AQP1慢病毒载体成功转染心肌细胞后加重体外循环后心肌细胞水肿

Lentiviral-AQPl vector successfully transfect the myocytes and aggravate the myocardiac edema after cardiopulmonary bypass

目的探讨水通道蛋白-1（AQP1）慢病毒载体可否成功转染心肌细胞，转染成功的心肌细胞在体外循环结束后，不同时点AQP1表达变化及与心肌水肿的关系。方法健康成年羊36只，分为空载体组和AQP1慢病毒转染组。根据羊AQP1mRNA全基因设计酶切引物，克隆到慢病毒表达载体中，与其它包装载体共转染293T细胞产生病毒载体并测定病毒滴度。体外循环期间将空载体或AQP1病毒悬液注射入左心室心肌组织，体外循环结束后不同时间点（2、6、12、24、48、72h）取材，每个时间点3只。应用reahimePCR、WesternBlot、ELASE、流式细胞仪（FACS）和干湿重法等检测AQP1慢病毒载体是否成功转染心肌细胞，成功转染体外循环后不同时间点AQP1表达变化和心肌水肿程度。结果成功构建AQP1慢病毒载体并转染心肌细胞，体外循环后不同时间点AQP1表达变化趋势和心肌水肿程度与空载体组心肌表达变化趋势一致，但程度较高，差异有统计学意义（P〈0．05）。结论AQP1慢病毒载体可成功转染心肌细胞，AQP1高表达的心肌组织在体外循环后不同时间点AQP1的表达和水肿程度变化趋势不变，但均较空载体转染组程度高。AQP1表达量与心肌水肿程度正相关。

Objective Construct the lentiviral AQP1 vector and explore whether it can transfect the myocyte or not,then test the law of the AQP1 expression and the edema in the successfully transducted myocytes after cardiopulmonary bypass in sheeps. Methods Design cleavage primer according to ovine AQP1 mRNA, clone it into expressing vector and transducated in- to the 293T cells with other packing vectors to produce the lentiviral AQP1 vector, then test the virus titer. 36 adult healthy sheeps are randomly divided into blank or AQP1-lentiviral transfected group, blank or AQP1-lentiviral vector suspension was in- jected in the ventricle tissue of healthy adult sheeps during cardiopulmonary bypass and take specimen in different time points （2, 6, 12, 24, 48, 72 h）after extracorporeal circulation, 3 in each group. Realtime-PCR WesternBlot ELISA FACS immu- mofluorescent and Dry/Wet methods are emploied to detect the expression of AQP1 and the according degree of edema. Results lentiviral AQP1 vector was successfully construeed and transducated into the myoeytes. The trandueated groups have the same trend of AQP1 of expression and cardiac edema after cardiopulmonary bypass compared to the blank vector group, but the degree is heavier（ P 〈 0.05 ）. Conclusion Lentivral AQP1 vector can successfully transfeet the myoetyes , and the overex- pressed myocardial tissue have the same trend of AQP1 expression and edema after cardiopulmonary bypass, but with a heaver degree. The expression of aquaporins was positively relevant to the edema.