AAV2-BPI700-Fcγ1700重组病毒转染小鼠结肠上皮离子转运的改变机理

The change mechanism of colonic epithelium transport in AAV2-BPI700-Fcγ1700 transferred mice

目的 应用Ⅱ型腺相关病毒-杀菌渗透增强蛋白嵌合基因（AAV2-BPI700-Fcγl700）转染的小鼠,给予感染最小致死剂量的大肠杆菌后,观察和探究结肠上皮电阻和离子转运的变化和机制.方法 应用腺相关病毒嵌合基因转染、短路电流记录、ELISA等方法测定小鼠结肠粘膜的离子转运信息.结果 基因转染小鼠与对照组相比,结肠上皮基础电阻和短路电流明显增加,在上皮的顶膜侧加入钠离子通道阻断剂阿米洛利后,则可以明显抑制增加的电流;应用上皮离子促分泌物5-HT后,可以明显增加结肠上皮的短路电流,这种短路电流可以被Cl-通道阻断剂DPC、Na＋-K＋-2Cl-共转运体抑制剂bumetanide明显抑制;并且5-HT作用结肠粘膜后,上皮cAMP的含量明显高于对照组.应用5-HT4受体拮抗剂GRI 13808加入到粘膜组织的基底侧进行预处理,可以明显抑制基因转染组5-HT诱导的cAMP增加,基因转染小鼠5-HT4受体免疫阳性明显高于对照组.结论 5-HT通过5-HT4受体诱导基因转染小鼠cAMP依赖性的Cl-离子转运增加,从而促进了肠道排毒反应.

Objective To explore the changes and mechanism of colonic epithelial resistance and ion transport, with adeno-associated virus 2 （AAV2）-bactericidal permeability increasing protein 700 （BPI700）-fragment crystallizable gamma one 700 （Fcγ1700） chimeric gene-transferred mice were infected by the minimal lethal dose of E.coli..Methods Gene transfection, the short circuit current（Isc） record, ELISA and other methods were used to determine the colonic ion transport information.Results The basal membrane resistance and Isc of colonic mucosa in gene-transferred mice were significantly increased, but the apical addition of amiloride, a blocker of epithelial sodium channels, significantly inhibited the ISC-5-HT significantly increased the change in colonic ISC, which was much stronger in gene-transferred mice.The ISC was significantly inhibited by both the apical addition of diphenylamine-2, 2＆#39;-dicarboxylicacid （DPC）, a blocker of the Cl-channel, and the basolateral application of bumetanide, an inhibitor of Na＋-K＋-2Cl-co-transporter （NKCC）.Furthermore, the 5-HT-induced increased in intracellular cAMP levels was higher in gene-transferred mice.Pretreatment with 5-HT4 receptor antagonists GRI 13808 significantly inhibited 5-HT induced cAMP increases in transgenic mice.Expression of 5-HT4 receptor in gene-transferred mice was significantly higher than that in the control mice.Conclusions The study demonstrates that 5-HT enhances cAMP-dependent Cl-secretion of colonic mucosa via 5-HT4 receptor in AAV2-BPI700-Fcγ1700 transgenic mice.