中国修复重建外科杂志

中国修复重建外科杂志

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

查看全文

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 mL/kg)后,在超声频率 1 MHz、超声强度 0.5 W/cm2、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(P<0.05);3、4、5 min 组间差异无统计学意义(P>0.05)。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/cm2、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。

Objective To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein (EGFP) gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction. Methods Thirty 3-month-old New Zealand rabbits (2.5-3.0 kg in weight) were randomly divided into 5 groups (n=6) and bone defect models were made on the right ulna. At 10 days after modeling, suspension of microbubbles and EGFP plasmids were locally injected (0.3 mL/kg) and then ultrasound was performed on defect at a frequency of 1 MHz, a intensity of 0.5 W/cm2, and a duty ratio of 20% for 1, 2, 3, 4, and 5 minutes respectively (in 1, 2, 3, 4, and 5 minutes groups respectively). The survival condition was observed. Rabbits were sacrificed for gross observation at 7 days after transfer. The gene expression was observed by fluorescence staining. HE staining and transmission electron microscopy were used to observe the local tissue damage. Results The animals all survived. New soft tissue formed in bone defects area at 1 week after transfer, the surrounding muscle tissue was partly filled in it. Green fluorescence expression was observed in all rabbits. The expression was the strongest in 2 minutes group, and was the weakest in 1 minute group. The absorbance (A) value showed significant differences when compared 1 minute and 2 minutes groups with other groups (P<0.05), but no significant difference was found between 3, 4, and 5 minutes groups (P>0.05). Tissue damage was observed in all groups and it was aggravated with the increase of irradiation time. Conclusion EGFP transfection efficiency in bone defect by ultrasound-mediated microbubble destruction is related to irradiation time. EGFP gene can be efficiently transfected without obvious toxicity at 1 MHz, 0.5W/cm2, and duty ratio of 20% for 2 minutes in bone defects of rabbits.

关键词: 骨缺损; 超声微泡转基因技术; 基因转染; 增强型绿色荧光蛋白;

Key words: Bone defect; ultrasound-mediated microbubble destruction; gene transfer; enhanced green fluorescent protein; rabbit

引用本文: 李世伟, 谢晓丽, 杨晓东, 刘利君, 唐学阳. 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. 中国修复重建外科杂志, 2017, 31(4): 437-442. doi: 10.7507/1002-1892.201611059 复制

登录后 ,请手动点击刷新查看全文内容。 没有账号,
登录后 ,请手动点击刷新查看图表内容。 没有账号,
1. Wiese A, Pape HC. Bone defects caused by high-energy injuries, bone loss, infected nonunions, and nonunions. Orthop Clin North Am, 2010, 41(1): 1-4.
2. Karger C, Kishi T, Schneider L,et al. Treatment of posttraumatic bone defects by the induced membrane technique. Orthop Traumatol Surg Res, 2012, 98(1): 97-102.
3. Wang Y, Van Manh N, Wang H,et al. Synergistic intra-fibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to promote the regeneration of bone defects. Int J Nanomedicine, 2016, 11: 2053-2067.
4. Kolambkar YM, Dupont KM, Boerckel JD,et al. An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects. Biomaterials, 2011, 32(1): 65-74.
5. Van der Stok J, Van der Jagt OP, Amin Yavari S,et al. Selective laser melting-produced porous titanium scaffolds regenerate bone in critical size cortical bone defects. J Orthop Res, 2013, 31(5): 792-799.
6. 殷渠东, 孙振中, 顾三军, 等. 骨搬运与骨短缩-延长治疗胫骨骨缺损合并软组织缺损的疗效比较. 中国修复重建外科杂志, 2014, 28(7): 818-822.
7. Long J, Li P, Du H,et al. Effects of bone morphogenetic protein 2 gene therapy on new bone formation during mandibular distraction osteogenesis at rapid rate in rabbits. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2011, 112(1): 50-57.
8. Gugala Z, Olmsted-Davis EA, Gannon FH,et al. Osteoinduction byex vivo adenovirus-mediated BMP2 delivery is independent of cell type. Gene Ther, 2003, 10(16): 1289-1296.
9. Qiu L, Zhang L, Wang L,et al. Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendonsin vivo. Gene Ther, 2012, 19(7): 703-710.
10. Osawa K, Okubo Y, Nakao K,et al. Osteoinduction by micro-bubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2. J Med, 2009, 11(7): 633-641.
11. Chen ZY, Yang F, Lin Y,et al. New development and application of ultrasound targeted microbubble destruction in gene therapy and drug delivery. Curr Gene Ther, 2013, 13(4): 250-274.
12. Song S, Shen Z, Chen L,et al. Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver. Gene Ther, 2011, 18(10): 1006-1014.
13. Fujii H, Li SH, Wu J,et al. Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair. Eur Heart J, 2011, 32(16): 2075-2084.
14. Zhang Y, Ye C, Wang G,et al. Kidney-targeted transplantation of mesenchymal stem cells by ultrasound-targeted microbubble destruction promotes kidney repair in diabetic nephropathy rats. Biomed Res Int, 2013, 2013: 526367.
15. Tinkov S, Bekeredjian R, Winter G,et al. Microbubbles as ultrasound triggered drug carriers. J Pharm Sci, 2009, 98(6): 1935-1961.
16. 陈智毅, 谢明星, 王新房, 等. 治疗性超声介导体外基因转染的参数优化. 中国医学影像技术, 2008, 24(10): 1511-1514.
17. Liang HD, Lu QL, Xue SA,et al. Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells. Ultrasound Med Biol, 2004, 30(11): 1523-1529.
18. Koch S, Pohl P, Cobet U,et al. Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects. Ultrasound Med Biol, 2000, 26(5): 897-903.
19. Delalande A, Bouakaz A, Renault G,et al. Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype. J Control Release, 2011, 156(2): 223-230.
20. Zarnitsyn VG, Prausnitz MR. Physical parameters influencing optimization of ultrasound-mediated DNA transfection. Ultrasound Med Biol, 2004, 30(4): 527-538.
21. Wang X, Liang HD, Dong B,et al. Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents. Radiology, 2005, 237(1): 224-229.
22. 马钊, 胡向东, 费琦, 等. 超声微泡介导基因治疗在骨科领域的应用进展. 临床和实验医学杂志, 2014, 13(6): 505-508.