肝细胞生长基因转染诱导肺高压模型动物肺血管生成的实验研究

In vivo transfection of hepatocyte growth factor gene induces pulmonary angiogenesis in rabbits with hyperkinetic pulmonary hypertension

目的在家兔高动力性肺动脉高压模型上经气管途径转染携带人肝细胞生长因子基因的重组腺病毒（Ad—HGF），探讨外源HGF基因转染诱导侧支肺血管生成的可行性。方法1月龄幼兔正中开胸，行左无名动脉与主肺动脉吻合，通过持续左向右分流，3个月后建立高动力性肺动脉高压模型。将模型动物随机分为对照组、GFP转染组、单次HGF治疗组和重复HGF治疗组。HGF治疗组（单次或1周后重复）通过气管内滴入的方法转染Ad-HGF。2周后，通过RT-PCR和免疫组织化学检测HGF基因和蛋白表达。分别在基因转染后14d和30d通过免疫组织化学检测肺微血管和小动脉密度。1个月时，肺血管造影观测侧支血管建立情况。结果气管内滴入Ad—HGF后2周，肺组织RNA提取后凝胶电泳显示有484bp长的特异性片段出现，免疫组化可检测到肺血管内皮、肺泡上皮细胞内HGF表达。肺组织病理切片观察可见HGF基因治疗组肺血管密度（单次HGF治疗组和重复HGF治疗组）明显高于对照组和GFP转染组，增多的血管以微血管为主，1个月后，肌性肺动脉明显增多。肺血管造影证实HGF基因治疗组侧支血管较对照组和GFP转染组丰富。结论外源性HGF基因经气管转染，早期（2周内）以诱导肺微血管新生为主，后期（1个月时）可促进肺小动脉生成。

Objective To investigate the effect of adenoviral-mediated exogenous HGF （Ad-HGF） gene transfer on lung angiogenesis in the rabbit lung in rabbits with hyperkinetic pulmonary artery hypertension. Methods A thoracotomy was performed through a midsternal incision in 1-month-old immature rabbit and an anastomosis between the left inominate artery and the pulmonary trunk was made to establish a chronic patent left to right shunt. Three months later, animals were randomly assigned to receive either Ad-HGF（2 × 10^9 Pfu in 0. 2 ml PBS, H1 group）, repeated administration of Ad-HGF after one week （H 2 group）, Ad-GFP （2 X 109 Pfu in 0. 2 ml PBS, G group）, or PBS （0.2 ml, C group） by the intratracheal method of gene transfection. After two weeks, reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemical examination was performed to identify HGFmRNA and HGF protein expression. The capillary density and small pulmonary artery density were determined by immunostained with antibodies against factor Ⅷ and α-SMA. After 1 month, the collateral vessels were evaluated by angiogram under digital subtraction angiography （DSA）. Results Two weeks after Ad-HGF transfection, 484 bp bands could be found by RT-PCR in H1 and H2 groups, but not in other groups. The expression of HGF protein could be detected on alveolar epithelium and pulmonary vessel endothelium by immunohistochemistry examination. The number of factor Ⅷ-positive pulmonary capillaries was also significantly increased in the H1 and H2 groups compared with the C and G groups （ P 〈 0.05 ）. The capillary density reached （ 17.0 ±3.3 ） mm2 and （ 19.7 ± 2.8 ） mm2 in the HI and H2 group, respectively, whereas it remained （ 13.2 ± 3.2） mm2 in the C group and （13.5 ±2.4） mm2 in the G group （P〈0.05）. One month after Ad-HGF transfection, the number of small pulmonary arteries was significantly increased in H1 and H2 group compared with control groups （ P 〈 0. 05 ）. The collateral vessels were more abundant in HGF transfection groups than that in the two control groups reviewed by angiogram under digital subtraction angiography （DSA）. Conclusion In vivo gene transfection with HGF by means of the intra-tracheal injection could induce pulmonary angiogensis in the early stage and small pulmonary arterial angiogensis in later stage.