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Systemic disposition and induction of antitumor immunity with murine gene-modified dendritic cells

小鼠基因修饰树突状细胞的全身配置和抗肿瘤免疫诱导

基本信息

批准号：
17591323
负责人：
KATANO Hisako
金额：
$ 1.79万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Administration of therapeutic gene-modified dendritic cells (DCs) is a promising approach for cancer immunotherapy. For its practical application, comprehensive examinations of safety are necessary, but not enough work has been done on the in vivo behavior of DC after administration. Thus, as a pre-clinical study, we developed a system to evaluate characteristics of gene-modified DCs including the disposition after administration in a mouse model.Mouse Interleukin-12 (mIL-12) that induced anti-tumor immunoreactions was selected as a therapeutic gene and recombinant adenoviral vector expressing mIL- l2 (Ad-mIL-12) was constructed. On the other hand, mouse bone marrow-derived immature dendritic cells (m-iDCs) ware generated in culture containing GM-CSF. This m-iDCs were infected with Ad-mIL-12 with centrifugation method, and mIL-12 gene-transduced dendritic cells (mIL-12-iDCs) are used for analysis of surface antigen and measurements of IL-12 concentrations in the supernatants. As a result, the surface expressions of CD80 and CD86 as well as the expression of mIL-12 protein were confirmed, suggesting that these mIL-12-iDCs had immunostimulating properties.Next, this mIL-12-iDC was administered to mice with direct intrahepatic injection. Serum mIL-12 concentration, values of hematological-biochemical tests, weight of the spleen, and nuclear cell count were measured. There were no significant difference among mIL-12-iDC group, non-treatment group and sham-operated group. In addition, a real-time PCR method was developed to quantify viral DNA as adenovirus E4 copy number. In some samples, viral DNA was detected in lung and spleen derived from mIL-12-iDC injected mice, suggesting the possibility that mIL-12-iDC administered to the liver has reached another organs through the blood stream.This system is regarded as an effective method to examine the safety in gene therapy using therapeutic gene-modified cells.

治疗性基因修饰树突状细胞（dc）是一种很有前途的癌症免疫治疗方法。为了实际应用，对其安全性进行全面的检验是必要的，但对其给药后的体内行为研究还不够。因此，作为一项临床前研究，我们开发了一个系统来评估基因修饰dc的特征，包括在小鼠模型中给药后的处置。选择诱导抗肿瘤免疫反应的小鼠白细胞介素-12 （mIL-12）作为治疗基因，构建表达mIL- l2的重组腺病毒载体（Ad-mIL-12）。另一方面，小鼠骨髓来源的未成熟树突状细胞（m-iDCs）在含有GM-CSF的培养物中产生。用Ad-mIL-12离心法感染m-iDCs，用mIL-12基因转导的树突状细胞（mIL-12- idcs）分析表面抗原和测量上清液中IL-12的浓度。结果证实了CD80和CD86的表面表达以及mIL-12蛋白的表达，表明这些mIL-12- idcs具有免疫刺激特性。然后，将mIL-12-iDC直接注射给小鼠肝内。测定血清mIL-12浓度、血液学生化试验值、脾脏重量和核细胞计数。mIL-12-iDC组与非治疗组、假手术组间差异无统计学意义。此外，还建立了实时PCR方法，将病毒DNA定量为腺病毒E4拷贝数。在一些样本中，在注射了mIL-12-iDC的小鼠的肺和脾脏中检测到病毒DNA，这表明给药于肝脏的mIL-12-iDC有可能通过血流到达其他器官。该系统被认为是检验治疗性基因修饰细胞基因治疗安全性的有效方法。