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目的:观察肌肉转染腺相关病毒(AAV)介导的CD151基因在大鼠后肢缺血模型中促血管再生作用并进行功能评价。方法:实验于2003-01/2005-10在华中科技大学同济医学院附属同济医院心血管研究室暨基因治疗研究中心完成。①分别包装携带有CD151基因和GFP基因的重组腺相关病毒(rAAV-CD151,rAAV-GFP)。②Wistar大鼠12只,采用随机数字法分为实验组和对照组,每组各6只。③实验组大鼠右下肢肌肉注射局部转染rAAV-CD151,对照组注射rAAV-GFP(1×1010pfu/只)。④基因转染2周后行股动脉切除术建立右侧大鼠后肢缺血模型。心内注入红四氮唑观察大鼠下肢变化,根据组织染色的部位或染色的深浅程度判断后肢缺血模型建立是否成功。⑤于模型建立术前、术后2d、术后1,2,3,4周进行缺血后肢的损伤评分,分为0～3分,3分为患肢爬行托拽,或者肢体有缺血性坏疽;0分为抗牵拉时患侧足底与对侧肢体外观无明显区别。⑥采用Westernblot检测两组大鼠局部缺血肢体CD151的表达。⑦股动脉切除术4周后,采用非致冷红外热成像仪分别检测大鼠后肢皮肤温度、免疫组织化学检测缺血肌肉组织毛细血管密度,观察缺血肢体血管再生情况。结果:12只大鼠全部进入结果分析。①股动脉切除术4周(基因转染6周)后,实验组大鼠缺血后肢平均皮肤温度高于对照组((32.92±0.63),(31.22±0.87)℃,P<0.05)。②Westernblot检测结果显示实验组大鼠缺血侧肌肉组织CD151表达显著高于对照组。③实验组缺血后肢肌肉组织毛细血管密度显著大于对照组(609.00±83.89),(517.00±70.65)/mm2,P<0.05)。④术后4周肢体功能的损伤评分实验组显著低于对照组(0.67±0.21,1.67±0.32,P<0.05)。结论:局部高表达CD151基因能够提高缺血组织的血管再生能力,促进缺血肢体的血运重建和功能恢复。
OBJECTIVE: To observe the angiogenic effect of muscle-derived adeno-associated virus (AAV) -mediated CD151 gene in rat hind limb ischemia model and to evaluate its function. METHODS: The experiment was performed at Cardiovascular Research Center and Gene Therapy Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 2003 to October 2005. ① Package the recombinant adeno-associated virus (rAAV-CD151, rAAV-GFP) carrying CD151 gene and GFP gene respectively. Twist Wistar rats were randomly divided into experimental group and control group with 6 in each group. (3) rAAV-CD151 was injected intramuscularly in the right lower extremity and rAAV-GFP (1 × 1010pfu / only) in the control group. ④ The right hindlimb ischemia model was established by femoral artery resection 2 weeks after gene transfection. Intra-heart infusion of red tetrazolium observed changes in the lower extremities of rats, according to the degree of tissue staining or the degree of staining to determine the success of hindlimb ischemia model. ⑤ in the model established preoperative, postoperative 2d, 1, 2, 3, 4 weeks after the ischemic hindlimb injury score, divided into 0 to 3 minutes, 3 minutes for the limbs crawling traction, or limb ischemia Sex gangrene; 0 points for anti-traction ipsilateral plantar contralateral limb appearance and no significant difference. ⑥Western blot was used to detect the expression of CD151 in ischemic limbs of the two groups. ⑦ After 4 weeks of femoral artery resection, the skin temperature of the hind limbs of rats were detected by un-cooled infrared thermal imager respectively. The capillary density of ischemic muscle tissue was detected by immunohistochemistry and the angiogenesis of ischemic limbs was observed. Results: All 12 rats entered the result analysis. ① After 4 weeks of femoral artery resection (6 weeks after gene transfection), the mean skin temperature of ischemic hindlimb in experimental group was significantly higher than that of control group (32.92 ± 0.63, 31.22 ± 0.87 ℃, P <0.05). Western blot results showed that the expression of CD151 in the ischemic side of the experimental group was significantly higher than that of the control group. ③ The capillary density of ischemic hindlimb muscle in the experimental group was significantly larger than that in the control group (609.00 ± 83.89, (517.00 ± 70.65) / mm2, P <0.05). ④ At 4 weeks after operation, the injury scores of limb function in experimental group were significantly lower than those in control group (0.67 ± 0.21, 1.67 ± 0.32, P <0.05). CONCLUSION: Local high expression of CD151 gene can improve the angiogenesis of ischemic tissue and promote the revascularization and functional recovery of ischemic limbs.