pUDKH基因治疗犬下肢缺血后对血管、肌肉组织的影响(一)

详细内容

作者：哈小琴，赵治华，吕同德，李元敏，任建平，吴祖泽

【关键词】 pUDKH基因；血管；肌肉；组织病理

　　【Abstract】 AIM: To observe the effects of pUDKH administration locally on blood vessels and muscles in dogs with hindlimb ischemia. METHODS: Dogs were anesthetized with an intravenous injection of sodium pentobarbital (30 mg/kg). Then the left external iliac artery as well as all of the above arteries were ligated pletely with silk thread. Thus the pletely ischemic hindlimb model was established. The dogs with pletely ischemic hindlimb were randomly divided into 2 groups: control group (control vector pUDK) and HGF transferred group (pUDKH), 4-6 dogs each. Then pUDKH or pUDK was injected directly into the left ischemic limb muscles. Three months (90 d) after injection, left external iliac arteriography was performed, blood flow rate of the left femoral artery was quantified and the temperature of distal muscle in hindlimb was measured. At the same time,the left femoral artery and femoral around muscles at the sites of injection of pUDKH and pUDK plasmids were removed and stained with HE to observe their histopathological changes by light microscopic examination. RESULTS: The degree of augmentation of collateral vessel formation was significantly greater than that treated by control vector in hindlimb ischemia models. In addition, the blood flow rate of femoral artery in dogs treated with pUDKH was recovered on day 90, while the flow rate was only 1/5 to 1/3 in control dogs. Degeneration and breakage of muscle fibers and edema among muscle bundles were observed in ischemic limb of control dogs, but in ischemic hindlimb transfected with pUDKH, no significant pathological changes were found. For control dogs, collapse of the left femoral artery, thickening of vessel wall and stenosis even olusion of small blood vessel among muscle bundles were observed, few new blood vessels were found. But in pUDKH dogs, above all were almost normal. CONCLUSION: pUDKH has a protective effect on blood vessels and muscles in dogs with hindlimb ischemia.

　　【Keywords】 pUDKH；artery；muscle；histopathology

　　【摘要】 目的: 观察pUDKH(携带肝细胞生长因子基因的质粒)基因治疗犬下肢缺血后对其血管和肌肉组织病理学的影响. 方法: 杂种犬在静脉内注射苯巴比妥钠麻醉下，于左后侧腹股沟在股动脉由髂外动脉分支处的起始端进行全结扎，造成肢体缺血模型. 模型犬被随机分为对照组和pUDKH处理组，并在结扎后即刻分别注射pUDK（空白质粒）或pUDKH. 于手术后3 mo，行左髂外动脉造影术，测定股动脉血流量和下肢远端局部温度，并从被结扎的后肢股动脉及内外侧肌肉取组织标本，经常规组织病理切片染色，光学显微镜下观察. 结果: 3 mo时血管造影pUDKH组可见明显血管网的形成，而转移空质粒的对照组仅见少量新生血管；此时转移pUDKH组犬股动脉血流量已恢复到结扎前的水平，而对照组股动脉血流量仅为结扎前的1/5~1/3；转移pUDKH组结扎侧肌肉温度与健侧相比无明显差异，而对照组两肢体肌肉温度却相差1~2℃. 组织学观察对照犬可见结扎点远端股动脉塌陷，肌束间动脉管壁变厚，管腔变小甚至消失，少见新生的小血管；pUDKH处理组犬结扎点远端股动脉管腔开放，肌束间小动脉壁均普遍未见增厚，管腔亦未缩小；对照犬结扎侧肌纤维变性、断裂、肌束内水肿、脂肪性变，pUDKH处理组结扎侧的肌肉组织中上述病变不明显. 结论： pUDKH的局部注射对犬下肢缺血后血管和肌肉具有保护作用.

　　【关键词】 pUDKH基因；血管；肌肉；组织病理

　　0引言

　　下肢缺血主要是血栓闭塞性脉管炎或糖尿病性下肢中小血管病变、周围神经病变等所造成的下肢慢性缺血，涉及血管、神经、肌肉等组织的病变，当病变进展而减少了动脉的横截面积至80%或更多时，患者表现间歇性跛行、功能障碍,甚至肢体坏死（坏疽）. 目前对此尚无理想的药物治疗方法，最终需行截肢手术而致残，预后较差〔1-4〕. 近年来基因治疗为其开辟了一条新的途径，通过合适的载体介导，转移促血管生长因子的基因于缺血组织，可促进新血管在局部的产生，形成血管“网”和血管“桥”，建立“分子搭桥”机制.

　　人肝细胞生长因子（hepatocyte growth factor, HGF）是一多功能生长因子，除促进血管新生外〔4〕，对器官/组织的损伤有一定的修复作用〔5〕，包括肌肉、神经等. 本文利用犬后肢结扎股动脉造成血管闭塞性疾病的动物模型，采用股动脉周围骨骼肌肉内多点注射携带HGF基因的裸质粒pUDKH方法，探讨其治疗犬肢体缺血时对其血管和肌肉组织病理学的影响，为局部pUDKH基因治疗的实际应用提供实验依据.

　　1材料和方法

　　1.1材料

　　动物：河北地区草狗18只，雄性，年龄为1.5~3岁，体质量12~15 kg，由军事医学科学院动物实验中心提供. pUDKH: 携带人肝细胞生长因子基因的重组裸质粒, pUDK: 未携带人肝细胞生长因子基因的空质粒, 军事医学科学院放射医学研究所三室提供.

　　1.2方法

　　1.2.1动物模型的建立犬经15 g/L戊巴比妥钠静脉麻醉（30 mg/kg）后. 在无菌条件下分离左侧髂外动脉远端及分支和股动脉近端，将髂外动脉远端及股动脉分支全部结扎，造成左下肢血管完全闭塞性血管病模型. 在股动脉处安置血流量计探头，左外侧肌肉插入THRNST针型体温度电极，测定股动脉完全闭塞前、后10 min内股动脉血流量及左、右外侧肌肉温度，以判断左侧髂外动脉远端已完全闭塞. 右侧下肢动脉不予结扎.

　　1.2.2基因转移将模型动物随机分为4组：模型对照组（给予不携带HGF基因的空质粒pUDK 0.30 mg/kg (6只）,pUDKH 0.15 mg/kg组(4只),pUDKH 0.30 mg/kg组(4只)和pUDKH 0.60 mg/kg组(4只). 按每公斤体质量给犬的药量，将pUDK或pUDKH稀释在1 mL生理盐水中，于手术后10 min内，在左大腿肌内侧注射3点，左大腿肌外侧注射2点，每点注射体积为0.2 mL，最后逐层关闭伤口. 以后的饲养过程中不再进行基因转移.

　　1.2.3动脉血管造影每组全部犬的术后90 d，于右侧股动脉内插管行至髂总动脉端，行左髂外动脉造影术，导管尾端连接一高压注射器快速注入泛影葡胺，拍摄X光片以观察新生血管和侧支循环的形成情况.

　　1.2.4股动脉血流量及肢体温度测定在术前和术后0 d，90 d各测定股动脉血流量和下肢远端局部温度一次.