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目的:探讨AREG蛋白在前列腺素E2(prostaglandin E2,PGE2)促进人胆管上皮癌细胞CCLP1增殖能力中的作用和机制。方法:用PGE2、4种EP受体激动剂(17-phenyltrinor Prostaglandin E2、Butaprost、Sulprostone和Prostaglandin E1 Alcohol)、EP2受体拮抗剂AH6809、PKA抑制剂H89、AC抑制剂SQ22536处理CCLP1细胞,通过Western blot、WST等实验检测AREG蛋白表达水平以及CCLP1细胞增殖能力的变化。结果:10μmol/L PGE2处理CCLP1细胞后,AREG蛋白的表达水平与对照组相比升高了44.2%(P<0.05);20μmol/L AREG中和抗体Mab262和SAB1402118分别处理1 h后的WST实验结果显示,PGE2诱导的CCLP1细胞增殖可被AREG中和抗体抑制,分别下降了18%、20%(P<0.01)。10μmol/L 4种EP受体激动剂处理CCLP1细胞的结果表明,EP2受体激动剂具有明显促进AREG蛋白表达的作用(表达水平上调了20.1%,P<0.05),而EP1、EP3、EP4无明显促进表达作用;10μmol/L EP2受体拮抗剂AH6809处理CCLP1细胞后,AREG蛋白的表达水平较EP2受体激动剂处理组下调了20%(P<0.05)。用PKA抑制剂H89处理后,AREG蛋白的表达水平和CREB蛋白的磷酸化水平较PGE2处理组分别下降了54.4%、45%(P<0.05);用CMV500-DN-CREB质粒转染CCLP1细胞抑制了CREB的表达和磷酸化后,AREG的表达量较对照组明显下降约65%(P<0.01)。结论:PGE2可通过EP2受体激活cAMP-PKA-CREB信号转导通路上调CCLP1细胞AREG的表达,从而促进CCLP1细胞的增殖。
OBJECTIVE: To investigate the role and mechanism of AREG protein in the proliferation of human cholangiocarcinoma CCLP1 cells by prostaglandin E2 (PGE2). Methods: CCLP1 cells were treated with PGE2, 4 kinds of EP receptor agonists (Butaprost, Sulprostone and Prostaglandin E1 Alcohol), EP2 receptor antagonist AH6809, PKA inhibitor H89 and AC inhibitor SQ22536. blot, WST and other experiments to detect AREG protein expression and CCLP1 cell proliferation. Results: The expression of AREG protein in CCLP1 cells treated with 10μmol / L PGE2 was increased by 44.2% (P <0.05) compared with that of the control group. The WST assay was performed after treated with 20μmol / L AREG neutralizing antibodies Mab262 and SAB1402118 The results showed that the proliferation of CCLP1 cells induced by PGE2 could be inhibited by AREG neutralizing antibody and decreased by 18% and 20%, respectively (P <0.01). The results showed that EP2 receptor agonist could significantly enhance AREG protein expression (up-regulated by 20.1%, P <0.05), whereas EP1, EP3 and EP4 had no effect on CCLP1 cells treated with 10μmol / L EP agonist (P <0.05). After treatment with 10μmol / L EP2 antagonist AH6809 for CCLP1 cells, AREG protein expression was down-regulated by 20% (P <0.05) compared with EP2 receptor agonist treatment group. After treatment with PKA inhibitor H89, the expression level of AREG protein and phosphorylation of CREB protein were decreased by 54.4% and 45%, respectively (P <0.05) compared with PGE2 treatment group. CCLP1 cells were transfected with CMV500-DN-CREB plasmid After CREB expression and phosphorylation, AREG expression was significantly decreased by about 65% (P <0.01) compared with the control group. CONCLUSION: PGE2 can up-regulate the expression of AREG in CCLP1 cells via EP2 receptor-activated cAMP-PKA-CREB signaling pathway, thereby promoting the proliferation of CCLP1 cells.