CYP19A1对兔卵巢颗粒细胞增殖和凋亡的影响

Effects of CYP19A1 on Proliferation and Apoptosis of Rabbit Ovary Granulosa Cells

旨在探究P450芳香化酶基因(cytochrome P450,family 19,subfamily A,polypeptide 1,CYP19A1)对兔卵巢颗粒细胞(granulosa cells,GCs)增殖和凋亡的影响。本研究选择健康状况良好的8月龄新西兰白兔母兔,采集卵巢组织,分离并鉴定GCs;利用一步克隆法获得兔CYP19A1基因全长序列,进行生物信息学分析;利用qRT-PCR、CCK8、FITC/PI等技术与方法,分析CYP19A1基因过表达和干扰对类固醇激素合成相关基因及GCs增殖和凋亡的影响。利用FSHR免疫荧光染色,鉴定分离的原代细胞是GCs,FSHR表达呈阳性。克隆CYP19A1基因,发现其cDNA全长为1512 bp,编码503个氨基酸。经生物信息学预测发现,CYP19A1蛋白是一种稳定的亲水性蛋白,含有一个P450家族结构域,与其它物种同源性较高。在GCs中,CYP19A1基因表达量的改变显著或极显著影响了类固醇激素合成相关基因IGF1R、CYP1B1、CYP1A1、BMP6、CYP17A1的表达(P<0.05或P<0.01)。过表达/干扰CYP19A1后,极显著上调/下调GCs增殖,极显著抑制/促进GCs凋亡水平(P<0.01)。综上所述,CYP19A1可以促进GCs增殖,抑制GCs凋亡,调控类固醇激素合成相关基因的表达,进而参与母兔卵泡发育进程。

The study aimed to investigate the effect of P450 aromatase(cytochrome P450,family 19,subfamily A,polypeptide 1,CYP19A1)on the proliferation and apoptosis of rabbit ovarian granulosa cells(GCs).Healthy 8-month-old New Zealand white rabbit female rabbits were selected to collect ovarian tissue,isolate and identify GCs.The full-length sequence of rabbit CYP 19A1 gene was obtained by one-step cloning method and analyzed by bioinformatics.The effects of CYP 19A1 gene overexpression and interference on steroid hormone synthesis-related genes,proliferation and apoptosis of ovarian GCs were analyzed using qRT-PCR,CCK8 and FITC/PI methods.Using FSHR immunofluorescence staining,isolated primary cells were identified as GCs with positive FSHR expression.The CYP 19A1 gene was cloned and the full length of cDNA was 1512 bp in,encoding 503 amino acids.Bioinformatics prediction revealed that CYP19A1 protein was a stable hydrophilic protein,containing a P450 family structural domain with high homology to other species.In GCs,the change of expression of CYP 19A1 gene significantly or extremely significantly affected the expression of steroid hormone synthesis-related genes IGF 1R,CYP 1B1,CYP 1A1,BMP6,CYP 17A1 expression(P<0.05 or P<0.01).Overexpression/knockdown of CYP 19A1 extremely significantly upregulated/downregulated the proliferation level of GCs,and highly significantly inhibited/promoted the apoptosis level of GCs(P<0.01).In conclusion,CYP19A1 can promote GCs proliferation,inhibit GCs apoptosis,and regulate the expression of steroid hormone synthesis-related genes to participate in the follicular development process in female rabbits.