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Development of hepatocyte bank and hepatocyte transplantation with Decoy receptor 3 gene transfer

通过诱饵受体3基因转移开发肝细胞库和肝细胞移植

基本信息

批准号：
15390381
负责人：
KANEMATSU Takashi
金额：
$ 6.85万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

We cinfirmed that the isolation of hepatocytes from resected human liver tissues in the clinics. In addition, small hepatocytes were also isolated from the fraction of non-parenchymal hepatocytes as described by Mitaka et al. We characterized these cells and investigated the possibility of gene therapy.1)Isolation of matured hepatocytes and small hepatocytesLiver tissues digested by collagenase were filtrated and centrifuged at 50xg. As a result, matured hepatocytes were precipitated, however, small hepatocytes located in the supernatant fraction.Result ; Small hepatocytes that could grow for more than 3 months were obtained which produced alpha1 anti-trypsin continuously.2)Optimal ingredients of cryopreservative solution for isolated human hepatocytesVarious type of cryopreservatice solution including ; a)Cell banker, b)10%DMSO+UW, c)10%DMSO+10%FBS+UW, d)10%DMSO+FBS, e)UW were compared.Results ; The highest viability (60%) of freezing and thawing hepatocytes were observed in 10%DMSO+UW group. However these freeze-thawed hepatocytes tended to lose adhesive capacity to the cell culture dishes, suggesting that the integrities of these cellular membranes were deteriorated.3)HVJ-envelope vector mediated gene transfer into hepatocytesWe initially investigated the efficacy of the conventional HVJ-liposome for gene transfer into hepatocytes in both rats and pigs. Based on these results, we analyzed the transfection efficiencies of HVJ-envelope vector into the hepatocytes by using LacZ and luciferase reporter genes. Also histological and biochemical examination of these transfected organs in the animals were performed.Results ; Subcapsular injection of the reporter genes shoed the highest transfection efficiency compared with portal vein injection. Other organs were not damaged by this transfection process according to the findings of histology.

我们在临床上证实了从切除的人肝组织中分离出肝细胞。此外，如Mitaka等人所述，小肝细胞也是从非实质肝细胞组分中分离出来的。1)成熟肝细胞和小肝细胞的分离。胶原酶消化的肝组织过滤，50xg离心。结果：获得可持续生长3个月以上的小肝细胞，可持续产生α1抗胰蛋白酶。不同类型的冻存液包括：a)细胞培养液，b)10%DMSO+UW，c)10%DMSO+10%FBS+UW，d)10%DMSO+FBS，e)UW。然而，这些冻融的肝细胞往往会失去与细胞培养皿的黏附能力，这表明这些细胞膜的完整性被恶化。3)HVJ-包膜载体介导的基因转移到肝细胞中我们初步考察了常规的HVJ-脂质体对大鼠和猪的肝细胞基因转移的效果。在此基础上，我们利用LacZ和荧光素酶报告基因分析了HVJ包膜载体对肝细胞的转染率。结果：与门静脉注射相比，包膜下注射报告基因的转染率最高。根据组织学的发现，这种转基因过程没有对其他器官造成损害。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Selective suppression of initial cytokine response facilitates liver regeneration after extensive hepatectomy in rats.

选择性抑制初始细胞因子反应可促进大鼠广泛肝切除术后的肝再生。

DOI：
发表时间：
2004
期刊：
Hepatogastroenterology 51(57)
影响因子：
0
作者：
Kitagawa Y;Kitano S;Kubota T;Kumai K;Otani Y;Saikawa Y;Yoshida M;Kitajima M;Tsutsumi R.
通讯作者：
Tsutsumi R.

The present status and future prospects of organ storage.

器官保存现状及未来展望.

DOI：
发表时间：
2003
期刊：
Surgery(in Japanese) 66
影响因子：
0
作者：
Ishibashi S;_Sonoda K;Fujii K;Ishikawa K;Shiraishi N;Kitano S;Kamohara Y.
通讯作者：
Kamohara Y.

Eradication of hepatocellular carcinoma xenografts by radiolabelled, lipiodol-inducible gene therapy

DOI：
10.1038/sj.gt.3302531
发表时间：
2005-11-01
期刊：
GENE THERAPY
影响因子：
5.1
作者：
Kawashita, Y;Ohtsuru, A;Yamashita, S
通讯作者：
Yamashita, S

Influence of serum from rats with fulminant hepatic failure on hepatocytes in a bioartificial liver system

暴发性肝衰竭大鼠血清对生物人工肝系统中肝细胞的影响

DOI：
发表时间：
2004
期刊：
Int J Artif Organs 27(4)
影响因子：
0
作者：
Kawashita Y;Ohtsuru A;Miki F;Kuroda H;Morishita M;Kaneda Y;Hatsushiba K;Kanematsu T;Yamashita S.;Ito Y
通讯作者：
Ito Y

臓器保存の現況と展望

器官保存现状与展望