应用转血管内皮生长因子基因治疗肢体缺血的研究

目的研究肌肉内转血管内皮生长因子(VEGF)基因治疗肢体缺血的可行性,比较各种治疗方法的疗效. 方法雄性新西兰大白兔50只,完全切除股动脉后随机分为3组.实验组为明胶海绵携载法转基因组(n=18)和肌肉内注射转基因组(n=18);只注射pcDNA3为对照组(n=14).通过直接注射法及明胶海绵携载法将构建的质粒pcDNA3-VEGF121基因转入缺血肌肉内,立即测定各组肢体髂内动脉血流量,在术后2天,1、2、3和4周应用逆转录-聚合酶链反应(RT-PCR)技术测定基因表达,术后30天通过测定缺血肢体髂内动脉血流量、动脉血管造影及组织学观察测定血管密度,评价侧支循环变化. 结果术后2天,转VEGF基因治疗的两实验组均测到基因表达,并均维持2周.术后立即测定的髂内动脉血流量各组间无明显差异.术后30天,缺血肢体髂内动脉血流量、肢体血管造影血管数目和肌肉组织血管密度转VEGF基因的两实验组均比对照组明显增高,差异有统计学意义(P<0.01),明胶海绵携载组较直接注射组亦有明显增高,差异有统计学意义(P＜0.05). 结论肌肉内转VEGF基因治疗可促进急性缺血肢体侧支循环、改善血供,明胶海绵携载法较直接注射法有更好的疗效.

EXPERIMENTAL STUDY ON TREATMENT OF ACUTE LIMB ISCHEMIA WITH VASCULAR ENDOTHELIAL GROWTH FACTOR-121 GENE TRANSFER

OBJECTIVE: To investigate the feasibility of intramuscular gene therapy for acute arterial ischemic diseases by use of plasmid pcDNA3-VEGF121 and to evaluate therapeutic efficiency of vascular endothelial growth factor (VEGF) by different routes of administration. METHODS: Fifty New Zealand White rabbits were randomly assigned to either gelation sponge carrying-pcDNA3-VEGF121 (n = 18), intramuscular injection-pcDNA3-VEGF121 (n = 18), or pcDNA3 (as control group, n = 14). After ligation of the external iliac artery and complete excision of the femoral artery, 500 micrograms of the plasmid pcDNA3-VEGF121 were transfected into the muscles of the ischemic limb by gelation sponge carrying or direct intramuscular-injection. Immediately after gene transfection, blood flow of the internal iliac artery were measured. VEGF121 gene expression was detected by RT-PCR after 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks of transfection. After 30 days, blood flow of the internal iliac artery, angiographic score and histological vessels of ischemic hindlimbs were measured respectively. RESULTS: In the two VEGF-treated groups, VEGF121 mRNA expressed in the transfected ischemic muscles after 2 days and lasted 2 weeks. Immediately after gene transfection, blood flow of the internal iliac artery had no significant difference between three groups. After 30 days, blood flow of the internal iliac artery, angiographic score and capillary density were significantly greater in both VEGF-treated groups than in control group. Complexity of vascular branching and vessel density of gelation sponge-VEGF treated limbs were significantly greater when compared with the intramuscular-injection limbs. CONCLUSION: These findings suggest the feasibility of employing gene therapy of pcDNA3-VEGF121 could augment collateral development and tissue perfusion in an animal model of hindlimb ischemia, and gelation sponge carrying VEGF gene may respect a potential therapy methods.