重组腺病毒载体介导外源基因转移至脑血管的实验研究

Experimental study on adenovirus-mediated gene transfer to cerebral blood vessels in normal rabbit

目的 基因载体直接体内转移血管壁为血管病的治疗提供一种新方法 ,本研究探讨腺病毒载体介导外源基因转移至颅内血管的可行性。方法 2 5 0 μl含巨细胞病毒启动子的编码报告基因 -绿色荧光蛋白 ( GFP)基因的复制缺陷型重组腺病毒载体 ( Ad5CMV5GFP) ,滴度为 2 .5× 10 9pfu/ m l,经枕大池穿刺法注入正常兔的脑池内。注入腺病毒载体后 1～ 7d,采用荧光显微镜、免疫组织化学和逆转录聚合酶链式反应方法从形态学和分子生物学方面检测颅内血管和血管周围组织中外源基因 GFP的表达情况。结果 直接体内转染后 1d,在颅内大血管如基底动脉的外膜可见外源基因的表达 ,3d时减弱 ,7d后消失 ,而血管中膜和内膜未见外源基因的表达 ;注入腺病毒载体后1～ 7d,外源基因可以有效转移至颅底软脑膜细胞 ,小血管外膜和平滑肌层也可见外源基因的表达 ;注入腺病毒载体后兔体温未见升高 ,但是脑脊液中的白细胞数明显升高 ,血管壁中可见炎性细胞浸润。结论 经脑池内注入重组腺病毒载体可以将外源基因转移至正常兔颅内血管和软脑膜中 。

Objective Gene transfering to blood vessels in vivooffers a new tool with potential for treatment of vascular disease.In this study,we examined transgene expression after injection of replication-deficient adenovirus into cerebrospinal fluid of rabbit,with an attempt to transfer gene to normal cerebral blood vessels.Methods Recombinant adenovirus(2.5×10 9pfu/ml) expressing green fluorescent protein(GFP,reporter gene) under the control of the cytomegalovirus promotor (Ad 5CMV 5GFP) was injected into the cisterna magna of normal rabbits.1 to 7 days after injection of adenovirus,expression of GFP on the basilar artery and perivascular tissue was examined by fluorescence microscope,immunohistochemical staining,and reverse transcriptase-PCR.Results GFP was expressed in adventitia of large blood vessels suchas basilar artery on 1d and 3d after injection but was undetectable on 7d,transgene was not expressed in vascular muscle or endothelium.During 7 days,GFPwas expressed in leptomeninges over the brain stem,cortex and cerebral arteriesand smooth muscle cells of small vessels wereoccasionlly transduced.Conclusion Intracisternal injection of recombinant adenovirus provides gene transfer in vivo to cerebral vessels and perivascular tissue in normal rabbit.