Development of artificial endocrine organs composed of cultured skin cells

本研究的目的是通过将激素或细胞因子基因导入人表皮角质形成细胞，形成人工内分泌器官。(1)为了提高逆转录病毒载体的传染性，我们构建了一个腺病毒载体，其中克隆了两性逆转录病毒受体的基因。虽然逆转录病毒载体对K562细胞的感染性增强，但感染腺病毒后对人表皮角质形成细胞的感染性没有改变。(2)构建了一种逆转录病毒载体，克隆了增强型绿色荧光蛋白（GFP）基因和人胰岛素前原基因cDNA。逆转录病毒载体感染后，采用FACS分选法筛选GFP阳性的人表皮角质形成细胞。该方法使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。然而，当细胞移植到免疫缺陷小鼠体内时，GFP和胰岛素原的表达在4周内就完全消失了。(3)将GFP和胰岛素前原基因克隆到LTR序列改变的MSCV逆转录病毒载体中。为了检测引入基因在体内的稳定性，用载体感染人角质形成细胞并将其移植到免疫缺陷小鼠体内。然而，在移植后4周，无法检测到引入的基因的表达。(4)利用一种基于复制缺陷的水泡性口炎病毒糖蛋白G伪型hiv -1载体编码增强的GFP，将基因转移到人角化细胞中。慢病毒载体感染后，10%的细胞呈GFP阳性。这些结果表明，使用GFP标记和FACS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低和引入基因在体内表达不稳定的问题。我们认为基于HIV-1的慢病毒载体是一种有效和稳定的基因转移到人表皮角质形成细胞的有希望的替代方法。少

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