超声微泡造影剂促腺病毒介导MDR1基因转染兔骨髓单个核细胞的作用机制及安全性研究  

Research on the mechanism and security of MDRI gene transfection into the bone marrow mononuclear cells of rabbit with adenovirus vector promoted by ultrasonic microbubble

作　　者：郭振华[1] 王佚[1] 金先庆[1] 郭玉霞[1] 罗庆[1] 王志刚[2] 

机构地区：[1]重庆医科大学附属儿童医院普外科,400014 [2]重庆医科大学超声研究所

出　　处：《中华超声影像学杂志》2010年第3期262-265,共4页Chinese Journal of Ultrasonography

基　　金：基金项目：国家自然科学基金（30600579）;重庆市教育委员会科学技术研究项目（市教委KJ060304）

摘　　要：目的探讨超声微泡造影剂促进以腺病毒为载体介导的多药耐药基因1（MDRl）体外转染兔骨髓单个核细胞的作用机制和安全性。方法根据是否加入超声微泡造影剂和进行超声辐照将分选的兔骨髓单个核细胞分为常规培养组（A）、腺病毒转染组（B）、腺病毒转染＋超声辐照组（c）、腺病毒微泡造影剂转染组（D）和腺病毒微泡造影剂转染＋超声辐照组（E）。不同实验因素处理后，采用流式细胞仪检测各组细胞外源性MDRI基因的转染率，同时观测转染后不同时间各组细胞体外扩增情况；台盼蓝拒染法计数各组细胞的存活率；分别用扫描电镜和透射电镜观察超声辐照后细胞超微结构的改变。结果流式细胞仪检测各组细胞MDRl基因的转染率分别为0．39％±0．11％、5．03％±0．35％、4．93％±（0．38％、5．25％±0．80％、19．93％±1．51％，E组明显高于其余4组（P〈0．05）；试验各组细胞住转染后不同时期体外增殖情况差异无统计学意义（P〉0．05），各组细胞存活率差异亦无统计学意义（P〉0．05）；一定强度的超声辐照后，骨髓单个核细胞胞膜出现一过性的小孔，细胞器发生可逆性肿胀改变。结论超声微泡造影剂可使兔骨髓单个核细胞通透性暂时性提高，促进腺病毒载体介导的MDRI基因转染；同时不会对细胞的增殖能力、存活率及超微结构造成严重的不可逆损害，证明其是一种安全可行的基因转移新方法。Objective To explore the mechanism and safety of mihidrug resistance gene 1 （MDR1） transfection into the bone marrow mononuelear cells of rabbit in vitro with the adenovirus vector promoted by ultrasonic mierobubble. Methods Bone marrow mononuclear cells of rabbits were collected and divided into 5 groups after cultured in the 6 well plate according to the different experimental conditions （MDR1 gene was transferred into the cells with or without ultrasound irradiation and microbubbles）： conventional culture group （A）, Ad5-MDR1 group（B）, Ad5-MDR1 ＋ ultrasound irradiation group（C）, Ad5-MDR1 ＋ microbubbles group（ D）, AdS MDR1 ＋ ultrasound irradiation ＋ microbubbles group（E）. The positive transfection rate of MDR1 gene in mononuclear cells of different groups were tested by flow eytometry, and the survival rate of cells in different periods were tested by trypan blue exclusion method. Moreover, the appearance and ultramicrostructure of cells were observed by electronmicroscope. Results （1）The transfection rate of MDR1 gene in different groups were 0.39%±0. 11,5.03% ±0. 35 % , 4. 93 % ± 0.38%,5.25% ＋ 0.80% and 19.93% ± 1.51% respectively. The transfection rate of MDR1 gene in group E was higher than those in other groups（ P 〈0. 05）. （2）Compared with those in control groups（group A, B, C and D）, the transfection rate in group E was significant raised by ultrasound irradiation and mierobubbles. However, there were no significant difference in survival rate of cells between the five groups（P 〉0.05）. （DAfter ultrasonic irradiation, there were transient holes on the cell membrane, which could disappear after irradiation by ultrasound for 24 hours. And the temporary swelling of organelles was reversible. Conclusions Microbubbles irradiated by ultrasound can cause small transient holes on cell membrane and increase permeability of it,and enhance the transfection of MDR1 gene in bone marrow mononuclear cells with the adenovirus vector,which is safe and av

关 键 词：超声检查 微气泡 P糖蛋白 

分 类 号：R730.5[医药卫生—肿瘤]