胰岛素样生长因子2通过磷酯酰肌醇-3激酶/蛋白激酶B信号通路调控人卵巢颗粒细胞增殖的机制

doi:10.16098/j.issn.0529-1356.2021.04.011

解剖学报 ›› 2021, Vol. 52 ›› Issue (4): 567-573.doi: 10.16098/j.issn.0529-1356.2021.04.011

胰岛素样生长因子2通过磷酯酰肌醇-3激酶/蛋白激酶B信号通路调控人卵巢颗粒细胞增殖的机制

丁祥云¹ 马永臻^1* 尹华伟¹ 段君君¹ 李真¹ 庄园¹ 张琳²

1.山东医学高等专科学校组织学胚胎学教研室, 山东临沂 276000;2.临沂市妇幼保健院妇科，山东临沂 276002

收稿日期:2020-04-30 修回日期:2020-06-18 出版日期:2021-08-06 发布日期:2021-08-06
通讯作者: 马永臻 E-mail:myz0818@163.com
基金资助:
山东省高等学校科技计划项目

Mechanism of insulin-like growth factor 2 regulating human ovarian granulosa cell proliferation through the phosphatidylinositol 3-kinase/ protein kinase B signaling pathway

DING Xiang-yun¹ MA Yong-zhen^1* YIN Hua-wei¹ DUAN Jun-jun¹ LI Zhen¹ ZHUANG Yuan¹ZHANG Lin²

1. Department of Histology and Embryology, Shandong Medical College, Shandong Linyin 276000,China; 2. Department of Gynecology, Linyi Women and Children’s Health Care Hospital, Shandong Linyin 276000,China

Received:2020-04-30 Revised:2020-06-18 Online:2021-08-06 Published:2021-08-06
Contact: MA Yong-zhen E-mail:myz0818@163.com

摘要/Abstract

摘要：

目的检测胰岛素样生长因子2（IGF2）对人卵巢颗粒细胞（KGN细胞）增殖的调控作用及作用机制。方法将体外培养的KGN细胞，分不同浓度IGF2处理组（对照组和25 μg/L、50 μg/L、100 μg/L IGF2组)和磷酯酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)信号通路干预组（以LY294002干预处理将细胞分对照组、100μg/L IGF2组、LY294002组和IGF2+LY294002组）。采用MTS和5-乙炔基-2’-脱氧尿苷(EdU)法检测IGF2对KGN细胞增殖的影响，ELISA法检测细胞培养液上清中雌激素、孕激素的含量，Western blotting法检测各组胰岛素样生长因子受体1（IGF1R）、蛋白激酶B（Akt）、磷酸化的蛋白激酶B（p-Akt）及CYP19A1蛋白表达。结果随着IGF2的浓度梯度增高，KGN细胞的增殖率及雌激素、孕激素的分泌量逐渐升高，以 100 μg/L IGF2处理组的细胞增殖率和激素水平最高 (P<0.01)，而抑制PI3K/Akt信号通路，细胞增殖率和激素的分泌量均明显降低(P<0.01)；Western blotting结果显示，IGF1R、p-Akt及CYP19A1 蛋白表达水平随着IGF2的浓度梯度逐渐升高(P<0.05)，而干预PI3K/Akt信号通路可影响上述蛋白的表达，与对照组相比，IGF2组和IGF2+ LY294002组IGF1R及p-Akt蛋白表达均明显升高（P<0.01），CYP19A1在IGF2组明显升高（P<0.01），LY294002组p-Akt及 CYP19A1蛋白明显降低（P<0.05），IGF1R的表达差异无统计学意义。与IGF2组相比，IGF2+LY294002组p-Akt及CYP19A1蛋白表达降低（P<0.01），IGF1R的表达差异无统计学意义，LY294002组p-Akt 及CYP19A1蛋白表达量均显著降低（P<0.01）。结论 IGF2 可能由IGF1R介导通过PI3K/Akt 信号通路促进人卵巢颗粒细胞的增殖及分泌功能。

关键词: 胰岛素样生长因子2, 磷酯酰肌醇-3激酶/蛋白激酶B信号通路, 人卵巢颗粒细胞, 免疫印迹法

Abstract:

Objective To explore the effect and mechanism of insulin-like growth factor 2(IGF2) on the proliferation of human ovarian granulosa cells (KGN). Methods KGN cells cultured in vitro and treated with different concentrations of IGF2 were divided into control group and IGF2 group (25 μg/L, 50 μg/L, 100 μg/L), and then cells were divided into control group, 100 μg/L IGF2 group, LY294002 group, and IGF2 +LY294002 group after intervened the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway with LY294002. MTS and 5-ethynyl-2’-deoxyuridine (EdU) method was used to detect the effect of IGF2 on KGN cell proliferation, and enzyme linked immunosorbent assay was used to detect the contents of estrogen and progesterone in cell culture supernatant. The expressions of insulin like growth factor 1 receptor (IGF1R), protein kinase B (Akt), phosphorylated protein kinase B(p-Akt) and CYP19A1 protein in each group were detected by Western blotting. Results With the concentration gradient of IGF2, the proliferation rate of KGN cells and the secretion of estrogen and progesterone gradually increased. The cell proliferation rate and hormone level in the group treated with 100μg/L IGF2 were the highest (P<0.01), while the PI3K/Akt signaling pathway was inhibited, and the cell proliferation rate and hormone secretion decreased significantly (P<0.01).The protein expression levels of IGF1R, p-Akt and CYP19A1 in different concentration groups increased significantly (P<0.05). While the expression of the above proteins were affected by intervened the PI3K/Akt signaling pathway.Compared with the control group, the protein expression of IGF1R and p-Akt increased significantly in IGF2 group and IGF2 +LY294002 group(P<0.01), CYP19A1 increased significantly in IGF2 group(P<0.01), the protein expression of p-Akt and CYP19A1 decreased significantly in LY294002 group(P<0.05), there was no significant difference in the protein expression of IGF1R.Compared with the IF2 group, the protein expression of p-Akt and CYP19A1 decreased in IGF2 +LY294002 group (P<0.01), there was no statistically significant difference in the protein expression of IGF1R, and the expression levels of IGF1R, p-Akt and CYP19A1 were significantly reduced in LY294002 group (P<0.01). Conclusion IGF2 may promote the proliferation and secretion of human ovarian granulosa cells through the PI3K/Akt signaling pathway mediated by IGF1R.

Key words: Insulin-like growth factor 2, Phosphatidylinositol 3-kinase/protein kinase B signaling pathway, Human ovarian granulosa cell, Western blotting

中图分类号:

R329.2

丁祥云马永臻尹华伟段君君李真庄园张琳. 胰岛素样生长因子2通过磷酯酰肌醇-3激酶/蛋白激酶B信号通路调控人卵巢颗粒细胞增殖的机制[J]. 解剖学报, 2021, 52(4): 567-573.

DING Xiang-yun MA Yong-zhen YIN Hua-wei DUAN Jun-jun LI Zhen ZHUANG Yuan ZHANG Lin. Mechanism of insulin-like growth factor 2 regulating human ovarian granulosa cell proliferation through the phosphatidylinositol 3-kinase/ protein kinase B signaling pathway[J]. Acta Anatomica Sinica, 2021, 52(4): 567-573.

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胰岛素样生长因子2通过磷酯酰肌醇-3激酶/蛋白激酶B信号通路调控人卵巢颗粒细胞增殖的机制

Mechanism of insulin-like growth factor 2 regulating human ovarian granulosa cell proliferation through the phosphatidylinositol 3-kinase/ protein kinase B signaling pathway

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