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

将携带人IGF-1 cDNA全长的腺病毒载体（AxIGF-1）在293细胞中进行增殖，并在氯化铯阶梯中进行连续超离心浓缩纯化。将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293细胞的终末细胞病变效应实验来测定AxIGF-1的感染性或AxIGF-1的滴度。利用从感染了axigf -1的HepG2培养物中收集的条件培养基进行免疫印迹，以检验感染了axigf -1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。在加载条件培养基的色谱柱中识别出与成熟IGF-1蛋白相同的分子大小带。用感染了axigf -1的293细胞培养物的条件培养基进行同样的实验，显示出与成熟的IGF-1蛋白相同的分子大小带。因此，我们发现细胞内修饰过程产生成熟的IGF-1蛋白存在于广泛的细胞类型中，而不仅仅存在于肝细胞中。此外，利用人肠细胞系“肠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HepG2的条件培养基显著促进了肠407细胞的增殖，并显著增加了流式细胞仪检测到的s期细胞。结果提示，利用AxIGF-1将IGF-1基因转移至肝脏可促进肠上皮细胞增殖，改善肠大切除后的营养损伤。在研究期间，我们在2003年的《Journal of Surgical research》上发表了携带人胰岛素基因的腺病毒载体脾内注射对去肝的糖尿病大鼠的肝损伤和基因转移效果优于门内注射或静脉注射的数据。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