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Research on gene transfer of hepatocyte growth factor in hepato-spleno-surgery

肝细胞生长因子基因转移在肝脾外科中的研究

基本信息

批准号：
11671273
负责人：
YAMAGUCHI Masahiko
金额：
$ 2.43万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671273/
关键词：
adenovirus vector hepatocyte growth factor liver regeneration Insulin 肝傷害脾自家移植

项目摘要

We manufactured adenovirus vector containing hepatocyte growth factor (HGF), of which cDNA was generously provided by Dr.T.Nakamura (Biomedical Research Center, Osaka University School of Medicine), according to the protocol Dr.I Saito (Institute of Medical Science, University of Tokyo) invented.After adenovirus vector containing HGF (AxHGF) was transfected into Hela cells. we collected cultured media which contained 1000-3000 ng/ml of HGF.Bioactivity of HGF produced by AxHGF-transfected Hela cells were proved by proliferative effects on cultured vascular endothelial cells and protective effects on radical-induced hepatocyte injury.AxHGF was propagated in 293 cellsand concentrated with purification by sequential ultracentrifugation in cesium chloride step gradients. In vivo injection of concentrated AxHGF improved histological changes and serum levels of AST, ALT and LDH in the dimethylnitrosoamine-induced cirrhotic rats. Hepatic ischemia-reperfusion injury was also reduced in the AxHGF-transfected rats.Increased angiogenesis could not be observed in the splenic autotransplantation with AxHGF injection. Methods of the experiment and evaluation needed to be reconsidered.Another study utilyzing insulin gene adenovirus vector (AxIns) which we made, demonstrated that AxIns injection promoted liver regeneration and increased body weight after hepatectomy in diabetic rats. One shot injection of AxIns prolonged mean survival periods for 30 days in total pancreatectomized dogs without daily insulin, and FK506, an immunosuppressant, increased the amounts and periods of insulin gene expression by AxIns and prolonged survival periods in AxIns-transfected total pancreatectomized dogs.

根据东京大学医学院生物医学研究所T.T.Nakamura博士慷慨提供的方法，制备了含肝细胞生长因子(HGF)的腺病毒载体，并将其导入Hela细胞。收集含1000-3000 ng/mlHGF的Hela细胞培养上清液，通过对培养的血管内皮细胞的增殖作用和对自由基诱导的肝细胞损伤的保护作用，证实了AxHGF的生物活性。AxHGF在293细胞中增殖并经氯化铯分级超速离心法纯化。体内注射浓缩AxHGF可改善二甲基亚硝胺诱导的肝硬变大鼠的组织学改变及血清AST、ALT和LDH水平。转染AxHGF的大鼠肝脏缺血再灌注损伤明显减轻，注射AxHGF的自体脾移植未见明显的血管生成增加。另一项利用我们研制的胰岛素基因腺病毒载体(AxIns)的研究表明，AxIns注射促进了糖尿病大鼠肝切除后的肝再生和体重增加。一次注射AxIns可使未注射胰岛素的全胰腺切除犬的平均存活时间延长30天，免疫抑制剂FK506可增加AxIns表达胰岛素基因的数量和时间，延长转AxIns的全胰腺切除犬的存活时间。