Gene carrier system by means of alveolar macrophage to the lung : an attempt of gene therapy 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离体转染目的基因的同系活细胞，在一系列的初步研究中，通过非病毒方法，即磷酸钙共沉淀、电极化和脂质体转染，用报告基因转染同系肺泡巨噬细胞的尝试都失败了，下一步，用同系成纤维细胞代替肺泡巨噬细胞显示基因转染的高效率。最后，通过钙-磷酸盐共沉淀法将具有在β-肌动蛋白启动子控制下的细菌β-半乳糖苷酶（β-gal）基因的质粒转染到已建立的Balb/c小鼠来源的成纤维细胞系A3 1中。将细胞在体外增殖并通过气管内（I.T.）或静脉内（I. V.）法给药后取肺、心、肾、肝、脑、脾等脏器，ELISA法检测各脏器中β-gal活性，肺组织中β-gal表达持续2周左右，给药后2 h达高峰。比较评估了I.T.和I. V.给药之间基因表达的动力学差异。当通过ELISA和RT-PCR评估时，除心脏外的所有其他器官在I.T.或I. V.施用后均未显示可检测的表达。用β-gal染色的组织学检查显示施用的细胞的鉴定和定位，所述细胞显示在肺中引入的基因的高表达。其他组织学检查和苏木精-伊红染色显示肺部无明显病变，如炎症和纤维化。总之，本研究证明了该基因递送系统用于肺部疾病的潜在临床能力和安全性。

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.