Smad7介导TGF-β信号通路对绵羊卵泡颗粒细胞增殖的影响

doi:10.3864/j.issn.0578-1752.2023.13.013

摘要/Abstract

摘要：

【目的】卵泡发育是一个复杂的调控过程，其中绵羊卵泡颗粒细胞（granulosa cells, GCs）的增殖，分化会直接影响卵泡的发育状况。TGF-β信号通路在绵羊卵巢发育和卵泡生长过程中起着重要作用，Smad7作为TGF-β信号通路关键性的抑制基因也发挥重要作用，通过探究Smad7介导TGF-β信号通路对绵羊卵泡 GCs增殖凋亡的影响，为进一步研究Smad7在卵泡GCs增殖凋亡过程中的调控作用提供依据。【方法】选取4—6月龄未怀孕的晋中健康杜湖杂交母羊20只，屠宰后采集双侧卵巢组织，分离培养卵泡颗粒细胞，FSHR细胞免疫荧光鉴定，Smad7细胞免疫荧光的表达定位，CCK8法测定细胞增殖情况，绘制生长曲线；细胞传代后外源添加不同浓度Smad7激动剂积雪草苷（asiaticoside，AS）（0、100、200、400、600 ng·mL^-1）培养绵羊卵泡GCs，选择AS最佳作用浓度处理二代颗粒细胞，采用实时荧光定量PCR和Western blotting检测TGF-β信号通路中关键基因及细胞凋亡和周期相关基因表达水平；合成3个siSmad7，转染至卵泡GCs中，选择干扰效果最佳的，采用实时荧光定量PCR和Western blotting检测TGF-β信号通路中关键基因及细胞凋亡和周期相关基因表达水平。【结果】 Smad7在绵羊卵泡GCs中有表达；200 ng·mL^-1 AS能极显著上调Smad7在GCs的表达（P <0.01），siSmad7-1对绵羊卵泡GCs的转染效果最佳（P<0.01）；Smad7上调能显著增加TGF-β信号通路Smad4，TFDP-1和EP300的表达量（P<0.05），极显著增加TGF-β信号通路SP1的表达（P<0.01），显著增加凋亡相关基因Caspase8的表达量（P<0.05），极显著增加凋亡相关基因Caspase3和Bim的表达量（P<0.01），显著升高细胞周期相关基因P21和P27的表达量（P<0.05），极显著升高细胞周期相关基因CCND2的表达量（P<0.01），而CCND1的表达量显著降低（P<0.05）；Smad7 siRNA显著降低TGF-β信号通路Smad4，TFDP-1和EP300的表达量（P<0.05），极显著降低TGF-β信号通路SP1的表达量（P<0.01），显著降低凋亡相关基因Caspase8的表达量（P<0.05），极显著降低凋亡相关基因Caspase3和Bim的表达量（P<0.01），极显著降低细胞周期相关基因P21，P27和CCND2的表达量（P<0.01），极显著升高CCND1的表达量（P<0.01）；CCK8分析显示，Smad7上调，GCs的增殖率在24和48 h显著下调（P<0.01），Smad7 siRNA在24和72 h显著升高GCs的增殖率（P<0.05），在48 h极显著升高GCs的增殖率（P<0.01）。【结论】Smad7介导TGF-β信号通路抑制绵羊卵泡GCs增殖和促进细胞凋亡。

关键词: 积雪草苷, siRNA, Smad7, TGF-β信号通路, 绵羊, 颗粒细胞

Abstract:

