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Experimental analysis for reconsruction of inferior vena cava and portal vein in abdominal surgery : endothelial seeding on artificial grafts and insulin gene transfer into endothelial cells.

腹部手术中下腔静脉和门静脉重建的实验分析：人工移植物上的内皮种植和胰岛素基因转移到内皮细胞中。

基本信息

批准号：
07457280
负责人：
KUMADA Kaoru
金额：
$ 1.15万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Purpose of the study is to investigate the effectiveness of endothelial seeding on the the artificial graft and insulin gene transfer to endothelial cells on the graft, which was interposed in the canine infreior vena cava and portal vein for vascular reconstruction in hepato-biliary-pancreatic surgery.We could not find any significant difference in patency of the vein grafts, ePTFE grfats, or endothelialized grafts, which were interposed in the IVC or PV.We performed insulin gene transfer using adenovirus vector carrying human insulin gene (AxIns). AxIns was constructed as follows ; human preproinsulin gene genomic DNA (1.3-kb) containing coding sequence, was inserted into the SwaI site of the full-length replication-deficient adenovirus genome cloned in a cassette cosmid. The cosmid was then co-transfected to 293 cells together with the adenovirus DNA-terminal protein complex digested at several sites with EcoT22I.AxIns was generated by overlapping recombination. AxIns was propagated in 293 cells and concentrated with purification using sequential ultracentrifugation in CsCl step gradients. Transfection of AxIns into endothelial cells induced the expression of preproinsulin mRNA and secret proinsulin. One shot injection of AxIns into the spleen transfered human insulin gene mainly in the liver and produced human insulin enough to decrease blood glucose levels in streptozotosin-induced diabetic rats and to restore the serum levels of total protein and albumin, which were impaired in diabetic rats. AxIns injection into the spleen, but not periodic insulin administration, controled blood glucose levels, restored serum total protein levels, and prolonged survival periods after total pacreatectomy in canine models. Thus we confirmed availability of AxIns for in vivo insulin gene transfer.

本研究的目的是探讨人工移植物上的内皮种植和胰岛素基因转移对移植物上内皮细胞的有效性，将移植物插入犬下腔静脉和门静脉用于肝胆胰手术中的血管重建，我们没有发现静脉移植物、ePTFE grfats和内皮化移植物的通畅性有任何显著差异，用腺病毒载体介导的人胰岛素基因（AxIns）进行胰岛素基因转移。如下构建AxIns：将含有编码序列的人前胰岛素原基因组DNA（1.3-kb）插入克隆在盒式粘粒中的全长复制缺陷型腺病毒基因组的SwaI位点。将重组质粒与腺病毒DNA末端蛋白复合物共转染293细胞，经EcoT 22 Ⅰ酶切后，重叠重组获得AxIns。AxIns在293细胞中增殖，并在CsCl阶梯梯度中使用连续超离心纯化进行浓缩。AxIns转染内皮细胞可诱导前胰岛素原mRNA和分泌胰岛素原的表达。一次性注射AxIns到脾脏中，主要在肝脏中转移人胰岛素基因，并产生足够的人胰岛素，以降低链脲佐菌素诱导的糖尿病大鼠的血糖水平，并恢复糖尿病大鼠受损的血清总蛋白和白蛋白水平。AxIns注射到脾脏中，但不定期胰岛素给药，控制血糖水平，恢复血清总蛋白水平，并延长犬模型全胰腺切除术后的生存期。因此，我们证实了AxIns可用于体内胰岛素基因转移。