CAJ | 학술논문

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璎.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14 d和30 d通过免疫组织化学榆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484 bp长的特异性片段出现,免疫组化町检测到肺血管内皮、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.
목적 재가토고동력성폐동맥고압모형상경기관도경전염휴대인간세포생장인자기인적중조선병독(Ad-HGF),탐토외원HGF기인전염유도측지폐혈관생성적가행성.방법 1월령유토정중개흉,행좌무명동맥여주폐동맥문합,통과지속좌향우분류,3개월후건립고동력성폐동맥고압모영.장모형동물수궤분위대조조、GFP전염조、단차HGF치료조화중복HGF치료조.HGF치료조(단차혹1주후중복)통과기관내적입적방법전염Ad-HGF.2주후,통과RT-PCR화면역조직화학검측HGF기인화단백표체.분별재기인전염후14 d화30 d통과면역조직화학유측폐미혈관화소동맥밀도.1개월시,폐혈관조영관측측지혈관건립정황.결과 기관내적입Ad-HGF후2주,폐조직RNA제취후응효전영현시유484 bp장적특이성편단출현,면역조화정검측도폐혈관내피、폐포상피세포내HGF표체.폐조직병리절편관찰가견HGF기인치료조폐혈관밀도(단차HGF치료조화중복HGF치료조)명현고우대조조화GFP전염조,증다적혈관이미혈관위주,1개월후,기성폐동맥명현증다.폐혈관조영증실HGF기인치료조측지혈관교대조조화GFP전염조봉부.결론 외원성HGF기인경기관전염,조기(2주내)이유도폐미혈관신생위주,후기(1개월시)가촉진폐소동맥생성.
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 wag 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 mortths later,animals were randomly assigned to receive either Ad-HGF(2×109Pfu in 0.2 ml PBS,H1 group),repeated administration of Ad-HGF after one week (H 2 group),Ad-GFP(2×109 Pfu in 0.2 ml PBS,G group),or PBS(0.2 ml,C group)by the intratraeheal 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 a-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 H l and H2 groups,but not in other groups.The expression of HGF protein could be detected on alveolag 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 H1 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 transfeetion.the numbeT of small pulmonary arteries was significantly increased in Hl 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.