外周血间充质干细胞移植对球囊损伤兔血管平滑肌细胞凋亡的影响

Effects of Peripheral Blood Mesenchyme Stem Cells Transplantation on Vascular Smooth Muscle Cell Apoptosis after Balloon-induced Artery Injury in Rabbits

探讨外周血间充质干细胞(MSCs)移植对模型兔颈动脉球囊成形术后平滑肌细胞凋亡的影响。粒细胞集落刺激因子(G-CSF)动员5 d后采集外周血,用密度梯度离心法结合反复贴壁法分离培养获得纯化的MSCs,以增强型绿色荧光蛋白(GFP)报告基因标记备用。建立兔动脉粥样硬化狭窄模型,随机分为MSCs移植组及对照组,于颈动脉球囊成形术后分别静脉注入MSCs或等体积培养液。术后7、14、28 d取球囊损伤血管标本经TUNEL法(TdT-mediated d-UTP nick end labeling)测定血管平滑肌细胞凋亡率;同时进行归巢MSCs的鉴定;此外,测定术后28 d的内膜、中膜面积,再狭窄率及再内皮化情况。术后7、14、28 d,与对照组比较,移植组平滑肌细胞凋亡率明显增高;仅在移植组血管组织中有GFP阳性细胞分布;术后28 d,移植组内膜面积、内膜中膜面积之比及再狭窄率显著低于对照组,而再内皮化程度则明显优于对照组。静脉移植外周血MSCs能够促进模型兔球囊损伤血管快速内皮化,抑制新内膜增生、减少再狭窄率;这一作用可能与损伤血管局部平滑肌细胞凋亡增加有关。

This study is purposed to investigate the effects of peripheral blood mesenchymal stem cells（PBMSCs） transplantation on vascular smooth muscle cell apoptosis in model rabbits after carotid balloon angioplasty.Bone marrow cells were mobilized by granulocyte colony-stimulating factor（G-CSF） subcutaneously for 5 days.The peripheral blood was gathered in the sixth day.Purified PBMSCs were obtained by density gradient centrifugation and adherent culture,and labeled by enhancement type green fluorescent protein（GFP） reporter genes.Rabbits undergoing carotid atherosclerostenosis were randomly divided into two groups,transplanted group（n = 30） was received intravenous transplantation of PBMSCs and the control group（n = 30） was only received intravenous culture solutions following right common carotid balloon angioplasty.The animals were killed 7、14、28 days after operation,respectively and the blood vessels specimen was taken for in situ detection of smooth muscle cell apoptosis.The TUNEL-technique（TdT-mediated d-UTP nick end labeling） was used.Simultaneously,the transplanted cells’homing to the injured area were detected by immunostaining of green fluorescent protein（GFP）.Morphometric analysis of injured carotid artery intima hyperplasia and lumina stenosis,immunohistochemisty analysis of injured carotid artery reendothelialization were performed 28 days after operation.There were only GFP-positive cells in the injured carotid artery area in the transplanted group and vascular smooth muscle cell apoptosis ratio significantly increased compared with the control group（P 0.01） in every time section.Neointimal thickening was observed in all balloon-injury vessels.Transplanted group neointimal area and restenosis ratio significantly reduced versus the control group 28 days after the operation（P 0.01）.Compared with the control group the injured carotid artery,reendothelialization significantly increased 28 days after the operation（P 0.01）.Intravenous transplantation of PBMSCs might accelerate reendothelialization and attenuate neointimal thickening and restenosis in rabbits after carotid balloon angioplasty.The effects probably attributed to the increase of smooth muscle cell apoptosis during injured vessels reconditioning.