Shh信号对奶牛卵泡颗粒细胞增殖与分化的影响

doi:10.13560/j.cnki.biotech.bull.1985.2025-0333

摘要/Abstract

摘要：

目的探究音猬因子（sonic hedgehog, Shh）信号对奶牛卵泡颗粒细胞增殖与分化的影响。方法采集发情期奶牛卵巢，通过IHC检测Shh在卵巢组织中的表达特征。分离并培养奶牛卵泡颗粒细胞，试验处理为：对照组、Ad-Shh组（转染Shh过表达腺病毒，MOI=50处理48 h）、Ad-Shh+Cyc组（在过表达Shh的基础上添加Shh信号通路抑制剂环巴胺（cyclopamine, Cyc，5 μmol/L）处理24 h）。免疫荧光、MTS、EdU法分析细胞的增殖活力；流式细胞术分析细胞周期变化；Western blot检测Shh、Ki67、增殖和分化相关蛋白的表达。结果 IHC结果显示，Shh在奶牛卵巢组织中高表达；免疫荧光结果显示，过表达Shh促进颗粒细胞中Ki67的表达，Cyc逆转了这种变化（P<0.01）；MTS和EdU结果表明，Cyc逆转了过表达Shh对细胞增殖活力的增强作用（P<0.01）；流式细胞术结果显示，过表达Shh促进颗粒细胞的细胞周期由G1期向S期转变（G1期细胞比例由91.42%降低至71.58%，降低19.84%，S期细胞比例由4.88%升高至22.22%，升高17.34%），Cyc减缓了细胞周期进程（P<0.01）；Western blot结果显示，过表达Shh促进Ki67、细胞增殖、细胞分化相关蛋白的表达（P<0.01），Cyc逆转了过表达Shh对Ki67、细胞增殖、细胞分化相关蛋白表达的促进作用（P<0.01）。结论 Shh信号正向调节奶牛卵泡颗粒细胞的增殖与分化。

关键词: Shh信号, 卵泡发育, 卵泡颗粒细胞, 增殖, 分化

Abstract:

Objective To investigate the effect of sonic hedgehog (Shh) signal on the proliferation and differentiation of bovine follicular granulosa cells. Method Ovaries from estrous dairy cows were collected and the expression characteristics of Shh in ovarian tissues were detected by IHC. Bovine follicular granulosa cells were isolated and cultured, and divided into: control group, Ad-Shh group (transfected with Shh overexpression adenovirus, MOI = 50 for 48 h), and Ad-Shh + Cyc group (treated with Shh pathway inhibitor cyclopamine (Cyc, 5 μmol/L) for 24 h on the basis of overexpression of Shh). The proliferation activities of cells was analyzed by immunofluorescence, MTS, and EdU methods; the cell cycle variations were analyzed by flow cytometry; and the expressions of Shh, Ki67, and proliferation and differentiation-related proteins were detected by Western blot. Result IHC results showed that Shh was highly expressed in the bovine ovarian tissues; immunofluorescence results indicated that the overexpression of Shh promoted the expression of Ki67 in granulosa cells, and Cyc reversed this change (P<0.01); MTS and EdU results demonstrated that Cyc reversed the enhancing effect of overexpression of Shh on cell proliferation activity (P<0.01). Flow cytometry results showed that the overexpression of Shh promoted the transition of granulosa cells from G1 phase to S phase (the proportion of G1 phase cells decreased from 91.42% to 71.58%, a decrease of 19.84%, and the proportion of S phase cells increased from 4.88% to 22.22%, an increase of 17.34%), and Cyc slowed down the cell cycle process (P<0.01). Western blot results showed that the overexpression of Shh promoted the expression of Ki67, cell proliferation, and cell differentiation-related proteins (P<0.01), and Cyc reversed the promoting effect of overexpression of Shh on the expression of Ki67, cell proliferation, and cell differentiation-related proteins (P<0.05). Conclusion Shh signal positively regulates the proliferation and differentiation of bovine follicular granulosa cells.

