Gene transfer into adult rat spinal cord using naked plasmid DNA and ultrasound microbubbles
Gene transfer into adult rat spinal cord using naked plasmid DNA and ultrasound microbubbles
复制标题
DOI：
10.1002/jgm.793
复制
发表时间：
2005-11-01
影响因子：
3.5
通讯作者：
Morishita, R
中科院分区：
文献类型：
--
作者：
Shimamura, M;Sato, N;Morishita, R
背景：虽然基因治疗可能成为治疗脊髓损伤的一种有前途的方法，但安全性问题是人类基因治疗中一个重要的考虑因素。质粒DNA转移比病毒载体更安全，但转染率较高。效率相当低。为了克服这一问题，我们将超声微泡介导的转染法应用于成年大鼠的脊髓，因为超声微泡被报道能有效地提高各种组织的转染率。方法通过腰椎穿刺术暴露T9-10脊髓后，将裸质粒DNA和微泡的混合物注入脑脊液中。结论0.4~0.5W/cm(2)的超声强度可显著提高T9-10脊髓中荧光素酶的表达水平，约为裸质粒DNA辐射水平的15~60倍。荧光素酶活性至少在转染后7d即可检测到，而表达水平在14d时几乎恢复到检测不到的水平。转基因细胞主要为损伤脊髓表面的脑膜细胞。与单独转染裸质粒DNA的大鼠或未转基因的大鼠相比，没有明显的证据表明神经功能障碍的恶化。同样，在脊髓损伤后的T9-10脊髓中也成功地实现了基因转移。总之，本研究证实了超声微泡介导的质粒DNA转移到脊髓靶点的可行性。版权所有(C)2005 John Wiley&Sons，Ltd.
Background Although gene therapy might become a promising approach to treat spinal cord injury, the safety issue is a serious consideration in human gene therapy. Plasmid DNA transfer is safer than viral vectors, but the transfection. efficiency is quite low. To overcome the problem, we applied the ultrasound microbubbles-mediated transfection method to the spinal cord in adult rats, since ultrasound microbubbles have been reported to be efficient to increase transfection efficiency in various tissues.Methods After exposing T9-10 spinal cord with a laminectomy, we injected a mixture of naked plasmid DNA and microbubbles into cerebrospinal fluid by lumbar puncture. Then, the T9-10 spinal cord was exposed to ultrasound.Conclusions An ultrasound intensity of 0.4-0.5 W/cm(2) significantly increased luciferase expression up to approximately 15-60-fold at the insonated level as compared to naked plasmid DNA alone. Luciferase activity could be detected at least up to 7 days after transfection, while the expression level was almost returned to undetectable level at 14 days after transfection. The transfected cells were mainly meningeal cells in the surface of insonated spinal cord. There was no obvious evidence of worsening of neurological deficits as compared to rats transfected with naked plasmid DNA alone or untransfected rats. Similarly, successful gene transfer was also achieved in the insonated T9-10 spinal cord after spinal cord injury. Overall, the present study demonstrated the feasibility of ultrasound microbubbles-mediated plasmid DNA transfer into the target level of the spinal cord. Copyright (C) 2005 John Wiley & Sons, Ltd.