肝细胞生长因子基因修饰的骨髓间充质干细胞对糖尿病大鼠下肢缺血肌肉损害的保护作用

Gene on Lower Limb Ischemia of Muscle Injury in Rats with Diabetes

目的观察携带人肝细胞生长因子(hepatocyte growth factor,HGF)基因的重组腺病毒(Ad-HGF)修饰后的骨髓间充质干细胞(mesenchymal stem cells,MSCs)对糖尿病大鼠下肢缺血肌肉组织的影响。方法体外分离、培养大鼠骨髓MSCs,以最佳感染强度转染Ad-HGF后备用。健康Wistar大鼠,给予高脂、高糖饲料饲养加小剂量STZ腹腔注射建立实验性2型糖尿病大鼠模型,经1%戊巴比妥钠麻醉后无菌手术暴露左侧后肢股动脉起始端及其分支并完全结扎,造成急性后肢缺血。并将模型大鼠随机分为Ad-HGF修饰的骨髓MSCs治疗组(A组),溶剂对照组(B组),每组8只。制备缺血模型后A组将Ad-HGF修饰的骨髓MSC、B组将PBS于术后10 min内行手术部位多点肌内注射。2组于手术注射后6周,取结扎后肢内与外侧肌肉组织,常规组织病理切片染色,在光学显微镜下观察其组织病理学变化。结果 B组大鼠因股动脉被结扎而使血运严重受到干扰的患肢肌肉组织都发生了显著的病理改变,肌纤维变性、断裂、肌束内水肿、脂肪性变,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;A组上述病变不明显。结论 Ad-HGF修饰的骨髓MSCs的局部应用可减轻或改善糖尿病大鼠下肢缺血后肌肉组织损伤,提示AdHGF修饰的骨髓MSCs对糖尿病大鼠下肢缺血性肌肉损伤具有明显的保护作用。

Objective To observe effects of mesenchymal stem cells （MSCs） transfected with recombinant of adenovirus vector （Ad-HGF） to human hepatocyte growth factor （HGF） gene on lower limb ischemia of muscle tissue in rats with diabetes. Methods Marrow MSCs of rats were isolated, cultured and transfected with the best infected inten- sion of Ad-HGF. Type 2 diabetic models were established with Wistar healthy rats, which were fed on high fat and glu- cose and given low dose of Streptozotocin （STZ） by intraperitoneal injection. Sixteen diabetic model rats were intrave- nously anesthetized with 1% Pentobarbital Sodium, the distal end and branches of the left femoral artery were completely ligated in aseptic operation, then acute left lower hindlimb ischemia model was established. The established model rats were randomly divided into marrow MSCs transfected with Ad-HGF group （ group A, n = 8 ） and phosphate buffered saline （PBS） control group （group B, n = 8）. After ischemia models were successfully established, the suspensions of marrow MSCs transfected with Ad-HGF （group A） and PBS （group B） undertook intramuscular injection in many operative sites respectively within postoperative 10 min. Six weeks after injection, muscle tissues of inside and lateral ligated hindlimbs undertook routine pathological section-staining, and histopathology changes were observed under optical microscope. Re- suits In group B, muscle tissues of ligated hindlimbs had significant histopathology changes because of severely interfe- rential hemokinesis, such as myofibrosis, breakage, edema in muscle bundles, fat deterioration, thickening arterial ves- sel wall among muscle bundles, decrescent or disappeared lumens, and few neonatal small vessels ; in group A there were no significant pathological changes. Conclusion The local injection of marrow MSCs transfected with Ad-HGF therapy can relieve or improve the injuried muscle tissues of lower limb ischemia in rats with diabetes, which has obvious protec- tive effect on injured muscle tissues.