【Background】Follicle development is a complex regulatory process, in which the proliferation and differentiation of ovine follicular granulosa cells (Granulosa Cells, GCs) will directly affect the development of follicles. TGF-β signaling pathway plays an important role in sheep ovarian development and follicle growth, and Smad7 also plays an important role as a key inhibitor of TGF-β signaling pathway. 【Objective】In this study, the effect of Smad7-mediated TGF-β signaling pathway on the proliferation and apoptosis of ovine follicular GCs was explored to provide a basis for further research on the regulatory role of Smad7 in the process of follicular GCs proliferation and apoptosis. 【Method】20 non-pregnant Jinzhong Duhu hybrid ewes aged 4-6 months were selected. After slaughtering, bilateral ovaries were collected, and GCs were isolated and cultured in vitro. And then, FSHR cell and expression localization of Smad7 cells was identified by immunofluorescence, the cell proliferation was measured by CCK8 method, and the growth curve was drawn. After the cells were passaged, different concentrations of Smad7 agonist Asiaticoside AS (0, 100, 200, 400, 600 ng·mL^-1) were added exogenously, and the optimal concentration of AS was selected to treat the second generation GCs. The expression levels of key genes as well as apoptosis and cycle-related genes in the TGF-β signaling pathway were detected using qRT-PCR and Western blotting. Three siSmad7s were synthesized and transfected into follicular GCs, and the one with the best interference effect was selected. The expression levels of key genes in the TGF-β signaling pathway, apoptosis and cycle-related genes were detected by qRT-PCR and Western blotting. 【Result】Smad7 was expressed in ovine follicular GCs; 200 ng·mL^-1 AS could significantly up-regulate the expression of Smad7 in GCs (P<0.01), siSmad7-1 could effectively transfected ovine follicle GCs (P<0.01); Up-regulation of Smad7 could significantly increase the expressions of Smad4, TFDP-1 and EP300 in the TGF-β signaling pathway (P<0.05), extremely significantly increase the expression of SP1 in the TGF-β signaling pathway (P<0.01), and significantly increase the expression of apoptosis-related gene Caspase8 (P<0.05), extremely significantly increased the expressions of apoptosis-related genes Caspase3 and Bim (P<0.01), significantly increased the expressions of cell cycle-related genes P21 and P27 (P<0.05), extremely significantly increased the expression of cell cycle-related gene CCND2 (P<0.01), while the expression of CCND1 was significantly decreased (P<0.05); Smad7 siRNA significantly decreased the expressions of Smad4, TFDP-1 and EP300 in the TGF-β signaling pathway (P<0.05), extremely significantly decreased the expression of SP1 in the TGF-β signaling pathway (P<0.01), and significantly decreased the apoptosis-related gene Caspase8 (P<0.05), extremely significantly decreased the expressions of apoptosis-related genes Caspase3 and Bim (P<0.01), extremely significantly decreased the expressions of cell cycle-related genes P21, P27 and CCND2 (P<0.01), extremely significantly increased the expression of CCND1 (P<0.01); CCK8 analysis demonstrated into that when Smad7 was up-regulated, the proliferation rate of GCs was significantly down-regulated at 24 h and 48 h (P<0.01). Smad7 siRNA significantly increased the proliferation rate of GCs at 24 h and 72 h (P<0.05), which also extremely significantly increased the proliferation rate of GCs (P<0.01).【Conclusion】In summary, it was suggested that Smad7 mediated TGF-β signaling pathway to inhibit the proliferation of ovine follicular GCs and to promote cell apoptosis.

Key words: asiaticoside (AS), siRNA, Smad7, TGF-β signaling pathway, sheep, granulosa cells

郭泽媛, 杜张胜, 张雅琦, 陈春路, 马晓燕, 成颖, 王锴, 吕丽华. Smad7介导TGF-β信号通路对绵羊卵泡颗粒细胞增殖的影响[J]. 中国农业科学, 2023, 56(13): 2597-2608.

GUO ZeYuan, DU ZhangSheng, ZHANG YaQi, CHEN ChunLu, MA XiaoYan, CHENG Ying, WANG Kai, LÜ LiHua. Effects of Smad7-Mediated TGF-β Signaling Pathway on Proliferation of Sheep Granulosa Cells[J]. Scientia Agricultura Sinica, 2023, 56(13): 2597-2608.

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