超声微泡促腺病毒载体转染MDR1基因效率的体外实验研究

Ultrasonic microbubbles enhance efficiency of adenovirus vector-mediated MDR1 gene transfection in vitro

目的探讨超声微泡造影剂在体外促进腺病毒载体介导的MDR1基因转染兔骨髓单个核细胞的效率,并初步探讨其安全性。方法根据是否加入超声微泡造影剂和超声辐照将分选的兔骨髓单个核细胞分为常规培养组(A)、腺病毒MDR1基因转染组(B)、腺病毒MDR1基因转染+超声辐照组(C)、微泡造影剂+腺病毒MDR1基因转染组(D)、微泡造影剂+腺病毒MDR1基因转染+超声辐照组(E)。收集各组细胞,应用RT-PCR法、流式细胞技术等分别从基因、功能及细胞生物学特性等方面检测外源性MDR1基因在骨髓单个核细胞内的功能性表达及安全性。结果收获高滴度的Ad5-MDR1腺病毒载体并成功制备腺病毒微泡造影剂;RT-PCR结果显示,除A组外其余4组在194bp处均观察到特异性MDR1电泳条带,证实外源性MDR1基因通过腺病毒载体整合到细胞基因组中,且E组MDR1电泳条带的光密度比值明显高于B、C、D组(P<0.05);柔红霉素排出实验证实外源性MDR1基因表达产物P-gp有药物外排泵功能,且E组P-gp阳性率最高(P<0.05),而柔红霉素阳性率显著降低;一定强度的超声辐照及微泡造影剂对骨髓单个核细胞的细胞周期无明显影响。结论低频超声波击碎微泡造影剂,能促进以腺病毒为载体介导的外源性MDR1基因转染兔骨髓单个核细胞的效率,且安全可行。

Objective To explore the efficacy and safety of ultrasonic microbubbles to enhance the transfection of adenoviral vector encoding muhidrug-resistance gene 1 （ MDR1 ） into rabbit bone marrow mononuclear cells. Methods Adenoviral vector AdS-MDR1 encoding MDR1 gene were prepared and titrated. Bone marrow mononuclear cells were isolated and collected from rabbit bone marrow, and divided into 5 groups （4.0 × 10^6 to 5.0 × 10^6 cells in each group） in a 6-well plate, conventional culture group （A）, Ad5-MDR1 group （B）; AdS-MDR1 ＋ ultrasound group （ C ）, Ad5-MDR1 ＋ microbubbles group （D） and AdS-MDR1 ＋ rnicrobubbles ＋ ultrasound group （E）. MDR1 gene was transferred into the cells with or without ultrasound wave irradiation （665 kHz, 0.5 W/cm2, 15 s） , and cocultured with ultrasonic microbubbles and/or adenoviral vector. The expression of MDR1 gene in the mononuclear cells, the positive transfection rate and the P-gp function were tested by RT-PCR, and daunorubicin （DNR） efflux testing with flow cytometry. The cell cycle of the mononuclear cells after transfection was tested by flow cytometry. Results The Ad5-MDR1 vector with high titer was obtained, and ultrasonic microbubbles were prepared. RT-PCR showed the specific electrophoresis strips were at 194 bp in 4 groups except group A, indicating that the foreign MDR1 gene had been transferred successfully into the cells mediated by adenovirus. The optical density of the strip from group E was higher than that in group B , C and D （P 〈 0.05 ）. DNR efflux testing confirmed that the P-gp of transferred MDR1 gene maintained its biological function, and the function of P-gp in group E was higher than that in other groups （ P 〈 0.05 ）. The cell cycles of transferred mononuclear cells did not be affected by the gene transfection. Conclusion The transfer of foreign MDR1 gene into rabbit mononuclear cells with adenovirus vector is enhanced by the ultrasonic microbubbles, with safety and availability.