Retrovirus-mediated transduction of adult hepatocytes.
Retrovirus-mediated transduction of adult hepatocytes.
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逆转录病毒介导的成体肝细胞转导。
DOI：
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发表时间：
1988
影响因子：
11.1
通讯作者：
R. Mulligan
中科院分区：
文献类型：
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作者：
James M. Wilson;D. Jefferson;J. Chowdhury;P. Novikoff;D. E. Johnston;R. Mulligan
利用逆转录病毒介导的基因转移技术，建立了一种将基因导入成年大鼠肝细胞原代培养的方法。在激素定义的不同基质底物上培养的亚融合单层肝细胞，感染了复制缺陷逆转录病毒的无辅助子库存，这种逆转录病毒结构性地表达高水平的β-半乳糖苷酶。逆转录病毒介导的转导通过两种方法进行检测：(I)原位细胞化学染色，检测病毒表达的β-半乳糖苷酶的表达；(Ii)Southern印迹分析，测量整合前病毒的相对拷贝数。培养3d后感染细胞，转导效率最高可达25%，基质对转导效率影响不大。酶细胞化学(过氧化氢酶和葡萄糖6-磷酸酶)和过氧化物酶免疫细胞化学(去唾液酸糖蛋白和UDP-葡萄糖醛酸基转移酶)分析表明，95%以上的细胞是肝细胞。在表达病毒导向的β-半乳糖苷酶的细胞中发现了肝细胞特有的细胞器，为肝细胞的转导提供了明确的证据。这些方法对肝脏导向的体细胞基因治疗的发展和肝脏特异性基因调控的研究将是有用的。
Retrovirus-mediated gene transfer was used to develop a method for introducing genes into primary cultures of adult rat hepatocytes. Subconfluent monolayers of hepatocytes, cultured in hormonally defined media on different matrix substrata, were infected with helper-free stocks of a replication-defective retrovirus that constitutively expresses high levels of beta-galactosidase. Retrovirus-mediated transduction was measured by two methods: (i) an in situ cytochemical stain that specifically detects the expression of viral expressed beta-galactosidase, and (ii) Southern blot analysis, which measures the relative copy number of integrated provirus. Maximal transduction efficiency of approximately equal to 25% was achieved when the cells were infected after 3 days in culture; matrix had little effect on transduction efficiency. Enzyme cytochemical (catalase and glucose 6-phosphatase) and peroxidase immunocytochemical (asialoglycoprotein and UDP-glucuronosyltransferase) analyses of the cultures indicated that greater than 95% of cells were hepatocytes. The demonstration of hepatocyte-specific organelles in cells expressing the viral-directed beta-galactosidase provided unambiguous evidence for the transduction of hepatocytes. These methods should be useful in the development of liver-directed somatic gene therapy and in the study of liver-specific gene regulation.