pVAX1-microdystrophin重组质粒的构建及其治疗Duchenne型肌营养不良症的初步研究  

Construction of recombinant plasmid pVAXl-microdystrophin and preliminary study on the treatment to Duchelme muscular dystrophy

作　　者：熊符[1,3] 张成[2,3] 郑卉[2] 肖少波[4] 于美娟[3] 许勇峰[2] 刘正山[3] 周畅[2] 

机构地区：[1]南方医科大学医学遗传学教研室,广州510515 [2]中山大学附属第一医院神经内科 [3]中山大学干细胞与组织工程研究中心 [4]华中农业大学农业微生物国家重点实验室

出　　处：《中华医学遗传学杂志》2008年第6期624-628,共5页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（30170337）;南方医科大学基础医学院院长基金（JC0702）

摘　　要：目的构建含有人microdystmphin基因的重组质粒，体内、外研究其表达，为进一步应用此重组质粒来研究电转、静脉、动脉注射或加入其它基因来增强microdystrophin基因的表达等方法对Duchenne型肌营养不良（Duchennen musculardystrophy，OMD）进行基因治疗奠定基础。方法用NotI酶切含microdys—trophin基因的pBSK-MICRO质粒，获得microdystrophin基因。片段回收后定向插入真核表达质粒pVhX1，获得重组质粒pAMICDYS。然后将重组质粒pAMICDYS转染小鼠成纤维细胞3T3细胞，通过逆转录-PCR以及间接免疫荧光检测microdystmphin的转录及蛋白表达；最后将重组质粒pAMICDYS通过肌肉注射到DMD模型鼠胫前肌中，检测治疗肌肉的病理变化和microdystrophin的表达情况。结果成功构建了含有microdys—troth基因的重组质粒，并在体外得到了很好表达，体内研究表明microdystrophin基因在mdx鼠TA也有一定程度的表达，而且减少了所治疗肌肉的核中移数量，证实了其对mdx鼠有一定的治疗作用。结论该重组质粒的构建及体内、外得到成功表达，为下一步用该质粒进行电转、静脉、动脉注射或加入其它基因来增强microdystrophin基因的表达来治疗DMD疾病奠定了基础。Objective To construct the recombinant plasmid containing human microdystrophin eDNA, and study the microdystrophin expression in vivo and in vitro. Methods Microdystrophin eDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I , the product was inserted into plasmid pVAX1, resulting in pAMICDYS. And then 3T3 cells were transfected with pAMICDYS. Forty-eight hours after transfection, the expression of the mierodystrophin was detected by reverse transeription-polymerase chain reaction （RT-PCR） and im- munocytochemistry. Finally, TA muscles of mdx mice were injected with the recombinant plasmid pAMICDYS through i.m. and the pathological change of TA was evaluated by histology, and the expression of mierodystrophin in mdx TA was detected by immunohistochemical analysis. Results The recombinant plasmid containing human microdystrophin eDNA was constructed successfully. The recombinant plasmid was proved to be able to express mierodystrophin protein both in vivo and in vitro. Moreover, treatment of the TA of mdx mice with the recombinant plasmid could decrease the number of centrally nucleated myofibers. Conclusion Recombinant plasmid containing the rnierodystrophin gene was constructed successfully, and it could express microdystrophin protein both in vivo and in vitro. It provides basis for further study on mierodystrophin as a target gene to treat Duchenne muscular dystrophy （DMD） by electrotransfer, i. v, arterial injection and combining with other exogenous gene to enhance mierodystrophin expression.

关 键 词：微小dystrophin基因 重组质粒 DUCHENNE型肌营养不良症 

分 类 号：R686[医药卫生—骨科学]