HSD17B12对兔卵巢颗粒细胞增殖和凋亡的影响

Effect of HSD17B12 on Proliferation and Apoptosis of Granulosa Cells in Rabbit Ovary

本实验旨在研究羟基类固醇17-β脱氢酶12(17βHydroxysteroid Dehydrogenase Type 12,HSD17B12)对兔卵巢颗粒细胞增殖、凋亡的影响。首先克隆了兔HSD17B12基因,进行生物信息学分析;分离原代兔颗粒细胞(Granulosa Cells,GCs),并利用FSHR免疫荧光法进行鉴定;构建HSD17B12基因过表达质粒及干扰RNA,转染GCs,利用qRT-PCR检测HSD17B12对增殖、凋亡相关基因的调控作用,并利用流式细胞、CCK8等方法检测HSD17B12对GCs增殖、凋亡的影响。结果显示:①利用FSHR免疫荧光染色,鉴定分离原代细胞是GCs,FSHR表达呈阳性。②克隆HSD17B12基因,发现其cDNA全长为938 bp,编码312个氨基酸。进化树显示兔与黑猩猩(Pan Troglodytes)、猕猴(Macaca Mulatta)等聚在一起,鸡(Gallus Gallus)为外支。③过表达HSD17B12可以提高增殖相关基因AKT1-1的表达(P<0.01),降低凋亡相关基因Bad、Fas、FasL、P53(P<0.01)以及Bcl-2(P<0.05)的表达;干扰后,对基因的调控作用相反。④HSD17B12可促进GCs增殖,抑制GCs凋亡(P<0.01)。因此,HSD17B12可能通过调控AKT1、Bad、Fas、FasL、P53等基因的表达,显著促进兔GCs增殖,抑制其凋亡,为其在母兔卵巢发育中的作用机制研究提供理论依据。

This study aimed to study the effect of hydroxysteroid 17-βdehydrogenase 12(17βhydroxysteroid dehydrogenase type 12,HSD17B12)on the proliferation and apoptosis of granulosa cells in rabbit ovary.Rabbit HSD17B12 gene was cloned and bioinformatics analysis;primary rabbit granule cells(Granulosa cells,GCs)were isolated and identified by FSHR immunofluorescence;HSD17B12 gene overexpression plasmid and interfering RNA were constructed,transfected with GCs,the effect of HSD17B12 on proliferation and apoptotic gene expression was detected by qRT-PCR,and the effect of HSD17B12 on GCs proliferation and apoptosis was detected by cell flow and CCK 8.The results showed that:1 The isolated primary cells were identified as GCs and were positive for FSHR expression using FSHR immunofluorescence staining.2 cloned the HSD17B12 gene and found its cDNA of 938 bp encoding 312 amino acids.What about homology?3.Overexpression of HSD17B12 can significantly increase the expression of proliferation-related genes AKT 1-1(P<0.01),significantly reduce the expression of apoptosis-related genes Bad,Bad,Fas,FasL,P53(P<0.01),and significantly reduce the expression of Bcl-2(P<0.05);however,after interference.4.HSD17B12 can significantly promote the proliferation of GCs and inhibit the apoptosis of GCs(P<0.01).The conclusion is HSD17B12 may regulate the expression of AKT 1,Bad,Fas,FasL,P53 and other genes to significantly promote the proliferation of rabbit GCs and inhibit their apoptosis,and provide a theoretical basis for the mechanism of its role in the ovarian development of female rabbits.