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Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes 总被引:3,自引:1,他引:3
Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins. 相似文献
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S Greenacre V Ridger P Wilsoncroft SD Brain 《Clinical and experimental pharmacology & physiology》1997,24(11):880-882
1. Increased expression of inducible nitric oxide synthase (iNOS) and subsequent elevation of nitric oxide (NO) levels at inflammatory sites have led to the suggestion that peroxynitrite (the reaction product of superoxide and NO) is involved in proinflammatory processes. The present study has investigated the ability of peroxynitrite to induce oedema formation in the rat cutaneous microvasculature. 2. Peroxynitrite was synthesized from hydrogen peroxide and acidified nitrite. Spectrophotometry was used to measure the concentration and breakdown of peroxynitrite. It was also used to determine maximum amounts of hydrogen peroxide and sodium nitrite remaining after synthesis. 3. Oedema formation in response to intradermall. (i.d.) injected peroxynitrite, hydrogen peroxide and sodium nitrite was measured by the extra vascular accumulation of i.v. [125I]-albumin in the anaesthetized rat. 4. Peroxynitrite (40,100 and 200 nmol/site) acted in a dose-dependent manner to cause a mean (± SEM) increase in plasma extravasation of 24 ± 2,55 ± 5 and 69 ± 6 μL, respectivel. (n= 4), with resulting inflammatory oedema. Peroxynitrite induced significantly larger plasma extravasation than equivalent vehicle controls at doses of 100 (P > 0.05) and 200 nmol (P > 0.001). This increased extravasation appears to be a direct microvascular response to peroxynitrite administration and not due to either a raised pH, necessary to stabilize the peroxynitrite, or contaminating concentrations of hydrogen peroxide or sodium nitrite from which peroxynitrite is formed. 5. These results suggest that peroxynitrite acts to increase microvascular permeability and oedema formation. Therefore, peroxynitrite may mediate vascular pro-inflammatory effects in addition to its direct cytotoxic activity. 相似文献
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