In vivo gene transfer into the lung Application in lung transplantation and therapeutic potential of HGF

本研究的目的是1)确定HVJ脂质体系统再转染的影响，2)比较肺移植背景下经气管和移植入路基因表达的分布。方法：将β-半乳糖苷酶（β-gal）质粒DNA包被高迁移率组1 （HMG1）蛋白脂质体，通过hvj介导的膜融合导入肺组织。两组SD大鼠分别于第0天（Tb-1组，n = 4）和第0-2天（Tb-3组，n = 4）气管内滴注含30μg β-gal基因的HVJ脂质体溶液0.3 ml。另一组（Tx组n = 3）行左肺原位移植。所有同种异体移植物用20 ml PBS溶液冲洗，保存4小时。在收获前将含有β-gal基因的hvj -脂质体复合物（1.0 ml）加入冲洗液中。各自的对照组以相应的方式接受hvj脂质体和空的…更多基因磁带。β-gal基因给药2天后，将转染后的肺固定，并用X-Gal染色。通过计数染色细胞，定量分析基因在肺组织中的表达和分布。结果：对照动物无基因表达。与单次给药相比，三次经气道重复给药可使肺泡或气道上皮细胞的表达增加2- 4倍，表明hvj -脂质体系统重复转染不会导致基因转移效率降低，且无炎症反应。与经气管入路相比，使用冲洗液成功地将基因转移到肺内皮细胞中，并适度转染到气道和肺泡细胞中。肝细胞生长因子（HGF）的治疗潜力最初被确定为成熟肝细胞的有效丝裂原。最近广泛而多样的研究表明，HGF在再生和维持中具有“热带”作用。首先，HGF是一种生长因子，可促进细胞在缺血和药物毒性等损伤后的恢复。其次，在暴发性肝衰竭的实验模型中，HGF通过激发抗凋亡作用来消除fas诱导的肝细胞损伤。抗凋亡蛋白BAG-1 （Bcl-2功能伴侣）水平升高也与HGF受体表达增加有关，从而防止细胞死亡。第三，HGF对肝纤维化/肝硬化、慢性肾小球硬化和肺纤维化的抗纤维化作用可能具有治疗潜力。我们首先研究了HGF在小鼠代偿性肺生长中的可能作用。血浆中内源性HGF水平在pypinibectting后第3天达到峰值，增加2.5倍，早于第5天PCNA指数的峰值。其余肺中HGF的表达和蛋白水平也在此期间升高。在第3天，重组人(rH)-HGF处理组的PNCA指数显著高于对照组，抗体(a)-HGF处理组的PNCA指数显著低于对照组。这些结果表明，补充HGF可能会加速单瘤切除术后代偿肺泡再生，而中和HGF可能会延迟这一过程。少

In vivo gene transfer into the lungThe aims of this study are 1) to determine the effect of retransfection of HVJ liposome system and 2) to compare the distribution of gene expression in the transtracheal and transplanted approaches in the setting of lung transplantation. Methods: Plasmid DNA of β-galactosidase (β-gal) were co-encapsulated in liposomes with high mobility group 1 (HMG1) protein, and were introduced into lung tissues by HVJ-mediated membrane fusion. Two groups of SD rats received intratracheal instillation of 0.3 ml of HVJ liposome solution containing 30μg of β-gal gene once on Day 0 (Group Tb-1, n = 4) and 3 times on Day 0-2 (Group Tb-3, n = 4). In another group, (Group Tx n = 3) orthotopic left lung transplantation was performed All isografts were flushed with PBS solution of 20 ml and preserved for 4 hours. HVJ-liposome complex (1.0 ml) containing β-gal gene was added to the flushing solution just before harvesting. Respective controls received HVJ-liposome with empty … More gene cassettes in a corresponding fashion. Two days after administration of β-gal gene, the transfected lungs were fixed and stained with X-Gal. The gene expression and distribution in the lung tissue was quantified by counting the staining cells. Results: There were no gene expression in the control animals. Three repetitive administrations via airway increased the expression in alveolar or airway epithelial cells by 2- to 4 fold compared to the single administration, indicating that the repeated transfection using HVJ-liposome system did not result in reduction of gene transfer efficiency without any inflammatory reaction. Compared to the transtracheal approach, successful gene transfer into the pulmonary endothelial cells using flushing solution as well as moderate degree of transfection into the airway and alveolar cells.Therapeutic potential of HGFHepatocyte growth factor (HGF) was initially identified as a potent mitogen for mature hepatocytes. Recent extensive and diverse studies have demonstrated that HGF has "tropic" roles in regeneration and maintenance First, HGF is a growth factor which promotes cell recovery following damage such as that associated with ischemia and drug toxicity. Second, in an experimental model of fulminant hepatic failure, HGF abrogated Fas-induced hepatocyte damage by eliciting of anti-apoptotic effect. Increased level of anti-apoptic protein BAG-1 (Bcl-2 functional partner) was also found to be associated with increased expression of HGF receptor, which: prevents cell death. Third, HGF may have therapeutic potential by its antifibtotic effects for the liver-fibrosis/cirrhosis, chronic glomelurosclerosis and pulmonary fibrosis.We first investigated the possible role of HGF on compensatory lung growth in mice. The endogenous HGF level in plasma peaked with 2.5 fold increase at day 3 after pneynibectiny, prior to the peak of the PCNA index at day 5. The expression and protein level of HGF in the remaining lungs also increased during this period. The PNCA index was significantly higher at day 3 in the recombinant human (rH)-HGF-treated group and was significantly lower in the antibody (a)-HGF group than in the corresponding controls. These results suggest that the supplement of HGF may accelerate postoneumonectomy compensatory alveolar regeneration while neutralizing HGF may delay this process. Less