FST对猪卵巢颗粒细胞增殖凋亡及激素分泌的影响

doi:10.11843/j.issn.0366-6964.2025.03.024

摘要/Abstract

摘要：

旨在明确卵泡抑素(follistatin，FST)对猪卵巢颗粒细胞增殖、凋亡和激素分泌的影响，以期为探讨FST在猪卵泡生长过程中发挥的作用提供科学依据。本研究以永生化猪卵巢颗粒细胞为试验材料，将细胞分为过表达对照组、过表达组、干扰对照组和干扰组共4个试验组，每个试验组设置3个重复孔，根据FST基因的mRNA序列构建过表达质粒和设计siRNA，通过将其转染至颗粒细胞中，验证过表达及抑制效果。使用CCK-8和流式细胞法分析转染24、48和72 h后颗粒细胞增殖及凋亡情况，ELISA法检测细胞中雌二醇和孕酮含量变化，并进一步利用RT-PCR和WB法分别检测此过程中增殖、凋亡和激素合成相关基因的mRNA和蛋白表达水平。结果表明，构建的过表达质粒和siRNA可显著改变细胞中FST的表达水平(P < 0.01)，且转染至猪卵巢颗粒细胞后，FST抑制细胞增殖，诱导细胞凋亡，并可显著降低细胞中增殖凋亡相关基因FSHR、GDF9、BCL2、CDKN1B的mRNA(P < 0.05)和蛋白表达水平(P < 0.01)。而干扰FST后，卵巢颗粒细胞中雌二醇和孕酮的含量极显著增加(P < 0.01)，且STAR、CYP11A1、CYP19A1、3β-HSD、17β-HSD等激素合成相关基因的mRNA和蛋白表达量同样极显著升高(P < 0.01)。综上所述，在猪卵巢颗粒细胞中，FST基因表达水平的降低可有效促进细胞的增殖及雌二醇和孕酮的分泌，这一作用发挥可能与上调增殖凋亡相关基因和激素合成限速酶的表达有关。

关键词: FST, 猪, 颗粒细胞, 增殖凋亡, 雌二醇, 孕酮

Abstract:

The purpose of this study was to clarify the effect of FST gene on proliferation, apoptosis, and hormone secretion of porcine ovarian granulosa cells, and provide scientific reference for investigating the role of FST gene in the development of pig follicles. In this study, immortalized porcine ovarian granulosa cells were used as experimental materials, the granulosa cells were divided into 4 groups with 3 replications per group that included overexpression negative control group, overexpression group, inhibition negative control group and inhibition group. The overexpression plasmid and siRNA of FST gene were designed and constructed according to the mRNA sequence, and transfected into porcine ovarian granulosa cells to verify the overexpression and inhibitory effects. The CCK-8 and flow cytometry were used to analyze granulosa cell proliferation and apoptosis after transfection for 24, 48 and 72 h, ELISA was used to detect estradiol and progesterone content in granulosa cell. Furthermore, RT-PCR and Western blot were used to detect mRNA and protein expression level of cell proliferation, apoptosis, and hormone synthesis related genes. The results showed that, the mRNA and protein expression level of FST gene were significantly changed by overexpression plasmid and siRNA (P < 0.01); and FST gene could inhibit cell proliferation and induce cell apoptosis after transfecting overexpression plasmid and siRNA into porcine ovarian granulosa cells, and also significantly downregulated the mRNA (P < 0.05) and protein (P < 0.01) expression level of cell proliferation and apoptosis related genes FSHR, GDF9, BCL2 and CDKN1B. After interfering with FST expression, the estradiol and progesterone content in porcine ovarian granulosa cells were extremely significantly increased (P < 0.01), and the mRNA and protein expression level of hormone synthesis related genes STAR, CYP11A1, CYP19A1, 3β-HSD, and 17β-HSD were also extremely significantly increased (P < 0.01). In summary, in porcine ovarian granulosa cells, the decrease of FST gene expression level could accelerate cell proliferation, estradiol and progesterone secretion, and this function could be due to the upregulation of cell proliferation and apoptosis related genes and hormone synthesis rate limiting enzymes expression.

Key words: FST, pig, granulosa cells, proliferation and apoptosis, estradiol, progesterone

中图分类号:

S828.3

刘晨龙, 季华员, 卢丹, 万明春, 胡耀, 周泉勇. FST对猪卵巢颗粒细胞增殖凋亡及激素分泌的影响[J]. 畜牧兽医学报, 2025, 56(3): 1242-1251.

LIU Chenlong, JI Huayuan, LU Dan, WAN Mingchun, HU Yao, ZHOU Quanyong. Effect of FST on Proliferation, Apoptosis and Hormone Secretion of Porcine Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1242-1251.

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名称 Name	正义链(5'→3') Sense	反义链(5'→3') Antisense
S2-199	GGACUGAGGAGGACGUAAATT	UUUACGUCCUCCUCAGUCCTT
S3-440	CCUCAAGGCCAGAUGUAAATT	UUUACAUCUGGCCUUGAGGTT
S4-714	GGAAAGUGUAUCAAAGCAATT	UUGCUUUGAUACACUUUCCTT
S1-NC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

引物 Primer	引物序列(5'→3') Sequence	片段大小/bp Products size
FST	F: GCCGCTGCCAGGTCCTGTAT R: CCACGTTCTCGCACGTTTCTTT	177
FSHR	F: TGCCACCACTGGGAACAT R: CACATAAGGAACCGAGGGAC	81
GDF9	F: AGAGGGCTGTCTGAGGGTGTA R: CAGTAGCGAGGGTTGTATTTGTG	230
BCL2	F: GCGACTTTGCCGAGATGTC R: CCGAACTCAAAGAAGGCCAC	133
CDKN1B	F: TCAGGCCAACTCAGAGGACA R: CAGGTCGCTTCCTTATCCCA	104
STAR	F: TGGCTGGAAGTCCCTCAAAG R: GCAAAGTCCACCTGGGTCTG	115
CYP11A1	F: GCTCGGCAACTTGGAATCTGT R: GGCGGGATGTTGTATCGTTCT	99
CYP19A1	F: CTGGGTTGCAGTTCATTGGC R: TTGTCCAGGTGCTTGGTGATG	163
3β-HSD	F: TGAGCTTCCTGCTGAGTCCA R: AGACGGTGAACACGCTGTTG	87
17β-HSD	F: TGCTTTGCTTCATTTAGCCCTAC R: CTCATTCTTCCAGTTAGCCTTGC	111
GAPDH	F: ATTCCACCCACGGCAAGTT R: TTTGATGTTGGCGGGATCT	110

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