IN-VIVO ADENOVIRAL VECTOR-MEDIATED GENE-TRANSFER INTO BALLOON-INJURED RAT CAROTID ARTERIES
IN-VIVO ADENOVIRAL VECTOR-MEDIATED GENE-TRANSFER INTO BALLOON-INJURED RAT CAROTID ARTERIES
复制标题
DOI：
10.1161/01.res.73.5.797
复制
发表时间：
1993-11-01
影响因子：
20.1
通讯作者：
DICHEK, DA
中科院分区：
文献类型：
--
作者：
LEE, SW;TRAPNELL, BC;DICHEK, DA
我们研究了腺病毒载体实现基因转移到受损动脉的能力。构建表达核靶向β-半乳糖苷酶基因的重组腺病毒载体，并将其注入球囊损伤的大鼠颈动脉。基因转移后3天，通过（1）测量组织提取物中的β-半乳糖苷酶抗原和活性以及（2）组织化学染色和表达β-半乳糖苷酶的细胞计数，定量评估重组基因表达。将损伤的颈动脉暴露于增加浓度的载体（每毫升10（8）至10（10）空斑形成单位）导致β-半乳糖苷酶表达的剂量响应性增加，峰值表达约为43 mU或25 ng β-半乳糖苷酶/血管。组织化学染色动脉的显微镜检查表明基因转移仅限于血管介质;转导细胞经化学鉴定为平滑肌细胞。对培养基中组织化学染色细胞核和总细胞核的计数显示，损伤血管培养基中约30%的细胞被转导。基于细胞计数和组织提取物中β-半乳糖苷酶表达水平的计算表明，每10 mm血管中存在5000至10000个转导细胞。仅用载体、对照腺病毒载体或脂质体与载体质粒组合输注的动脉含有很少或没有β-半乳糖苷酶表达的证据。高水平的体内β-半乳糖苷酶表达持续至少7天后，基因转移，但下降显着的第14天。我们的结论是，腺病毒载体介导的基因转移到受伤的大鼠颈动脉的结果在有效的基因转移到血管介质中，重组蛋白的生产水平显着高于任何以前报道的动脉基因转移研究。腺病毒载体似乎是体内动脉基因转移的特别有用的试剂。
We studied the ability of adenoviral vectors to achieve gene transfer into injured arteries. A recombinant adenoviral vector expressing a nuclear-targeted beta-galactosidase gene was constructed and infused into balloon-injured rat carotid arteries. Three days after gene transfer, recombinant gene expression was assessed quantitatively by (1) measuring beta-galactosidase antigen and activity in tissue extracts and (2) histochemical staining and counting of cells expressing beta-galactosidase. Exposure of injured carotid arteries to increasing concentrations of the vector (10(8) to 10(10) plaque-forming units per milliliter) resulted in a dose-responsive increase in beta-galactosidase expression, with peak expression of approximately 43 mU or 25 ng beta-galactosidase per vessel. Microscopic examination of histochemically stained arteries demonstrated gene transfer limited to the vascular media; transduced cells were identified immunohistochemically as smooth muscle cells. Counting of both histochemically stained and total nuclei in the media revealed that approximately 30% of the cells in the media of the injured vessels were transduced. Calculations based on both counting cells and on the level of beta-galactosidase expression in tissue extracts suggested the presence of 5000 to 10 000 transduced cells per 10 mm of vessel. Arteries infused with either vehicle only, a control adenoviral vector, or liposomes combined with the vector plasmid contained little or no evidence of beta-galactosidase expression. High levels of in vivo beta-galactosidase expression persisted for at least 7 days after gene transfer but declined significantly by day 14. We conclude that adenoviral vector-mediated gene transfer into the injured rat carotid artery results in efficient gene transfer into the vascular media, with levels of recombinant protein production significantly higher than any previously reported in arterial gene transfer studies. Adenoviral vectors appear to be particularly useful agents for in vivo arterial gene transfer.