腺病毒介导人FRNK基因对结肠癌细胞Colo320WT中p190RhoGAP磷酸化和RhoA活性的影响

Effects of adenovirus vector expression hFRNK on phosphorylated 190RhoGAP and RhoA Activity in colo320WT colorectal cells

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乏受体／胃泌素受体（CCK-2R）的阳性克隆，逆转录一聚合酶链反应（RT—PCR）鉴定。用10～8mol／L胃泌素干预Colo320WT细胞12h和pAdhFRNK体外感染Colo320WT细胞2d后，再用10—8moL／L的胃泌素干预细胞12h，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12h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 study effects of hFRNK gene on phosphorylated plgORhoGAP expression and RhoA activation by mediated adenoviral vector in colorectal carcinoma cell Colo320WT stimulated with extrinsic gastrin17 in vitro. Methods AdEasyTMsystem was used to construct pAdhFRNK expressing human FRNK gene by recombination in E. coll. BJ5283. pCP,3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colonic carcinoma cell line Colo320 cells by LipofectamineTM2000 and expression stably CCK-2R clones was selected by G418（500 μg/mL）. The expression levels of gastrin receptor of Colo320 cells and the transfected cells Colo320WT were assayed by RT-PCR. Colo320WT cells were treated by 10 - 8 mol/L Gastrinl7 for Oh and 12 h;and after Colo320WT cells were infected by pAdhFRNK （MO1：100） for 2 d, the cells were treated by Gastrinl7 for 12 h again. The expression levels of phosphorylated pl90RhoGAP of Colo320WT cells were assayed by immuniprecipation and western blot. RhoA activation was assayed by pull-down, using GST- Rhotekin-RBD followed by RhoA blotting. Results When 10 - 8 mol/L Gastrinl7 stimulated Colo320WT cells for 12 h,the expression levels of phosphorylated p190RhoGAP increased apparently and RhoA activation diminished. When pAdhFRNK infected Colo320WT cells for 2 d and 10 - 8 mol/L Gastrinl7 treated the cells for 12 h ,the expression levels of phosphorylated p190RhoGAP decreased apparently and RhoA activity was elevated. Conclusion hFRNK can inhibit expression of phosphorylated p190RhoGAP and enhanced RhoA activity in the cells stimulated with Gastrin17 ,and its mechanism is probably that hFRNK can block FAK phosphorylation and FAK pathway.