Key words: Shh signal, follicular development, follicular granulosa cells, proliferation, differentiation

孙晗冰, 向念, 方欣馨, 岳俊秀, 许秋实, 马莉. Shh信号对奶牛卵泡颗粒细胞增殖与分化的影响[J]. 生物技术通报, 2025, 41(9): 326-334.

SUN Han-bing, XIANG Nian, FANG Xin-xin, YUE Jun-xiu, XU Qiu-shi, MA Li. Effects of Shh Signal on the Proliferation and Differentiation of Follicular Granulosa Cells in Dairy Cows[J]. Biotechnology Bulletin, 2025, 41(9): 326-334.

图/表 6

图1 Shh信号在奶牛卵巢组织中的表达特征A：Shh信号在奶牛卵巢组织中的表达特征。棕色表示Shh蛋白，蓝色表示细胞核；B：图A框选部分放大结果

Fig. 1 Expression characteristics of the Shh signal in the ovarian tissue of dairy cowsA:Expression characteristics of the Shh signaling pathway in the ovarian tissue of dairy cows. The brown color indicates Shh protein, and the blue color indicates the cell nucleus. B: Magnified results of the part enclosed in figure A

图2 奶牛卵泡GCs中Ki67的免疫荧光表达A：GCs中Ki67表达的免疫荧光结果；B：Ki67阳性细胞率统计分析；*P<0.05，**P<0.01，下同

Fig. 2 Immunofluorescence expression of Ki67 in the GCs of dairy cow folliclesA: The immunofluorescence results of Ki67 expression in GCs. B: Statistical analysis of Ki67 positive cell rate; *P<0.05, **P<0.01, the same below

图3 Shh信号对奶牛卵泡GCs增殖活力的影响A：MTS检测细胞增殖率；B：EdU检测细胞增殖结果；C：EdU阳性细胞率统计分析；D：Shh和增殖标志蛋白Ki67的蛋白表达灰度图；E：Shh的蛋白相对表达量；F：增殖标志蛋白Ki67的蛋白相对表达量

Fig. 3 Influence of Shh signal on the proliferation activity of GCs in dairy cow folliclesA: Cell proliferation rate was detected by MTS. B: EdU detects the results of cell proliferation. C: Statistical analysis of EdU positive cell rate. D: Grayscale images of protein expression of Shh and proliferation marker protein Ki67. E: The relative expression of Shh protein. F: The relative expression of the proliferation marker protein Ki67

图4 Shh信号对奶牛卵泡GCs增殖进程的影响A-C：奶牛卵泡GCs细胞周期进程的变化；D：细胞周期进程变化水平

Fig. 4 Influences of Shh signal on the proliferation process of GCs in dairy cow folliclesA-C: Variation in the cell cycle process of GCs in dairy cow follicles. D: Variation in the cell cycle progression

图5 Shh信号对奶牛卵泡GCs的细胞周期的影响A：细胞周期蛋白（CCND1、CCND2、CCND3、CCNE1）的蛋白表达灰度图；B：细胞周期蛋白的蛋白相对表达量；C：细胞周期蛋白依赖性激酶（CDK2、CDK4、CDK6）的蛋白表达灰度图；D：细胞周期蛋白依赖性激酶的蛋白相对表达量

Fig. 5 Influence of Shh signal on the cell cycle of GCs in dairy cow folliclesA: Grayscale images of the protein expressions of cyclins (CCND1, CCND2, CCND3, and CCNE1). B: Relative protein expression of cyclins. C: Grayscale images of the protein expressions of cyclin-dependent kinases (CDK2、CDK4、CDK6). D: Relative protein expression of cyclin-dependent kinases

图6 Shh信号对奶牛卵泡GCs中分化蛋白表达的影响A：细胞分化标志分子（StAR、CYP19A1、LHCGR）的蛋白表达灰度图；B-D：细胞分化标志分子（StAR、CYP19A1、LHCGR）的蛋白相对表达量

Fig. 6 Influence of Shh signal on the expression of differentiation proteins in GCs of dairy cow folliclesA: Grayscale images of the protein expressions of cell differentiation marker molecules (StAR, CYP19A1, and LHCGR). B-D:Relative protein expression of cell differentiation marker molecules (StAR, CYP19A1, and LHCGR)

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