脂质体介导报告基因Lac-Z导入大鼠原位移植肺细胞的实验研究

Experimental Study of ex vivo Liposome-mediated Gene Transfer in Orthotopic Rat Left Lung Transplantation

目的 通过体外途径的脂质体介导将报告基因 (Lac -Z基因 )转入移植肺并表达 ,证明将外源基因转入移植肺进行基因治疗的可行性。方法 首先建立大鼠左肺原位移植模型和经肺静脉逆行灌注技术。然后将 18只SD大鼠随机分为转基因组、脂质体组和空白对照组 ,每组各 6只。转基因组经肺静脉将质粒载体DNA(PSV -β -gal) /脂质体Lipofectin R○ (DOTMA/DOPE )复合物灌注入移植肺 ;脂质体组和空白对照组则分别灌注脂质体和 0 9%生理盐水。手术 1d以后获取肺脏 ,冰冻切片 ,经X -gal组织化学染色检测Lac -Z基因的转染和表达情况。结果 转基因组 3例都检测到Lac -Z基因的表达。其表达位于气管上皮细胞和肺泡上皮细胞内 ,呈点片状兰斑。而脂质体组和空白对照组则均未检测到Lac -Z基因的表达。结论 以质粒表达载体PSV -β -gal/脂质体Lipofectin R○ 复合物的形式 ,经肺血管 (静脉逆行 )灌注方式 ,把外源基因转入移植肺是可行的。本实验为进一步开展大鼠肺移植术后缺血 -再灌注损伤。

Objective To determine the feasibility of ex vivo liposome-mediated gene transfer ( Lac-Z gene ) to lung isografts and to get expression. Methods An orthotopic rat left lung transplant model was developed with the use of a modification of the “cuff” technique. 18 SD rats were divided into three groups randomly. Gene transfer group ( group 1 ): PSV-β-gal/ liposome ( Lipofectin R○ ) complex was transfered into the donor left lung via the left pulmonary vein route at the time of lung harvesting. Liposome group ( group 2 ) and Control group ( group 3 ) : Grafts underwent the same procedure but received liposome and 0 9% saline solution respectively. Donor lungs were harvested one day after transplantation. The transgene expression of Lac-Z gene was detected by histochemical staining. Results Transgene expression of β-gal in group 1 was detected in bronchial epithelial cells and alveolar epithelial cells. But no expression of β-gal was found in groups 2 and 3. Conclusion Ex vivo plasmid vector/liposome complex-mediated gene transfer to lung isografts via the left pulmonary vein route is feasible. This study provides the scientific proof to the feasibility of functional gene therapy of complications such as ischemia-reperfusion injury, rejection and infection after rat lung transplantation.