超声微泡造影剂促腺病毒转染mdr1基因的体外实验

Research on the gene transfection of mdr1 mediated by adenovirus enhanced by ultrasonic microbubbles in vitro

目的研究体外超声微泡造影剂促腺病毒转染mdr1基因的效率。方法扩增表达mdr1基因的重组腺病毒Ad5-mdr1并与微泡造影剂混合；密度梯度离心法分选兔骨髓单个核细胞，与腺病毒微泡造影剂混合，经超声辐照后常规培养；荧光显微镜下观察细胞内荧光强度表达；RT-PCR法检测骨髓单个核细胞基因组中外源性mdr1基因的表达；免疫组织化学法检测转染细胞中mdr1基因的表达产物。结果①转染后b（腺病毒转染组）、c（腺病毒转染＋超声辐照组）、d（腺病毒微泡造影剂转染组）及e（腺病毒微泡造影剂转染＋超声辐照组）组细胞内均有绿色荧光显示，a（常规培养组）组细胞内未见绿色荧光，e组细胞内荧光强度明显高于各对照组；②RTPCR法显示，除a组外其余4组在157bp处分别观察到特异性mdrl电泳条带，证实外源性mdr1基因通过腺病毒作为载体整合到细胞基因组中；③免疫组织化学法检测e组细胞P-gp表达阳性率（24％）显著高于a、b、Cxd（阳性率分别为0．5％、8．5％、10%、11．5％）各组（P〈0．05）。结论体外实验证实，超声微泡造影剂能促进以腺病毒为载体的mdr1基因转染和表达。

Objective To research the transfection efficiency of mdrl gene mediated by adenovirus enhanced by ultrasonic microbuhhles in vitro. Methods Recombinant adenovirus Ad5-mdr1 ,which could express mdr1 gene, was cultured and mixed with ultrasonic microhuhhles. Mononuclear cells in bone marrow which contained massive hematopoietic cells of the New Zealand rabbits were gathered and enriched by density gradient centrifugation,and mixed with ultrasonic microbubbles. The mixtures were cultured conventionally after ultrasound wave irradiation. The fluorescence intensity in cells was observed by fluorescence microscope, and the expression of the exogenous mdr1 gene in transferred bone marrow mononuclear cells was detected by RT-PCR,and the Poglycoprotein （expression product of mdr1 gene） in cells was detected by immunohistochemical staining. Results After the transfection, the green fluorescence had been observed in cells of adenovirus transfection group （group B）, adenovirus transfection and ultrasonic irradiation group （group C）, adenovirus transfection with ultrasonic microhuhbles group （group D） and adenovirus transfection with ultrasonic microhuhhles and ultrasonic irradiation （group E）, while not in adenovirus transfection group （group A）. Moreover, the fluorescence intensity in cells of group E was significantly higher than that of the other groups. The results of RT-PCR showed that the specific electrophoresis strips were observed at 157hp in 4 groups except group A,which showed that the mdr1 gene had been transferred into the genome of the cells mediated by adenovirus. The positive rate of P-gp expression in cells of group A, B, C, D and E was 0. 5%, 8. 5%,10%,11.5 % and 24% respectively , which were detected by immunohistochemical staining. The rate of expression of P-gp in group E was higher than that of the other groups （P〈0. 05）. Conclusions The results demonstrate that the efficiency of gene transfection and expression of mdr1 can be enhanced by adenovirus mediated ultrasonic microhuhhles in vitro.