【摘要】 目的 探索垂体生长激素腺瘤细胞增殖的分子机制。方法 收集12例肢端肥大症患者的功能性生长激素垂体腺瘤（fGH-PA）组织样本。qPCR法测定fGH-PA组织和大鼠RGC-5、MMQ、GH3细胞中cAMP直接激活的交换蛋白1(Epac1) mRNA表达水平。免疫组织化学测定fGH-PA组织中Epac1蛋白表达情况。Western blot测定MMQ、GH3细胞中Epac1蛋白表达情况。在GH3细胞中过表达或敲低Epac1，或敲低cAMP反应元件结合蛋白(CREB)，流式细胞术测定细胞周期变化；CCK-8法测定细胞增殖能力；Western blot测定细胞内p-CREB、CREB、Cyclin D1、CDK2、p21的表达情况。结果 qPCR和免疫组织化学结果显示，与旁侧正常组织相比，fGH-PA组织中Epac1 mRNA和蛋白表达水平明显增高（P<0.05）。qPCR和Western blot结果显示，与RGC-5细胞相比，MMQ和GH3细胞中Epac1 mRNA和蛋白表达水平明显增高（P<0.05）。在GH3细胞中过表达Epac1后，与Control组相比，Epac1-OE组G0/G1期和S期细胞占比明显减少（P<0.05）,G2/M期细胞占比明显增多（P<0.05）；细胞增殖能力明显增强（P<0.05）；细胞内p-CREB和Cyclin D1的表达水平明显升高（P<0.05）,CDK2和p21的表达水平明显降低（P<0.05），而CREB的表达水平无明显变化（P>0.05）。在GH3细胞中敲低Epac1或敲低CREB后，上述所有结果均发生逆转（P>0.05）。结论 垂体生长激素腺瘤细胞中Epac1过表达能够上调细胞内p-CREB的水平，并促进腺瘤细胞通过G1/S期检查点，发生细胞周期检查点失调和大量增殖，但不影响CREB的表达水平。更多还原

【Abstract】 Objective To explore the molecular mechanism of pituitary growth hormone adenoma cell proliferation.Methods Functional growth hormone-secreting pituitary adenoma(fGH-PA) tissue samples were collected from 12 patients with acromegaly. The exchange protein 1 directly activated by cAMP(Epac1) mRNA expression levels in fGH-PA tissues and rat RGC-5,MMQ and GH3 cells were determined by qPCR. The expression levels of Epac1 in fGH-PA tissues were determined by immunohistochemistry. Western blot was used to determine the expression levels of Epac1 in MMQ and GH3 cells. Overexpression or knockdown of Epac1, or knockdown of cAMP response element-binding protein(CREB) in GH3 cells, cell cycle changes were determined by flow cytometry, cell proliferation ability was determined by CCK-8 assay, and the expression levels of p-CREB, CREB, Cyclin D1, CDK2 and p21 in cells were determined by Western blot.Results qPCR and immunohistochemistry results showed that the expression levels of Epac1 mRNA and protein in fGH-PA tissues were significantly higher than those in adjacent normal tissues(P<0. 05). qPCR and Western blot results showed that compared with RGC-5cells, the expression levels of Epac1 mRNA and protein in MMQ and GH3 cells were significantly increased(P<0. 05). After overexpression of Epac1 in GH3 cells, compared with the Control group, the proportion of cells in G0/G1 phase and S phase in the Epac1-OE group were significantly reduced(P<0. 05), and the proportion of cells in G2/M phase were significantly increased(P<0. 05); the cell proliferation ability were significantly enhanced(P<0. 05); the expression levels of p-CREB and Cyclin D1 in cells were significantly increased(P<0. 05), the expression levels of CDK2 and p21 were significantly decreased(P<0. 05), while there was no significant change in the expression level of CREB between the two groups(P>0. 05). After knockdown of Epac1 or knockdown of CREB in GH3 cells, all of the above results were reversed(P<0. 05).Conclusion The overexpression of Epac1 in pituitary growth hormone adenoma cells can up-regulate the levels of p-CREB in cells, and promote adenoma cells to pass through G1/S phase checkpoint, resulting in cell cycle checkpoint disorder and massive proliferation, but it does not affect the expression levels of CREB.更多还原