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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

将携带全长人IGF-1 cDNA的腺病毒载体（AxIGF-1）在293细胞中增殖，并通过在氯化铯分步梯度中使用连续超离心进行纯化浓缩。将病毒带用磷酸盐缓冲盐水（PBS）-10%甘油透析过夜。AxIGF-1的感染性或AxIGF-1储备液的滴度通过使用96孔板中的293个细胞的终点细胞病变效应测定来确定。使用从AxIGF-1感染的HepG 2培养物收集的条件培养基进行免疫印迹，以测试从AxIGF-1感染的肝细胞产生的IGF-1是否确实通过细胞内修饰过程改变为成熟IGF-1蛋白。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。使用来自AxIGF-1感染的293细胞培养物的条件培养基的相同实验证明了与成熟IGF-1蛋白相同的分子大小条带。因此，我们 ...更多信息包括产生成熟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。使用人肠细胞系“肠407”进行细胞增殖测定，以测试AxIGF-1感染的细胞产生的IGF-1是否可以促进肠上皮细胞增殖。结果，来自AxIGF-1感染的HepG 2的条件培养基显著促进了肠407的细胞增殖，并且还显著增加了通过流式细胞仪测量的S期细胞。结论：应用AxIGF-1将IGF-1基因转移至肝脏，可促进肠上皮细胞增殖，改善肠段切除术后的营养障碍，在研究期间，我们发表了脾内注射携带人胰岛素基因的腺病毒载体上级门静脉内注射，或静脉内注射在肝损伤方面的研究以及使用肝切除的糖尿病大鼠的基因转移的功效，发表在2003年的“Journal of Surgical Research”上。少