SRD5A2对兔颗粒细胞增殖、凋亡和类固醇激素合成相关基因表达的影响

doi:10.11843/j.issn.0366-6964.2025.01.024

摘要/Abstract

摘要：

旨在探讨类固醇5α还原酶2(steroid 5 alpha reductase 2，SRD5A2)对新西兰母兔卵巢颗粒细胞(granulosa cells，GCs)的增殖、凋亡以及合成类固醇激素基因表达的影响。本研究选取健康的4~6月龄性成熟的雌性新西兰白兔, 分离兔卵巢GCs，利用FSHR进行鉴定，进行SRD5A2基因的过表达和干扰，利用CCK-8、流式细胞技术和qRT-PCR方法检测GCs增殖、凋亡和类固醇激素合成相关基因表达。结果发现，兔SRD5A2基因CDS全长为765 bp，编码252个氨基酸。在GCs中过表达/敲低SRD5A2后，qRT-PCR检测发现HIF1A的表达水平极显著降低/升高，INHBB、HSD17B1、GDF9、BMP15的表达水平极显著升高/降低(P＜0.01)。进一步，过表达/敲低SRD5A2后显著促进/抑制GCs增殖，并抑制/促进凋亡(P＜0.05)。结果表明，SRD5A2基因影响了合成类固醇激素基因的表达和颗粒细胞的增殖和凋亡，并参与新西兰兔卵泡发育过程。

关键词: SRD5A2, 颗粒细胞, 卵巢, 增殖, 凋亡

Abstract:

The purpose of this study was to investigate the effects of steroid 5 alpha reductase 2 (SRD5A2) on proliferation, apoptosis of granulosa cells, and the expression of genes related to steroid hormone synthesis in granulosa cells (GCs) of New Zealand White female rabbits. Healthy sexually mature female New Zealand White rabbits aged 4 to 6 months were selected. GCs were isolated from the rabbit ovaries and identified using follicle-stimulating hormone receptor (FSHR). Overexpression and knockdown of the SRD5A2 gene were performed. The proliferation, apoptosis of GCs, and the expression of genes related to steroid hormone synthesis were detected using CCK-8, flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) methods. The full-length coding sequence (CDS) of the rabbit SRD5A2 gene was 765 bp, encoding 252 amino acids. After overexpressing/knocking down SRD5A2 in GCs, qRT-PCR analysis revealed that the expression level of HIF1A was extremely significantly decreased/increased, while the expression levels of INHBB, HSD17B1, GDF9, and BMP15 were extremely significantly increased/decreased (P < 0.01). Furthermore, overexpressing/knocking down SRD5A2 significantly promoted/inhibited GCs proliferation and inhibited/promoted apoptosis (P < 0.05). These findings indicate that the SRD5A2 gene affects the expression of genes related to steroid hormone synthesis, as well as the proliferation and apoptosis of GCs, and is involved in the follicular development process in New Zealand White rabbits.

Key words: SRD5A2, granulosa cells, ovary, proliferation, apoptosis

中图分类号:

S829.1

王磊, 白少成, 王森, 鲍志远, 蔡佳炜, 刘燕, 赵博昊, 吴信生, 陈阳. SRD5A2对兔颗粒细胞增殖、凋亡和类固醇激素合成相关基因表达的影响[J]. 畜牧兽医学报, 2025, 56(1): 259-268.

WANG Lei, BAI Shaocheng, WANG Sen, BAO Zhiyuan, CAI Jiawei, LIU Yan, ZHAO Bohao, WU Xinsheng, CHEN Yang. Effect of SRD5A2 on the Expression of Genes Related to Proliferation, Apoptosis and Steroid Hormone Synthesis in Rabbit Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 259-268.

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序列名称 Sequence name	引物序列(5′→3′)Primer sequence
CDS-SRD5A2	F: cgtttaaacgggccctctagaATGCCTCTTAGGTGCCAGCA R: tagtccagtgtggtggaattcTTCGCGTTCTTTAAAAGATGAATG
Negative control	S: UUCUCCGAACGUGUCACGUTT A: ACGUGACACGUUCGGAGAATT
siRNA-295	S: GGGCCUCUUCUGCACGCAUTT A: AUGCGUGCAGAAGAGGCCCTT
siRNA-462	S: GGUACACGGAUCUAAGGUUTT A: AACCUUAGAUCCGUGUACCTT
siRNA-508	S: GGGAAUGGGCAUCAACAUUTT A: AAUGUUGAUGCCCAUUCCCTT

项目Project	用途Usage	网址/软件Website/Software
ProtParam^[17]	蛋白质的氨基酸序列	https://web.expasy.org/cgi-bin/protparam/
ProtScale^[17]	亲疏水性	https://web.expasy.org/cgi-bin/protscale/
SOPMA^[18]	蛋白质二级结构	https://npsa-prabi.ibcp.fr/
Swiss-Model^[19]	蛋白质三级结构	https://swissmodel.expasy.org/
PSORT Ⅱ	蛋白的亚细胞定位	https://psort.hgc.jp/
NetPhos	蛋白的磷酸化位点	http://www.cbs.dtu.dk/services/NetPhos/
CDD database^[20]	保守结构域	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
进化树分析Phylogenetic analysis	SRD5A2进化树分析	MEGA 5.1

基因 Gene	引物序列(5′→3′) Primers sequence	退火温度/℃ Annealing temperature
SRD5A2	F: TGCATCGGAAATGGGTTCCT R: GCCAAGGCATAGCCAATCCA	60
HSD17B1	F: GAGCTTCAAAGTGTACGCCAC R: TCCGAGTCCCTTACGTCCA	59
HIF1A	F: AGTGCCACAACATCACCACC R: GATGCTCATCTCCACCTCGG	60
INHBB	F: GGACCAACCTCTGTTGCAGG R: CCTCGCAGTAGTTGCCGTAG	60
GDF9	F: GTGGTTTTGCTGTCTTGCCT R: TACTGCAACGCTCTCGAGTC	59
BMP15	F: TGGGGCAGTCTTCTATTGCC R: TCCAACATGTATCGCAGGGG	60
GAPDH	F: CACCAGGGCTGCTTTTAACTCT R: CTTCCCGTTCTCAGCCTTGACC	61

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