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目的探讨超声介导白蛋白微泡破裂促进心肌营养素1(CT-1)基因转移骨髓间充质干细胞(MSC)表达的测定。方法从分离培养的新生大鼠心肌细胞中克隆CT-1基因,构建其表达载体,与白蛋白微泡混合后,在超声作用下介导CT-1基因转染大鼠MSC,荧光显微镜及流式细胞术检测报告基因EGFP的表达,Western blot检测MSC中CT-1表达。结果可获大鼠CT-1编码基因,并成功构建表达载体pIRES2-CT-1。体外实验结果示,以脉冲0.75 W/cm2作用30 s转染2次,可以有效地将CT-1基因转染MSC,经转染的MSC可以表达分泌CT-1及报告基因EGFP。结论超声介导白蛋白微泡破裂可以有效促进CT-1基因转染MSC,该技术可能为临床应用基因联合干细胞移植治疗急性心肌梗死提供新的方法。
OBJECTIVE: To investigate the effect of ultrasound-mediated albumin microbubble rupture on the expression of cardiotrophin-1 (CT-1) gene transfer bone marrow mesenchymal stem cells (MSC). Methods CT-1 gene was cloned from cardiomyocytes isolated from neonatal rats. The expression vector was constructed and mixed with albumin microbubbles. The CT-1 gene was transfected into rat MSCs under fluorescence microscope. The fluorescence microscopy and flow cytometry The expression of EGFP was detected by cytometry and the expression of CT-1 in MSC was detected by Western blot. Results Rat CT-1 encoding gene was obtained and the expression vector pIRES2-CT-1 was successfully constructed. The results of in vitro experiments showed that CT-1 gene could be transfected into MSCs effectively with 0.75 W / cm2 pulse for 30 s. The transfected MSCs could express secreted CT-1 and EGFP. Conclusion Ultrasound-mediated rupture of albumin microbubbles can effectively promote CT-1 gene transfection into MSCs. This technique may provide a new method for clinical application of gene combined with stem cell transplantation in the treatment of acute myocardial infarction.