腺病毒载体介导的人凝血因子 IX 基因的离体和活体表达

Expression of Adenovirus Mediated Human Coagulating Factor IX in Vitro and in Vivo

为血友病Ｂ的基因治疗探索了高效转移和表达载体，建立了重组腺病毒载体介导的基因转移系统（Ａｄｈｆｉｘ），并进行了离体和活体表达研究。结果显示：腺病毒离体基因转移效率可达９８％以上并可在多种细胞中高效表达，ｈＦＩＸ蛋白最高可达６．８６μｇ／１０６细胞／２４ｈｒ。小鼠血浆中ｈＦＩＸ蛋白含量最高可达１０７２ｎｇ／ｍｌ血浆，并可持续表达１０周左右；以腹腔注射效果最好；免疫抑制剂环磷酰胺可明显延长表达持续时间。结果表明重组腺病毒载体可介导ｈＦＩＸｃＤＮＡ在离体细胞和活体组织中高效转移和表达。

Recombinant adenoviral vector with human coagulating Factor IX(AdhFIX) was established and studied in vitro and in vivo. The plasmid pAdhFIX and pJM17 were homologously coinfected into 293 cells, then purified and comfirmed by PCR assay. The efficiency of gene transfer in human myoblasts, HT cells and rabbit fibroblasts in vitro was assayed, and the expressive duration and ultrastructure localization of hFIX protein were estimated. The expressive amount and duration of hFIX protein in vivo were estimated and compared by injection route of tail vein, intraperitoneum and muscle seperately. The results showed that the gene transfer efficiency of adenoviral vector was above 98%, and AdhFIX could express in multiple cells and has the peak amount of 6.86μg/10 6 cells/24hr. In vivo the peak amount in mice plasma was 1072ng/ml and the expressive duration was 10 weeks or so. Among the three injective strategies the intraperitoneal injection was the most efficient one, and the immunosupressant cyclophosphamide could prolonge the expressive duration. So the recombinant adenovirus mediated hFIX cDNA could expresse in vitro and in vivo with high efficiency.