Bosch R J, Woolf A S, Fine L G
Department of Medicine, University College and Middlesex School of Medicine, London, UK.
Exp Nephrol. 1993 Jan-Feb;1(1):49-54.
Gene transfer using retroviral vectors requires cell replication for insertion of the DNA provirus. Since the mitotic index of the mammalian kidney is very low, renal tubular cell replication was induced in adult rats as part of a regenerative response to the nephrotoxic injury induced by an intraperitoneal injection of folic acid. At 48 h, at the time of maximum 3H-thymidine incorporation, the left kidney was directly injected with a suspension of the Psi2 BAG retrovirus which transduces the beta-galactosidase gene. At 1-7 weeks after virus administration the left kidney was harvested. Using the polymerase chain reaction to amplify viral DNA, successful gene transfer was achieved in 8 of 15 kidneys. In 6 of 10 kidneys assessed histochemically positive staining for beta-galactosidase activity was detected in the cytoplasm of tubular epithelial cells. There was no evidence of gene expression in glomerular, vascular or endothelial cells. All analyses were negative in vehicle-injected kidneys and in the kidneys of animals which did not receive pretreatment with folic acid. These studies demonstrate the feasibility of gene transfer into the adult kidney provided that replication of specific cell types can be achieved.
使用逆转录病毒载体进行基因转移需要细胞复制以插入DNA原病毒。由于哺乳动物肾脏的有丝分裂指数非常低，因此在成年大鼠中诱导肾小管细胞复制，作为对腹腔注射叶酸诱导的肾毒性损伤的再生反应的一部分。在48小时，即3H-胸腺嘧啶掺入量最高时，将Psi2 BAG逆转录病毒悬浮液直接注射到左肾，该病毒可转导β-半乳糖苷酶基因。在病毒给药后1至7周收获左肾。使用聚合酶链反应扩增病毒DNA，在15个肾脏中的8个中成功实现了基因转移。在组织化学评估的10个肾脏中的6个中，在肾小管上皮细胞的细胞质中检测到β-半乳糖苷酶活性的阳性染色。在肾小球、血管或内皮细胞中没有基因表达的证据。在注射载体的肾脏和未接受叶酸预处理的动物的肾脏中，所有分析均为阴性。这些研究表明，只要能够实现特定细胞类型的复制，就可以将基因转移到成年肾脏中。
