骨髓间充质干细胞联合血管内皮生长因子移植治疗糖尿病兔下肢缺血的实验研究  

Experimental Study on the Treatment of Lower Limb Ischemia of Diabetic Rabbits by Injecting Bone Marrow Mesenchymal Stem Cells and Vascular Endothelial Growth Factor-loaded Nanoparticles

作　　者：杨亚冬[1] 张文元[1] 赵佳骏[1] 张科技[1] 房国坚[1] 

机构地区：[1]浙江省医学科学院生物工程研究所,杭州310013

出　　处：《医学研究杂志》2015年第6期47-51,共5页Journal of Medical Research

基　　金：浙江省医药卫生科学研究计划项目(2013KYA045);浙江省医学重点学科群项目(XKQ-010-001)生物医药重点学科群

摘　　要：目的用包裹血管内皮生长因子(VEGF)的纳米球与骨髓间充质干细胞(BMSCs)共同多点肌内注射到糖尿病兔下肢缺血部位,以期使糖尿病兔下肢缺血症状改善,缺血部位形成新的血管。方法通过密度梯度离心法及贴壁培养法分离纯化BMSCs。采用超声乳化-溶剂挥发法制备包载VEGF的PLGA纳米球,并检测其各指标。按100mg/kg耳缘静脉注射四氧嘧啶制备糖尿病兔模型,14天后空腹血糖>16mmol/L确定为糖尿病模型。成模的糖尿病兔双侧下肢股动脉结扎制作下肢缺血模型,并分4组在股动脉结扎离断区进行多点注射治疗。PBS组、BMSCs组、VEGF组和BMSCs+VEGF组,同时设正常对照组。主要观察指标:治疗后各组兔子活动情况、大体解剖情况及下肢缺血区血管形成情况。结果制备的PLGA纳米粒基本呈球形,分散较好,平均粒径为438.97±15.60nm。载VEGF的PLGA纳米粒包封率为87.4%,载药量为43.7μg/g。24h释放率55.48%。共选用28只雌兔制作糖尿病模型,死亡14只,成模12只,2只血糖没达到糖尿病模型标准。死亡率为50%,模型成功率为42.86%,不敏感率7.14%。移植后1个月发现注射PBS组的1只兔子胫骨有一1.0cm×1.5cm的溃疡,行走时呈拖行,另2只呈跛行。注射VEGF组3只兔子均呈跛行。注射MSC组有2只跛行,1只行走正常。注射BMSCs+VEGF组2只行走正常,活动自如,仅1只略呈跛行。从大体解剖结果及切片结果都可以看出移植BMSCs组、VEGF组及BMSCs+VEGF组在结扎股动脉后,可形成侧支循环,使股动脉远端保持充盈,血供良好,尤其是移植BMSCs+VEGF组形成的侧支较多。而PBS治疗组股动脉远端未见充盈,只有回流的股静脉增粗。结论用包裹VEGF的PLGA纳米球与BMSCs共同多点肌内注射到糖尿病兔下肢缺血部位,对糖尿病兔下肢缺血区血管有促进生成作用,能改善动物模型下肢缺血的症状。Objective To explore the treatment effects of transplantation of vascular endothelial growth factor （VEGF） and bone marrow mesenchymal stem cells （BMSCs） on the lower limb ischemia in diabetic rabbits. Methods Bone marrow mesenchymal stem cells （BMSCs） were isolated and purified by a combination of density gradient centrifugation and adhesive culture in vitro. PLGA nanop- articles loaded with VEGF were prepared by ultrasonic emulsification- solvent evaporation method. Alloxan was intravenously injected 100mg/kg body weight to make rabbit model with diabetes mellitus. The standard of diabetic model was fasting glucose 〉 16mmol/L on the 14th day. Then both sides of the diabetic rabbits＇ femoral arteries of lower limbs were ligated and cut to built hind limb ischemia model. Rabbits were randomly allocated into four groups for transplantation therapy, PBS group, VEGF group, BMSCs group, and VEGF ＋ BM- SCs group. Main outcome measures were, each rabbit＇s activity , gross anatomy and angiogenesis in lower limb ischemic regions. Results The VEGF-loaded PLGA nanoparticles were spherical in shape. The mean diameter was 438.97 ± 15.60nm. The entrapment efficiency was 87.4% and medicine loading was 43.7μg/g. Accumulative release quantity in 24 hour was 55.48%. In total of 28 rabbits, the mor- tality was 50% , the model-forming rate was 42.86% , the insensitive rate was 7.14% . In PBS group, there was a 1.0cm × 1.5cm ulcer on one rabbit＇s tibia 1 month after transplantation. Their hind limbs were weakness and only could drag their legs for walking or limp. In YEGF group and BMSCs group, all rabbits had a significant limp except that one could walk unrtestricted which injected with BMSCs. In BMSCs ＋ VEGF group, two rabbits walked normally, the other one walked a little limp. The gross anatomy and pathological sections ex-hibited that collateral vessels were significantly increased and distal femoral artery kept filling in BMSCs group, VEGF group, and BMSCs ＋ VEGF group compared with PBS group, esp

关 键 词：骨髓间充质干细胞 血管内皮生长因子(VEGF) 糖尿病 下肢缺血 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]