Delivery of human interferon-gamma via gene transfer in vitro: prolonged expression and induction of macrophage antimicrobial activity.
Delivery of human interferon-gamma via gene transfer in vitro: prolonged expression and induction of macrophage antimicrobial activity.
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通过体外基因转移递送人干扰素-γ：延长表达并诱导巨噬细胞抗菌活性。
DOI：
10.1089/jir.1996.16.1015
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Murray,HW
中科院分区：
文献类型：
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作者：
Stoeckle,MY;Falck-Pederson,E;Rubin,BY;Anderson,SL;Murray,HW
每日肠外注射外源性干扰素-γ (IFN-γ)可诱导或加速实验和人类感染的恢复。为了开发一种替代的递送系统，构建了一种表达人IFN-γ的复制缺陷重组腺病毒。利用RT-PCR扩增出IFN-γ的完整编码区，并在人巨细胞病毒启动子控制下插入腺病毒克隆载体。用线性化质粒和腺病毒基因组El区缺失片段共转染293细胞，分离到含有IFN-γ小基因的重组腺病毒（dave -IFN-γ）。在体外感染dAv-IFN-γ后，Northern blot和ELISA分别在人二倍体成纤维细胞和HeLa细胞培养中证实了IFN-γ mRNA的剂量依赖性和时间依赖性表达和可溶性蛋白的产生。初始转染后，细胞外蛋白分泌持续≥4周，分泌的IFN-γ诱导抗病毒活性（8000-25,000 U/ml）和巨噬细胞活化，杀死细胞内弓形虫和多诺瓦利什曼原虫。这些结果表明，dave -IFN-γ能够在体外长期分泌具有生物活性的IFN-γ，并提示该载体可能是细胞因子治疗的一种有用的传递系统。
Daily parenteral administration of exogenous interferon-γ (IFN-γ) induces or accelerates recovery in experimental and human infections. To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-γ was constructed. The complete coding region of IFN-γ was amplified by RT-PCR and inserted into an adenovirus cloning vector under the control of a human cytomegalovirus promoter. Recombinant adenovirus containing the IFN-γ minigene (dAv-IFN-γ) was isolated from 293 cells cotransfected with the linearized plasmid and an El region-deleted fragment of adenovirus genome. Followingin vitroinfection with dAv-IFN-γ, dose-dependent and time-dependent expression of IFN-γ mRNA and production of soluble protein were demonstrated in human diploid fibroblast and HeLa cell cultures by Northern blot and ELISA, respectively. Extracellular protein secretion persisted for ≥4 weeks following initial transfection, and secreted IFN-γ induced both antiviral activity (8000–25,000 U/ml) and macrophage activation with killing of intracellularToxoplasma gondiiandLeishmania donovani.These results establish that dAv-IFN-γ generates long-term secretion of biologically active IFN-γin vitroand suggest that this vector may be a useful delivery system for cytokine therapy.