骨髓间充质干细胞治疗慢性后肢缺血机理的初步研究

Mechanism of Mesenchymal Stem Cells Transplantation for Chronic Hindlimb Ischemia in Lewis Rat

目的利用细胞示踪技术探讨骨髓间充质干细胞(MSCs)治疗慢性后肢缺血的相关机理。方法采用密度梯度离心法结合直接贴壁法分离和培养大鼠MSCs,并以5-溴脱氧尿嘧啶核苷(BrdU)标记。采用线栓法制备8只Lewis大鼠慢性后肢缺血模型,将其随机均分为MSCs移植组和对照组,分别于患侧后肢肌肉注射MSCs和生理盐水。分别于移植术后第7天和第14天,对其进行临床观察、后肢血流量测定及后肢血管造影,再于相应时间点处死大鼠,取患侧后肢股四头肌和腓肠肌,行HE染色及BrdU免疫组化染色。结果移植术后14 d,8只大鼠全部成活,移植部位均无坏死和肿瘤形成;MSCs移植组大鼠患侧/健侧后肢的血流灌注比值明显增高(1.773比1.279),而血管造影结果提示2组大鼠的侧支血管数量比值未见显著增加(0.908比0.835)。HE染色结果示2组大鼠的股四头肌及腓肠肌并未发生特殊的病理学变化。BrdU免疫组化结果显示,阳性颗粒定位为股四头肌及腓肠肌的间质细胞和血管内皮细胞;且MSCs的分布存在差异,移植术后7 d腓肠肌内阳性细胞所占比例明显高于股四头肌,而14 d时则相反。结论 MSCs移植在术后早期可以提高血流灌注量,但这并非为增加了侧支血管数量使然,MSCs移植后所引起的旁分泌效应可能在术后早期起着重要的作用。

Objective To explore the mechanism of mesenchymal stem cells（MSCs）transplantation for chronic hindlimb ischemia in Lewis rats by using cell tracer technique.Methods MSCs were isolated and cultured by using density gradient centrifugation and adherence method respectively,then labeled by 5-bromo-2-deoxyuridine（BrdU）.Eight chronic hindlimb ischemia models of Lewis rats were prepared by using suture-occluded method and then divided randomly to MSCs transplantation group and control group,each group enrolled 4 rats,accepting MSCs transplantation and saline respectively.Then on 7 days and 14 days after transplantation,clinical observation,determination of blood flow,and angiography were performed on rats of the 2 groups.At the same time points after previous tests,rats of the 2 groups were sacrificed to get quadriceps tissues and gastrocnemius tissues to perform HE staining and BrdU immunohistochemistry.Results The 8 rats were all survived on 14 days after transplantation,with no tumor happened and necroses in the transplanted area.On 14 days after transplantation,the blood flow ratio of operated side to un-operated side in the hindlimb（1.773 vs.1.279）of rats in MSCs transplantation group and control group increased,and the angiography results