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Gene carrier system by means of alveolar macrophage to the lung : an attempt of gene therapy for lung diseases.

通过肺泡巨噬细胞到肺部的基因载体系统：肺部疾病基因治疗的尝试。

基本信息

批准号：
09670601
负责人：
KURIYAMA Takayuki
金额：
$ 1.98万
依托单位：
Chiba University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670601/
关键词：
Alveolar Macrophage Gene Vector Gene Transfer Gene Therapy Fibroblast

项目摘要

A novel gene delivery system to lung has been established in mice. The method employs intravenous injection of syngeneic viable cells that are transfected with a target gene in ex vivo.In a series of preliminary studies, attempts of transfecting syngeneic alveolar macrophages with a reporter gene by means of non-viral methods, i.e. calcium-phosphate co-precipitation, electropolation and lipofectin have failed, In the next step, syngeneic fibroblasts instead of alveolar macrophages were utilized to reveal high efficacy of gene transfection. Eventually, a plasmid that had bacterial beta-galactosidase (beta-gal) gene under the control of a beta-actin promoter was transfected to an established Balb/c mouse derived fibroblast cell line, A3 1, by the calcium-phosphate co-precipitation method in vitro. The cells were propagated in vitro and administrated to syngeneic Balb/c mice by intra tracheal (I.T.) or intra venous (I.V.) method. After the administrations, each organ, induding lung, heart, kidney, liver, brain and spleen was removed from the sacrificed mice, and the beta-gal activity in the each organ was assessed by ELISA.A high level of beta -gal expression was observed in lung tissues, lasting as long as about two weeks, with a peak at 2 hours after the administration. Kinetic differences of the gene expression between I.T.and I.V.administration were comparatively evaluated. All other organs, except for hearts, showed no detectable expression after either I.T.or I.V.administration, when evaluated by ELISA and RT-PCR.Histological examinations with beta -gal staining showed identification and localization of the administrated cells those showed high expression of the introduced gene in the lung. Other histological examination with hematoxylin-eosin staining disclosed no significant lesions such as inflammation and fibrosis in the lungs.In conclusion, this study demonstrated potential clinical capability and safety of this gene delivery system for lung diseases.

在小鼠体内建立了一种新的肺基因传递系统。该方法采用静脉注射在体外转染靶基因的同源活细胞。在一系列的前期研究中，通过非病毒方法，即磷酸钙共沉淀、电电泳和脂质体素转染同源肺泡巨噬细胞报告基因的尝试都失败了，下一步，利用同源成纤维细胞代替肺泡巨噬细胞来显示基因转染的高疗效。最后，通过体外磷酸钙共沉淀法，将含有β -肌动蛋白启动子控制的细菌β -半乳糖苷酶（β -gal）基因的质粒转染到已建立的Balb/c小鼠源性成纤维细胞系A3 1中。体外培养这些细胞，并通过气管内或静脉内给药。给药后，取小鼠肺、心、肾、肝、脑、脾等脏器，ELISA法测定各脏器β -半乳糖活性。在肺组织中观察到高水平的β -gal表达，持续时间长达约两周，在给药后2小时达到峰值。比较评价两组间基因表达的动力学差异。通过ELISA和RT-PCR评估，除心脏外，所有其他器官在给药或静脉注射后均未显示可检测到的表达。β -半乳糖染色的组织学检查显示，在肺中高表达引入基因的给药细胞得到了鉴定和定位。其他苏木精-伊红染色组织学检查未见明显病变，如肺部炎症和纤维化。总之，本研究证明了该基因传递系统治疗肺部疾病的潜在临床能力和安全性。