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

携带人胰岛素样生长因子-1全长c DNA的腺病毒载体(AxIGF-1)在293细胞中增殖并经氯化铯梯度超速离心法纯化。病毒带在磷酸盐缓冲盐水(PBS)-10%甘油中透析过夜。用96孔板中的293个细胞进行终点细胞病变试验，确定AxIGF-1的感染性或AxIGF-1股票的滴度。用AxIGF-1感染的HepG2细胞的条件培养液进行免疫印迹，以检测AxIGF-1感染的肝细胞产生的IGF-1是否一定会通过细胞内修饰过程转变为成熟的IGF-1蛋白。与成熟IGF-1蛋白分子大小相同的条带出现在加载条件培养液的柱子上。同样的实验使用了感染AxIGF-1的293细胞培养的条件培养液，显示出与成熟IGF-1蛋白相同的分子大小条带。因此，我们共同…更多的证据表明，产生成熟IGF-1蛋白的细胞内修饰过程存在于广泛的细胞类型中，而不仅仅是肝细胞。此外，以人肠上皮细胞株‘肠道407’进行细胞增殖实验，检测AxIGF-1感染细胞产生的IGF-1是否能促进肠上皮细胞增殖。结果表明，AxIGF-1感染的HepG2细胞的条件培养液显著促进了肠上皮细胞的增殖，并显著增加了S期细胞的数量。在本研究期间，我们在2003年的《外科研究》杂志上发表了携带人胰岛素基因的腺病毒载体脾内注射在肝损伤和基因转移疗效方面优于门静脉注射或静脉注射的数据。较少

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