10^(6)个细胞/孔)接种细胞呈现成纤维细胞样外观,细胞无聚集倾向;中密度组(2.0×10^(6)个细胞/孔)可观察到少量聚集的细胞团;高密度(3.0×10^(6)个细胞/孔)培养条件下,大多数细胞聚集成细胞团。低密度组(1.0×10^(6)个细胞/孔)中,CYP19A1及FSHR高表达,RGS2及VNN2低表达;高密度组(3.0×10^(6)个细胞/孔)表达量则相反。综上所述,低密度组(1.0×10^(6)个细胞/孔)细胞可作为具有E_(2)活性的牛颗粒细胞体外模型。Bovine granulosa cells(GCs) are the main source of estrogen(E_(2)) synthesis.The establishment of a bovine GCs model with E_(2) activity in vitro is an important tool to study the potential regulatory mechanisms of E_(2) synthesis and secretion,and the establishment of this cell model can provide theoretical and technical support for the study on the E_(2) synthesis molecular regulation mechanism and bovine ovarian follicle development mechanism.Cell seeding density is the key factor of the GCs culture model in vitro,and high density can cause significant changes in cell physiology and molecules.In this study,the optimal seeding density of bovine GCs culture in vitro was screened by morphological changes and gene expression under different seeding densities.Primary bovine GCs were cultured by the long term serum-free method,FSH and IGF-1 were added in culture medium to induce synthesis of E_(2).After 7 d of culture,images of cells with different seeding densities(low(1×10^(6) cells/well),medium(2×10^(6) cells/well),and high(3×10^(6) cells/well)) were collected for observation,and qRT-PCR was used to detect the expression of related genes in the three different seeding densities.The results showed that the low-density(1×10^(6) cells/well)inoculated cells showed a fibroblast-like appearance and no tendency for cell aggregation.A small number of aggregated cell clusters were observed in the medium-density group(2×10^(6) cells/well),and most of the cells were aggregated into cell clusters under high-density(3×10^(6) cells/well) culture conditions.In the low-density group(1×10^(6) cells/well),CYP19A1 and FSHR were highly expressed,RGS2 and VNN2 were lowly expressed,and the expression levels were opposite in the high-density group(3×10^(6) cells/well).In summary,the low-density group(1×10^(6) cells/well) of cells could be used as an in vitro model of bovine GCs with E_(2) activity.

关 键 词：牛 颗粒细胞 接种密度 无血清培养 细胞培养模型 

分 类 号：S823.148[农业科学—畜牧学]