HSD17B12基因对湖羊垂体细胞促性腺激素合成的影响

Effect of HSD17B12 Gene on Gonadotropin Synthesis in Hu Sheep Pituitary Cells

旨在探索湖羊垂体中17β-羟类固醇脱氢酶12(HSD17B12)基因对垂体激素分泌的影响。本研究挑选体重相近(40 kg左右)且健康的性成熟湖羊公羊(9月龄)3只,采集垂体组织进行HSD17 B12基因CDS区扩增及蛋白同源性分析,确定其CDS区序列。利用免疫组化分析HSD17B12在性成熟雄性湖羊垂体中的表达定位。体外分离湖羊垂体细胞,利用RNA干扰和细胞转染技术体外干扰HSD17B12,qPCR鉴定激素相关基因的表达水平,并利用流式细胞仪检测细胞增殖、凋亡、周期等的变化。结果表明,HSD17 B12基因CDS区长度为939 bp,并且在物种间保守性较高。此外,HSD17B12在湖羊垂体组织大部分细胞中均呈现阳性表达。在垂体细胞干扰HSD17 B12后,可以造成细胞增殖效率显著降低(P<0.05),细胞凋亡比率显著增加(P<0.05),细胞周期发生显著改变(P<0.05),且垂体促性腺激素合成相关基因FSHβ、LHβ和生长激素基因GH表达水平均显著降低(P<0.05)。结果提示,在湖羊垂体细胞中干扰HSD17 B12的表达,可通过影响细胞增殖、周期和凋亡水平的变化而显著降低促性腺激素及生长激素的分泌。本研究初步证明了HSD 17B12基因在湖羊垂体中的重要作用,为进一步探索其中的作用机制提供一定的基础。

This study aimed to explore the effect of 17β-hydroxysteroid dehydrogenase 12(HSD17B12)in Hu sheep pituitary on the secretion of pituitary hormones.Three healthy Hu sheep rams at sexually mature stage(9-month-old)with similar weight(about 40 kg)were selected.And the pituitary tissues were collected for HSD17 B12 gene CDS region amplification and protein homology analysis to determine the CDS region sequence.The location of HSD17B12 in sexually mature male Hu sheep pituitary was detected by immunohistochemistry.In addition,pituitary cells were isolated in vitro,and after down-regulated the expression of HSD17 B12 using RNA interference and cell transfection,the expression levels of hormone-related genes were measured by qPCR.Moreover,the changes of cell proliferation,apoptosis and cycle were analyzed by flow cytometry.The results showed that the CDS region of HSD17 B12 gene was 939 bp,and it was highly conserved among species.And the immunohistochemistry analysis indicated that HSD17B12 was positively expressed in most cells of pituitary tissue.After RNA interference of HSD17 B12 in vitro,a significant decrease in cell proliferation efficiency(P<0.05),a significant increase in cell apoptosis rate(P<0.05),and significant change in cell cycle(P<0.05)were detected.In addition,the expression levels of gonadotropin synthesis-related genes FSHβ,LHβand growth hormone gene GH were significantly reduced(P<0.05).The results suggested that interfering the expression of HSD17B12 in Hu sheep pituitary cells could significantly reduce the secretion of gonadotropins and growth hormone by affecting cell proliferation and apoptosis.This study preliminarily proved the important role of HSD17B12 in Hu sheep pituitary,and provided a basis for further exploration of its mechanism.