人受体活性修饰蛋白1基因修饰的骨髓间充质干细胞移植对兔血管成形术后新生内膜的影响

Effect of transplantation of hRAMP1 gene-modified bone marrow MSCs on postangioplasty neointima formation in rabbits

目的:探讨人受体活性修饰蛋白1(hRAMP1)基因修饰的骨髓间充质干细胞(MSCs)移植对动脉粥样硬化模型兔颈动脉球囊成形术后新生内膜增殖的影响。方法:密度梯度离心法加贴壁培养法获得MSCs,并以携带hRAMP1的腺病毒转染MSCs获得hRAMP1基因修饰的MSCs(hRAMP1-MSCs)。建立动脉粥样硬化狭窄并球囊损伤血管成形术的兔模型,随机分为hRAMP1-MSCs组、MSCs组和对照组,模型建立成功后经耳缘静脉分别注射携带pAd2-EGFP-hRAMP1或pAd2-EGFP的MSCs和PBS,于细胞移植后7 d、14 d和28 d分别处死动物取材,观察MSCs归巢、血管内皮化程度及内膜增生程度,同时检测血清血管内皮生长因子(VEGF)水平和血管局部内皮型一氧化氮合成酶(eNOS)表达。结果:细胞移植后不同时点hRAMP1-MSCs组和MSCs组损伤血管增生内膜均有CD31和增强型绿色荧光蛋白(EGFP)分布,但以hRAMP1-MSCs组内皮化程度明显,MSCs组次之,两者与对照组比较均有显著差异(P<0.05);细胞移植后不同时点hRAMP1-MSCs组和MSCs组内膜增生面积及再狭窄率均低于对照组(P<0.05),尤以hRAMP1-MSCs组降低明显;细胞移植后不同时点hRAMP1-MSCs组和MSCs组血清VEGF水平和血管eNOS表达较对照组增加(P<0.05),而hRAMP1-MSCs组又明显高于MSCs组(P<0.05);损伤血管新生内膜增殖细胞核抗原(PCNA)阳性表达在hRAMP1-MSCs组最少,对照组最多。结论:hRAMP1基因修饰的MSCs能更有效促进损伤内膜快速内皮化,重组hRAMP1腺病毒载体不影响MSCs向内皮细胞分化潜能。基因联合干细胞治疗能更好地促进损伤动脉快速内皮化,降低血管成形术后再狭窄。

AIM： To explore the effect of transplantation of human receptor activity-modifying protein 1（hRAMP1） gene-modified bone marrow mesenchymal stem cells（MSCs） on neointima formation after carotid balloon angioplasty in carotid atherosclerosis rabbits.METHODS： MSCs were collected through density gradient centrifugation and adherent culture.MSCs were transfected with adenovirus vector carrying hRAMP1 gene to generate hRAMP1 gene-modified MSCs（hRAMP1-MSCs）.All animals with carotid atherosclerosis and balloon angioplasty were randomly divided into hRAMP-MSCs group,MSCs group and control group.After the model was established,MSCs transfected with pAd2-EGFP-hRAMP1 or pAd2-EGFP and PBS were injected to the ear vein,respectively.The injured carotid arteries were harvested to detect the homing of MSCs,reendothelialization and neointima thickness 7 d,14 d and 28 d after cell transplantation.The plasma samples were collected for detecting vascular endothelial growth factor（VEGF） by ELISA.The expression of endothelial nitric oxide synthase（eNOS） in injured carotid arteries was measured by Western blotting.RESULTS： The expression of CD31 and EGFP was observed in the neointima at different time points in hRAMP1-MSCs group and MSCs group.Compared to control group,the reendothelialization of carotid significantly increased in both hRAMP1-MSCs group and MSCs group at different time points（P0.05）,and that in hRAMP1-MSCs group showed better than that in MSCs group（P0.05）.The area of neointima and the rate of restenosis were lower in hRAMP1-MSCs group and MSCs group than those in control group,and those in hRAMP1-MSCs group were significantly lower than those in MSCs group.The plasma level of VEGF and the expression of eNOS in the injured carotid arteries were significantly higher in both hRAMP1-MSCs group and MSCs group than those in control group at different time points（P0.05）,and those in hRAMP1-MSCs group were better than those in MSCs group（P0.05）.In the injured carotid arteries,the expression level of proliferating cell nuclear antigen（PCNA） in hRAMP1-MSCs group was the lowest,with the middle level in MSCs group and the highest level in control group.CONCLUSION： The hRAMP1 gene-modified MSCs are better in promoting reendothelialization and attenuating neointima than natural MSCs.The recombinant hRAMP1 adenovirus vectors don＇t affect the differentiation potential of MSCs into endothelial cells.These findings indicate that the modified stem cells have the potency of more effective reendothelialization to decrease restenosis after angioplasty.