Research on treatments after maasive bowel resection : Effects of IGF-1 gene transfer

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Adenovirus vector carrying human IGF-1 cDNA at full length (AxIGF-1) was propagated in 293 cells and concentrated with purification by using sequential ultra-centrifugation in cesium chloride step gradients. The virus band was dialyzed overnight against phosphate-buffered saline (PBS)-10% glycerol. The infectivity of AxIGF-1 or titers of AxIGF-1 stocks was determined by an end-point cytopathic effect assay that used 293 cells in 96-well plates. Immunoblotting was performed using conditioned media collected from AxIGF-1-infected HepG2 cultures in order to test whether IGF-1 produced from AxIGF-1-infected hepatocytes surely be changed to mature IGF-1 protein through intracellular modification process. The same molecular size band as mature IGF-1 protein was recognized in the column where the conditioned media were loaded. The same experiments using conditioned media from AxIGF-1-infected 293 cell cultures demonstrated the same molecular size band as mature IGF-1 protein. Therefore, we co … More ncluded that intracellular modification process producing mature IGF-1 protein exits in broad cell types, not only in hepatocytes. Futhermore, cell proliferation assay was performed using human intestinal cell line ‘intestine 407' to test whether the IGF-1 produced by AxIGF-1-infected cells can promote intestinal epithelial proliferation. As a result, conditioned media from AxIGF-1-infected HepG2 significantly promoted cell proliferation of Intestine 407, and also significantly increased S-phase cells measured by flowcytemeter.. It was suggested that IGF-1 gene transfer to the liver using AxIGF-1 might promote intestinal epithelial proliferation and improve the nutritional impairments after intestinal massive resection.During the research period, we published the data that intrasplenic injection of adenovirus vector carrying human insulin gene was superior to intraportal, or intravenous injection on the aspects of liver injury and efficacy of gene transfer using the hepatectomized diabetic rats in "Journal of Surgical Research" in 2003. Less