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

大肠切除术后的治疗研究：IGF-1 基因转移的影响

基本信息

批准号：
15591436
负责人：
YAMAGUCHI Masahiko
金额：
$ 1.98万
依托单位：
Dokkyo University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591436/
关键词：
Massive bowel resection IGF-1 gene transfer intestinal epithelial regeneration

项目摘要

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

携带全长人 IGF-1 cDNA (AxIGF-1) 的腺病毒载体在 293 细胞中繁殖，并通过使用氯化铯梯度梯度连续超速离心进行浓缩和纯化。病毒条带针对磷酸盐缓冲盐水 (PBS) 透析过夜。 )-10% 甘油 AxIGF-1 的感染性或 AxIGF-1 原液的滴度由终点细胞病变确定。使用从 AxIGF-1 感染的 HepG2 培养物中收集的条件培养基进行免疫印迹分析，以测试从 AxIGF-1 感染的肝细胞产生的 IGF-1 是否确实转变为成熟的 IGF-1。通过细胞内修饰过程，在加载条件培养基的柱中识别出与成熟 IGF-1 蛋白相同的分子大小条带。 AxIGF-1 感染的 293 细胞培养物显示出与成熟 IGF-1 蛋白相同的分子大小带，因此，我们得出结论，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，而不仅仅是肝细胞中。使用人肠细胞系‘intestine 407’进行细胞增殖实验，测试AxIGF-1感染细胞产生的IGF-1是否能够促进肠上皮增殖。结果，来自 AxIGF-1 感染的 HepG2 的条件培养基显着促进了 Intestine 407 的细胞增殖，并且通过流式细胞仪测量也显着增加了 S 期细胞。这表明使用 AxIGF-1 将 IGF-1 基因转移到肝脏可能促进肠上皮增殖，改善肠大量切除术后的营养损害。研究期间，我们发表了携带人胰岛素基因的腺病毒载体脾内注射优于的数据2003年《外科研究杂志》上对糖尿病大鼠肝切除后门静脉或静脉注射对肝损伤和基因转移效果的影响。