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Development of artificial endocrine organs composed of cultured skin cells

由培养的皮肤细胞组成的人工内分泌器官的开发

基本信息

批准号：
09557096
负责人：
INOKUCHI Sadaki
金额：
$ 7.36万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

A purpose of this study was to develop artificial endocrine organs by introducing genes of hormones or cytokines into human epidermal keratinocytes.(1) To increase infectivity of a retrovirus vectorwe constructed an adenovirus vector into which gene of an amphotropic retrovirus receptor is cloned. Although infectivity of a retrovirus vector to K562 cells were increased, infectivity to human epidermal keratinocytes was not changed after infection of the adenovirus.(2) We constructed a retrovirus vector into which both enhanced green fluorescent protein (GFP) gene and human preproinsulin cDNA were cloned. After infection of the retrovirus vector, GFP positive human epidermal keratinocytes were selected by FACS sorting. The procedure enriched GFP positive keratinocytes to10 times. These GFP positive cells stably produced proinsulin for 14 days in vitro. Although, when the cells were transplanted into immunodeficient mice, expression of both GFP and proinsulin were despaired within 4 weeks … More .(3) GFP and preproinsulin genes were cloned into MSCV retrovirus vector, which had altered LTR sequence. To measure the stability of introduced genes in vivo, human keratinocytes were infected by the vector and transplanted into immunodeficient mice. However, expression of the introduced genes could not be detected at 4 weeks after transplantation(4) A replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced GFP was used for gene transfer into human keratinocytes. Afier infection of the lentiviral vector, 10% of the cells showed GFP positive.These results showed that enrichment of gene introduced cells using GFP marker and FACS sorting was a useful procedure for human keratinocytes. However, two major problems of conventional recombinant retrovirus vectors, low infectivity and unstable expression of introduced gene in vivo, were still remained. We considered that HIV-1 based lentiviral vector was a promising alternative for efficient and stable gene transfer into human epidermal keratinocytes. Less

本研究的目的是通过将激素或细胞因子的基因导入人表皮角质形成细胞来构建人工内分泌器官。(1)为了提高逆转录病毒载体的感染性，我们构建了一个腺病毒载体，其中克隆了嗜异性逆转录病毒受体基因。尽管逆转录病毒载体对K562细胞的感染性增加，但腺病毒感染后对人表皮角质形成细胞的感染性没有改变。(2)我们构建了一个逆转录病毒载体，其中同时克隆了增强型绿色荧光蛋白（GFP）基因和人前胰岛素原cDNA。感染逆转录病毒载体后，通过FACS分选选择GFP阳性的人表皮角质形成细胞。该程序将GFP阳性角质形成细胞富集至10倍。这些GFP阳性细胞在体外稳定产生胰岛素原14天。尽管如此，当将细胞移植到免疫缺陷小鼠中时，GFP和胰岛素原的表达在4周内均消失 ...更多信息 . (3)将GFP和前胰岛素原基因克隆到改变了LTR序列的MSCV逆转录病毒载体中。为了测量引入的基因在体内的稳定性，用载体感染人角质形成细胞并移植到免疫缺陷小鼠中。然而，在移植后4周不能检测到导入基因的表达（4）使用编码增强的GFP的复制缺陷型水泡性口炎病毒糖蛋白G假型HIV-1基载体将基因转移到人角质形成细胞中。转染慢病毒载体后，10%的细胞显示GFP阳性，表明用GFP标记和流式细胞仪分选富集基因导入细胞是一种有效的方法。然而，传统的重组逆转录病毒载体仍然存在感染性差和导入基因在体内表达不稳定两个主要问题。我们认为，基于HIV-1的慢病毒载体是一个有前途的替代高效和稳定的基因转移到人表皮角质形成细胞。少