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

我们制作了含有肝细胞生长因子（HGF）的腺病毒载体，其cDNA由Dr.T.Nakamura（大阪大学医学院生物医学研究中心）慷慨提供，根据Dr.I . Saito（东京大学医学研究所）发明的方案。含HGF的腺病毒载体（AxHGF）转染Hela细胞后。我们收集了含有1000-3000 ng/ml HGF的培养基。通过对培养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转染Hela细胞所产生的HGF的生物活性。AxHGF在293细胞中增殖，经氯化铯阶梯梯度超离心浓缩纯化。体内注射浓缩的AxHGF可改善二甲亚硝胺诱导的肝硬化大鼠的组织学变化和血清中AST、ALT和LDH的水平。转染axhgf的大鼠肝缺血再灌注损伤也有所减轻。在自体脾移植中，注射AxHGF后血管生成未见明显增加。实验方法和评价方法需要重新考虑。另一项利用胰岛素基因腺病毒载体（AxIns）的研究表明，注射AxIns可以促进糖尿病大鼠肝切除术后的肝脏再生和体重增加。一针注射AxIns可延长未每日注射胰岛素的全胰切除犬的平均生存期30天，免疫抑制剂FK506可增加转染AxIns的全胰切除犬的胰岛素基因表达量和时间，延长生存期。

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.