原花青素对体外培养绵羊卵泡颗粒细胞增殖的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-1177

摘要/Abstract

摘要：

旨在研究原花青素对体外培养绵羊卵泡颗粒细胞增殖的影响。不同浓度梯度（20、30、40、50、60、70和80 μg/mL）原花青素的完全培养基中培养绵羊卵泡颗粒细胞,培养时间分别为24、48和72 h,通过MTT法测定颗粒细胞的增殖率;通过RT-qPCR法检测细胞周期相关基因p21、p27和细胞凋亡相关基因caspase-8的表达。结果表明,培养48 h,在原花青素浓度为50 μg/mL时,颗粒细胞的增殖率最高,细胞周期相关基因p21、p27和细胞凋亡相关基因caspase-8这3个基因的表达量均显著降低（P<0.01）。综上所述,原花青素对体外培养绵羊卵泡颗粒细胞有一定的促进增殖效应,并且在相关基因mRNA水平上得到验证。

关键词: 原花青素, 颗粒细胞, 绵羊, 体外培养, 增殖

Abstract:

The aim of this study is to investigate the effects of proanthocyanidin（PC）on the proliferation of sheep follicular granulosa cells in vitro. Sheep follicular granulosa cells（GCs）were cultured in complete medium with different concentrations of PC（20,30,40,50,60,70 and 80 μg/mL）for 24,48 and 72 h,respectively. The proliferation rate of GCs was determined by MTT method. And the mRNA abundances of cell cycle related genes p21,p27 and cell apoptosis-related gene caspase-8 were detected by RT-qPCR. The results showed that the proliferation rate of GCs was the highest when the concentration of PC was 50 μg/mL when cultured for 48 h in vitro. And the relative mRNA abundances of three genes（p21,p27 and caspase-8）significantly decreased,respectively（P<0.01）. In sum,PC could promote the proliferation of sheep follicular GCs in vitro,and which was verified at relevant mRNA level.

Key words: proanthocyanidins, granulosa cells, sheep, in vitro, proliferation

杨小峰, 秦小伟, 郭泽媛, 吕丽华. 原花青素对体外培养绵羊卵泡颗粒细胞增殖的影响[J]. 生物技术通报, 2022, 38(9): 258-263.

YANG Xiao-feng, QIN Xiao-wei, GUO Ze-yuan, LV Li-hua. Effects of Proanthocyanidin on the Proliferation of Sheep Follicular Granulosa Cells in vitro[J]. Biotechnology Bulletin, 2022, 38(9): 258-263.

图/表 4

表1 RT-qPCR引物序列

Table 1 Primer sequences of RT-qPCR

基因名称Gene name	引物序列 Primer sequence（5'-3'）
p21	F：ACGTCTCAGGAGGACCACTT, R：TGGTAGAAATCTGTCATGCTGGT
p27	F：TACTTGGGTCTCAGGCAAAC, R：GCTCTTTTGTTTTGAGGAGAGG
caspase-8	F：ACTGTGTGGAGCAGGTAACA, R：CCGGCTTAGGAACTTGAGGG
18S	F：GTAACCCGTTGAACCCCATT, R：CCATCCAATCGGTAGTAGCG

图1 不同浓度PC处理体外培养绵羊卵泡颗粒细胞不同时间后的生长状况（100×）

Fig. 1 Growth status of sheep follicular granulosa cells in vitro cultured with different concentrations of PC for different time（100×） A1：48 h,0 μg/mL;B1：48 h,20 μg/mL;B2：48 h,50 μg/mL;B3：48 h,70 μg/mL;A2：24 h, 0 μg/mL;A3：72 h, 0 μg/mL;C：24 h, 50 μg/mL;D：72 h, 50 μg/mL

图2 不同浓度PC处理体外培养颗粒细胞24、48、72 h后的OD值上标小写字母表示在0.05显著水平上的比较结果

Fig. 2 OD values of granulosa cells cultured in vitro after 24,48 and 72 h treatment with different concentration of PC Superscript lowercase letters indicate comparison results at 0.05 significance level

图3 绵羊颗粒细胞中增殖相关基因mRNA的表达上标大写字母表示在0.01显著水平上的比较结果

Fig. 3 mRNA expression of proliferation-related genes in sheep granulosa cells Superscript capital letters indicate comparison results at 0.01 significance level

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