超声微泡转基因技术促增强型绿色荧光蛋白基因在骨缺损处转染的实验研究

发布时间：2018-05-11 13:41

本文选题：骨缺损 + 超声微泡转基因技术　；参考：《中国修复重建外科杂志》2017年04期

【摘要】：目的探讨超声微泡转基因技术介导增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因在兔骨缺损处转染时,不同超声辐照时间对转染效率及局部组织的影响。方法 3月龄雄性新西兰大白兔30只,体质量2.5~3.0 kg,制备右尺骨骨缺损模型,并随机分为5组(n=6)。造模后第10天于骨缺损断端间注射EGFP质粒微泡混悬液(0.3 m L/kg)后,在超声频率1 MHz、超声强度0.5 W/cm~2、占空比20%条件下,对骨缺损部位分别进行1、2、3、4、5 min超声辐照(分别为1、2、3、4、5 min组)。观察动物存活情况;转染后1周取材,大体观察骨缺损处软组织形态;荧光染色观察基因表达情况;HE染色及透射电镜观察局部组织损伤情况。结果各组动物均存活至实验完成。转染后1周各组骨缺损处有软组织生长,周围肌肉组织部分内陷填充于其间。荧光显微镜下观察,各组兔骨缺损处均有绿色荧光表达,其中2 min组表达最强,1 min组表达最弱,其吸光度(A)值与其他各组比较差异有统计学意义(P0.05);3、4、5 min组间差异无统计学意义(P0.05)。HE染色及透射电镜观察示,各组骨缺损处局部均有不同程度组织损伤,损伤程度随辐照时间的延长而加重。结论超声微泡转基因技术介导EGFP质粒在兔骨缺损部位转染时,其转染效率和超声辐照时间相关。当超声参数为1 MHz、0.5 W/cm~2、20%占空比时,超声辐照2 min可获得最佳转染效率及相对较轻的组织损伤。
[Abstract]:Objective to investigate the effect of different ultrasound irradiation time on the transfection efficiency and local tissue of enhanced green fluorescent protein (EGFP) gene in rabbit bone defect mediated by ultrasound microbubble technology. Methods Thirty 3-month-old male New Zealand white rabbits with a body mass of 2.5 and 3.0 kg were used to establish a model of right ulnar bone defect and were randomly divided into 5 groups. On the 10th day after the model was established, the EGFP plasmid microbubble suspension was injected between the broken ends of the bone defect (0.3 mL / kg). Under the condition of ultrasound frequency 1 MHz, ultrasound intensity 0.5 W / cm ~ (2) and duty ratio 20%, the site of the bone defect was irradiated with 1 ~ (2) ~ 2 ~ 3 ~ (3) ~ 3 ~ 4 ~ (5) min ultrasound irradiation (12 ~ 3X ~ 4 ~ 4 ~ 5 min group, respectively). The survival of animals was observed, the soft tissue morphology of bone defect was observed 1 week after transfection, the expression of gene was observed by fluorescence staining and the local tissue injury was observed by transmission electron microscope (TEM) and HE staining. Results all the animals survived until the experiment was completed. At 1 week after transfection, soft tissue growth was found in the bone defect of each group, and part of the surrounding muscle tissue was filled with soft tissue. The expression of green fluorescence was observed under fluorescence microscope in all groups, and the expression of green fluorescence was the weakest in the 2 min group, and the strongest in the 2 min group, and the weakest in the 1 min group. Compared with other groups, there was significant difference in absorbance A) value. There was no significant difference between the two groups (P 0.05). He staining and transmission electron microscope observation showed that there were different degrees of tissue injury in the bone defect in each group. The degree of injury increased with the prolongation of irradiation time. Conclusion the transfection efficiency of EGFP plasmid mediated by ultrasound microbubble gene transfer in rabbit bone defect is correlated with the time of ultrasound irradiation. The optimal transfection efficiency and relatively light tissue damage could be obtained by ultrasound irradiation for 2 min when the ultrasonic parameters were 0.5 W / cm ~ (2) ~ (2) duty cycle ratio of 1 MHz / cm ~ (2) C ~ (2 +).
【作者单位】：四川大学华西医院小儿外科;广州市妇女儿童医疗中心小儿外科;
【基金】：国家自然科学基金资助项目(81071475)~~
【分类号】：R683

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