缺氧應激对HepG-2细胞AFP和ICAM-1表达的影响.docVIP

缺氧应激对HepG-2细胞AFP和ICAM-1表达的影响 卢根林 邓勇 【摘要】 目的 探讨缺氧应激对HepG-2细胞甲胎蛋白（alpha fetoprotein, AFP）和细胞间粘附分子（intercellular adhesion molecule，ICAM）-1表达的影响。方法 缺氧（5%O2、5%CO2、90%N2）和常氧培养HepG-2细胞。逆转录聚合酶链反应（RT-PCR）技术测定常氧组和缺氧12、24h组 HepG-2细胞AFPmRNA和ICAM-1mRNA 的表达量。流式细胞仪技术测定ICAM-1蛋白表达，放射免疫法 (RIA）测定HepG-2细胞培养上清AFP蛋白表达。结果随着缺氧时间的延长HepG-2细胞的AFP、ICAM-1 mRNA和蛋白表达量明显增高(P＜0.01）。结论 缺氧应激上调HepG-2细胞AFP、ICAM-1基因表达。 【关键词】 缺氧应激 HepG-2细胞 甲胎蛋白 细胞间粘附分子-1 Effects of hypoxia stress on expressions of AFP and ICAM-1 in HepG-2 cell LU Gen-lin *, DENG Yong (*The First Department of General Surgery, Yiwu central hospital of Zhejiang Province, Yiwu 332000, P. R. China) 【Abstract】Objective To investigate whether Hypoxia stress enhances expressions of AFP and ICAM-1 in HepG-2 cell or not . Methods HepG-2 cells were cultured under hypoxia (5%O2, 5%CO2 and 90%N2) for 12, 24 h or normoxia. The expressions of AFP and ICAM-1 mRNA in HepG-2 cell were determined by reverse transcription polymerase chain reaction（RT-PCR）.The expression of ICAM-1 protein in HepG-2 cell was studied by flow cytometry assay. The expression of AFP protein in HepG-2 cell was determined by radio-immunity assay (RIA）. Results With the hyopxia time inceasing, the expressions of AFP and ICAM-1 gene were dramatically enhanced (P＜0.01）. Conclusion Hypoxia stress upregulates the expression of AFP and ICAM-1 in 【Key words】 Hypoxia stress HepG-2 cell AFP ICAM-1 缺氧应激（5%O2）促进HepG2细胞增殖活性[1]、粘附、侵袭和迁移能力[2]，但其分子机制尚不明确。本实验探讨缺氧应激（5%O2）对HepG-2细胞AFP和ICAM-1表达的影响。 1.材料与方法 1.1细胞株和主要试剂：人肝癌细胞株HepG-2引自兰州军区总医院实验科（来源于美国国立肿瘤研究所）， 胎牛血清为杭州四季青生物工程公司产品， MTT、DMSO和胰蛋白酶均为美国Sigma产品， RT-PCR Kit 购于Takara公司，Trizol为Invitrogen公司产品。人β-肌动蛋白（β-actin[3]）、AFP [4]和ICAM-1 [5]基因聚合酶链反应引物序列如下：β-actin上游引物5′-GTACCACTGGCATCGTGATGGACT-3′，下游引物5′-ATCCACACGGAGTACTTGCGCTCA-3′，目的片段长度为600bp；AFP上游引物5′- GTTCCAGAACCTGTCAC AAG-3′，下引物5′-CTTTGTTTGGAAGCATTCAACTGC-3′，目的片段长度为193bp；ICAM-1上游引物5′- TGGTAGCAGCCGCAGTCATA-3′，下引物5′-CTCCTTCCTGGCTTAGT-3′，目的片段长度为377bp；由Invitrogen公司合成。兔抗人ICAM-1多克隆抗体，羊抗兔IgG-FITC