冠脉内导入重组β-半乳糖苷酶腺病毒的实验研究

目的 :研究经冠状动脉内导入重组 β-gal腺病毒后心肌的转染效率 ,证明冠脉内导入途径是否为重组基因导入心脏的有效的可行的途径。方法 :用定向克隆的方法构建重组 β -gal腺病毒载体 ,将 1 .53× 1 0 1 2 pfu重组 β-gal腺病毒经冠状动脉造影导管由左冠状动脉分别导入到 7只雄性Yorkshire猪心脏中 ,于术后 1周、2周、4周及 8周处死动物 ,取左右冠状动脉、3～ 5mm厚的不同部位的心肌及各实质脏器标本 ,经 1 .2 5 %戊二醛固定 2 0min ,用PBS洗涤 3次 ,在室温下用X -gal染液染色 1 6～ 2 4h ,脱水、石蜡包埋、切片及HE复染 ,光镜下计算出表达 β -gal的心肌细胞百分数即为转染效率。结果 :经冠状动脉内导入重组基因后 ,于术后 1周即有 β-gal表达 ,2周达高峰 ,心肌细胞的转染效率高达 45 .57% ,全层左冠状动脉壁、心肌间质中均有 β-gal表达 ,4周 β-gal表达下降 ,8周 β -gal恢复至对照组的基线水平。结论 :经冠状动脉内导入重组腺病毒 ,心肌细胞及冠脉壁重组基因表达效率高 ,说明冠状动脉内导入重组目的基因将是冠心病基因治疗中有效的可行的方法

Experimental study of intracoronary infusion of recombinant adenovirus coding for β-galactosidase gene

Objective To demonstrate the transfer efficiency of catheter-mediated intracoronary infusion of recombinant adenovirus coding for β-galactosidase gene to show the approach being efficient pathway.Methods Recombinant adenovirus vector containing β-galactosidase gene was constructed by directional cloning.Left coronary artery was infused with 1.53×10 12 pfu Ad-β-gal via an intracoronary catheter in 7 male Yorkshire swines.The swines were sacrificed one week,two weeks,four weeks and eight weeks after intracoronary infusion and their hearts were harvested.β-galactosidase gene expression was examined histochemically in their hearts.The efficiency of β-galactosidase gene expression was estimated by determining the fraction of cells expressing β-galactosidase gene in 10 high power fields.Results β-galactosidase gene expression was detected in both the myocardium and coronary arteries 1 week following the adenovirus infusion.The peak values of β-galactosidase gene expression were detected in both the myocardium and coronary arteries 2 weeks after the Ad-β-gal infusion,which were 45.57% in the myocardium and 26.11% in the coronary arteries.The β-gal expression was decreased 4 weeks later, and recovered to baseline at 8 weeks.Conclusion The experimental study indicates that the recombinant adenovirus-mediated gene expression is effective in both the myocardium and coronary arteries. The approach of intracoronary infusion is effective and feasible for gene therapy of cardiovascular diseases.