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目的利用腺病毒载体表达人FRNK基因,体外观察其对胃泌素干预下的结肠癌细胞Colo320WT中p190RhoGAP磷酸化和RhoA活性的影响。方法2005年10月至2006年9月,武汉大学人民医院消化内科在中国科学院,武汉病毒所病毒学国家重点实验室,利用AdEasyTM系统在大肠埃希菌内同源重组构建表达人FRNK基因的腺病毒载体pAdhFRNK。脂质体转染pCR3.1-GR质粒于结肠癌细胞Colo320中,G418抗生素筛选出稳定表达胆囊收缩素-2受体/胃泌素受体(CCK-2R)的阳性克隆,逆转录-聚合酶链反应(RT-PCR)鉴定。用10~8mol/L胃泌素干预Colo320WT细胞12 h和pAdhFRNK体外感染Colo320WT细胞2 d后,再用10~8 mol/L的胃泌素干预细胞12 h,然后用免疫沉淀方法检测磷酸化的p190RhoGAP的表达,pull-down测定RhoA的活性。结果在胃泌素干预12 h后的磷酸化的p190RhoGAP的表达量明显增加,RhoA活性降低,而用pAdhFRNK感染后胃泌素干预的细胞中磷酸化p190RhoGAP表达又降低,RhoA活性却增加。结论hFRNK基因可明显阻断外源性胃泌素引起Colo320WT细胞中p190RhoGAP磷酸化的表达和RhoA的活性。
Objective To express human FRNK gene by adenoviral vector and observe its effect on p190RhoGAP phosphorylation and RhoA activity in colorectal cancer cell line Colo320WT with gastrin intervention. Methods From October 2005 to September 2006, the gland of human FRNK gene was constructed by homologous recombination in Escherichia coli using the AdEasyTM system at the National Key Laboratory of Virology of Chinese Academy of Sciences and Wuhan University, Department of Gastroenterology, People’s Hospital of Wuhan University. Viral vector pAdhFRNK. Liposome-transfected pCR3.1-GR plasmid in colon cancer cells Colo320, G418 antibiotics screened positive expression of cholecystokinin-2 receptor / gastrin receptor (CCK-2R) positive clones, reverse transcription - polymerization Enzyme-linked reaction (RT-PCR) identification. Colo320WT cells were treated with 10 ~ 8mol / L gastrin for 12 h and pAdhFRNK was infected with Colo320WT cells in vitro for 2 days. Then the cells were treated with 10 ~ 8 mol / L gastrin for 12 h, and then phosphorylated by immunoprecipitation p190RhoGAP expression, pull-down assay RhoA activity. Results The expression of phosphorylated p190RhoGAP was significantly increased and the activity of RhoA was decreased 12 h after gastrin intervention. However, the expression of phosphorylated p190RhoGAP was decreased and the activity of RhoA was increased in cells treated with pAdhFRNK. Conclusion hFRNK gene can obviously block p190RhoGAP phosphorylation and RhoA activity induced by exogenous gastrin in Colo320WT cells.