GnIH对小鼠卵巢颗粒细胞增殖、凋亡和雌激素分泌的影响

doi:10.11843/j.issn.0366-6964.2025.05.022

摘要/Abstract

摘要：

旨在探究GnIH对小鼠颗粒细胞增殖、凋亡以及雌激素分泌水平的影响。本研究选取健康的21~23 d雌性ICR小鼠，分离小鼠卵巢GCs，利用FSHR进行鉴定，并采用qRT-PCR对小鼠颗粒细胞中GnIH和GnIH-R基因进行检测。构建pshRNA-1, 2, 3, 4干扰质粒载体和pLV-GnIH过表达质粒载体对GnIH基因分别进行干扰和过表达(GnIH干扰组分为：pshNC组和pshRNA1~4组，GnIH过表达组分为：PLV-NC组和PLV-GnIH组)。采用慢病毒滴度检测和qRT-PCR方法检测干扰和过表达效率，利用MTT法检测干扰和过表达GnIH后小鼠颗粒细胞增殖变化情况，采用Annexin V-FITC/PI流式细胞仪检测小鼠卵巢颗粒细胞的凋亡情况，并采用ELISA检测GCs培养液上清中孕酮和雌二醇的分泌水平，以上结果均重复3次。结果发现，在转染48 h和96 h时pshRNA-1, 2, 3, 4均能够极显著地干扰GnIH的表达(P＜0.01)，而在72 h时只有pshRNA-4能够极显著地下调GnIH的表达(P＜0.01)；72 h时pLV-GnIH能够极显著上调GnIH的表达(P＜0.001)。过表达GnIH后颗粒细胞增殖活力极显著下调(P＜0.001)，且能促进细胞凋亡(P＜0.001)，极显著抑制小鼠颗粒细胞雌激素的分泌(P＜0.001)。而转染pshRNA-2, 4 72 h和96 h时则极显著上调颗粒细胞增殖活力(P＜0.1)和雌激素的分泌(P＜0.05)，且显著抑制颗粒细胞凋亡(P＜0.05和P＜0.01)。本研究发现，GnIH能够抑制小鼠卵巢颗粒细胞增殖，促进颗粒细胞凋亡，降低雌激素的分泌水平。

关键词: GnIH, 颗粒细胞, 增殖, 凋亡, 雌激素分泌

Abstract:

The experiment aimed to investigate the effects of interfering and overexpressing GnIH on the proliferation, apoptosis and estrogen secretion in mouse ovarian granulosa cells. In this study, healthy female ICR mouse of 21-23 days old were selected. GCs were isolated from the mouse ovaries and identified using follicle-stimulating hormone receptor (FSHR). qRT-PCR was used to detect the expression of GnIH and GnIH-R genes in mouse granulosa cells. pshRNA-1, 2, 3, 4 interfering plasmid vectors and pLV-GnIH overexpressed plasmid vectors were constructed to overexpress and interfere with GnIH gene in mouse granulosa cells, respectively (GnIH interference groups were divided into pshNC group and pshRNA1-4 groups, and GnIH overexpression groups were divided into PLV-NC group and PLV-GnIH group). The interference and overexpression efficiency were detected by lentivirus titer detection and RT-qPCR, and the effects of psh-RNA and pLV-RNA on the proliferation and apoptosis of granulosa cells were detected by MTT and Annexin V-FITC/PI flow cytometry. The concentrations of progesterone and estradiol were detected by ELISA, all the above results were repeated 3 times. The results showed that pshRNA-1, 2, 3, 4 significantly interfered with GnIH expression at 48 h and 96 h (P < 0.01), but only pshRNA-4 significantly decreased GnIH expression at 72 h (P < 0.01). At 72 h, pLV-GnIH significantly up-regulated the expression of GnIH (P < 0.001). Overexpression of GnIH significantly down-regulated the proliferation activity of granulosa cells and promoted cell apoptosis (P < 0.001), and significantly inhibited granulosa cell estrogen secretion (P < 0.001). After transfection with pshRNA-2, 4 for 72 and 96 h, the proliferative activity of granulosa cells was extremely significantly increased (P < 0.01), the secretion of estrogen was significantly enhanced (P < 0.05), and apoptosis was significantly decreased(P < 0.05 and P < 0.01). In conclusion, inhibition of GnIH expression can promote the proliferation of granulosa cells and the secretion of estrogen, and down-regulate cell apoptosis, while promotion of GnIH expression can inhibit the proliferation of granulosa cells and the secretion of estrogen, and promote cell apoptosis.

Key words: GnIH, granulosa cells, proliferation, apoptosis, estrogen secretion

中图分类号:

S814.1

高正婕, 罗萍, 李博成, 王水莲. GnIH对小鼠卵巢颗粒细胞增殖、凋亡和雌激素分泌的影响[J]. 畜牧兽医学报, 2025, 56(5): 2230-2242.

GAO Zhengjie, LUO Ping, LI Bocheng, WANG Shuilian. Effects of GnIH on Proliferation, Apoptosis and Estrogen Secretion of Mouse Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2230-2242.

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基因 Gene	引物序列 Primer sequence	产物大小/bp Product length
musGnIH	F: ATGCCTATCACATTGCTG R: CAAGGTGAAGACGGAAGC	114
musGnIH-R	F: CAGAAAGCAGGTGGATGG R: CTCCAGGTCCCCAAATCC	247
musGAPDH	F: CCGTGTTCCTACCCCCAATG R: AGCCCAAGATGCCCTTCAGT	128

序列名称 Sequence name	序列信息 Sequence information
GnIH-siRNA1	GCAACTTCAAGCTTCTTAACA
GnIH-siRNA2	GCAGGGACCAGGAGCCATTTC
GnIH-siRNA3	GCTCCAGCGAGTTGCTCTACG
GnIH-siRNA4	GCGAGTTGCTCTACGTCATGA

基因 Gene	引物序列 Primer sequence	产物大小/bp Product length
musGnIH	F: CTGGCTAGGATGGCGAATGG R: GCTCATGGTGGACCTTCCTA	111
musGAPDH	F: CCAGGTGGTCTCCTCTGA R: GCTGTAGCCAAATCGTTGT	127

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