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Establishment of an efficient gene transfer system to normal epithelial cells and its application for cancer research

正常上皮细胞高效基因转移系统的建立及其在癌症研究中的应用

基本信息

批准号：
15590358
负责人：
AKAGI Tsuyoshi
金额：
$ 2.3万
依托单位：
Osaka Bioscience Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Determination of optimal condition for retroviral vector infection of normal epithelial cells.We found that culturing in Epilife medium (Cascade), serum free and low-calcium medium, under 3% Oxygen condition significantly improve the growth of normal human epithelial cells, and thereby the efficiency of infection with, retroviral vectors. Under this condition, we have achieved around 70-80% efficiency of infection. Using this condition, we first introduced ecotropic retrovirus receptor gene making human cells susceptible to ecotropic retrovirus whose host range is limited to rodent cells. This enabled us to introduce oncogenes using ecotropic retroviral vector into human epithelial cells, providing a good system in terms of biosafety. We succeeded in making such human epithelial cells expressing ecotropic receptor using epidermal keratinocytes, bronchial epithelial cells.Establishment of immortalized human epidermal keratinocytes.Using the system described above, we consecutively introduced hTERT(human catalytic subunit of telomerase), Cdk4, and dominant negative mutant of p53 in normal human epidermal keratinocytes. The resulting cells seemed to be immortalized but still retain normal function of differentiation in response to calcium and serum.Expression of dominant acting oncogenes in human epithelial cells.Next, we analyzed the effect of dominant acting oncogenes in these immortalized human epidermal keratinocytes. Expression of activated H-Ras mutant (H-Ras V12) resulted in the formation of many vacuolated cells which died subsequently. Expression of N-terminally truncated active mutant of b-catenin reduced the growth rate. Both of these oncogenes could not induce malignant transformation of the immortalized human epidermal keratinocytes.

确定逆转录病毒载体感染正常上皮细胞的最佳条件。我们发现在Epilife培养基（Cascade）、无血清低钙培养基、3%氧气条件下培养可显着提高正常人上皮细胞的生长，从而提高逆转录病毒载体的感染效率。在这种情况下，我们的感染效率达到了70-80%左右。利用这种条件，我们首先引入了亲嗜性逆转录病毒受体基因，使人类细胞对亲嗜性逆转录病毒敏感，其宿主范围仅限于啮齿动物细胞。这使得我们能够利用亲嗜性逆转录病毒载体将癌基因引入人类上皮细胞，在生物安全方面提供了良好的系统。我们成功地利用表皮角质形成细胞、支气管上皮细胞制作了表达共亲性受体的人类上皮细胞。建立了永生化的人类表皮角质形成细胞。使用上述系统，我们连续将hTERT（人类端粒酶催化亚基）、Cdk4和p53的显性失活突变体引入正常人体内表皮角质形成细胞。所得细胞似乎是永生化的，但仍保留对钙和血清反应的正常分化功能。人上皮细胞中显性作用癌基因的表达。接下来，我们分析了这些永生化人表皮角质形成细胞中显性作用癌基因的作用。激活的 H-Ras 突变体 (H-Ras V12) 的表达导致许多空泡细胞的形成，这些细胞随后死亡。 N-末端截短的β-连环蛋白活性突变体的表达降低了生长速率。这两种癌基因均不能诱导永生化人表皮角质形成细胞的恶性转化。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation

DOI：
10.1073/pnas.1834876100
发表时间：
2003-11-11
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Akagi, T;Sasai, K;Hanafusa, H
通讯作者：
Hanafusa, H

Human diploid fibroblasts are refractory to oncogene-mediated transformation

人二倍体成纤维细胞对癌基因介导的转化具有抵抗力