分泌生长激素和催乳素的奶牛腺垂体细胞的分离鉴定及特征分析

Isolation,identification and character analysis of dairy cow anterior pituitary cells secreting growth hormone and prolactin

目的获得具有生长激素(growth hormone,GH)和催乳素(prolactin,PRL)分泌功能的奶牛腺垂体细胞(dairy cow anterior pituitary cells,DCAPCs),并对其进行鉴定和特征分析。方法采用免疫组织化学方法对奶牛腺垂体中GH和PRL分泌细胞进行定位,利用酶消化法对其所在部位进行消化,获得的细胞经分离培养后,用形态学方法和免疫组织化学方法进行鉴定,并分析其传代性能;采用实时荧光定量PCR及ELISA方法对其GH和PRL合成和分泌特征进行分析。结果 GH分泌细胞主要位于腺垂体侧翼区下缘,而PRL分泌细胞主要位于腺垂体侧翼区上缘。获得的DCAPCs接种培养2 d后,可见形状规则的铺路石样上皮细胞,经免疫组织化学法鉴定,可分泌GH和PRL,经多次传代后仍可表达GH和PRL,但表达水平随代次增加而降低。结论成功建立了DCAPCs分离培养方法,可用于体外研究奶牛GH和PRL合成及分泌的调控机制。

Objective The isolate and identify the dairy cow anterior pituitary cells（DCAPCs）secreting growth hormone（GH） and prolactin（PRL） and analyze their characters.Methods The locations of dairy cow anterior pituitary cells secreting GH and PRL were investigated by immunohistochemical assay,and digested with various enzymes.The obtained cells were isolated,cultured,identified by morphological and immunohistochemcial methods,and analyzed for the performance of subculture.The synthesis and secretion levels of GH and PRL were analyzed by real-time fluorescent quantitative PCR and ELISA.Results The cells secreting GH were mainly located within the posterolateral wings of the anterior pituitary,while those secreting PRL within the anterolateral wings.Regular cobblestone-like epitheliums were observed in DCAPCs after culture for 2 d,which secreted GH and PRL as proved by immunohistochemcial assay.Both GH and PRL were still expressed in DCAPCs after several passages,while the expression levels decreased with the increasing passage.Conclusion The method for isolation and culture of DCAPCs was developed successfully,which might be used for the in vitro study on synthesis and secretion of GH and PRL in dairy cow anterior pituitary cells.