自体骨髓来源内皮祖细胞移植对移植静脉桥血管再内皮化及内膜增生的影响

Influences of autologous bone marrow derived endothelial progenitor cell transplantation on reendothelialization and intimal hyperplasia of vein grafts

目的:已有实验研究证明,移植骨髓来源内皮祖细胞可以促进球囊损伤后血管内皮的修复及抑制内膜的增生.那么移植骨髓来源内皮祖细胞对自体静脉移植术后静脉血管内皮修复和内膜增生是否起同样的作用?观察自体骨髓来源内皮祖细胞移植对自体静脉移植术后静脉桥血管再内皮化及内膜增生的作用。方法:实验于2007-01/08在北华大学附属医院内科实验室完成。实验室级别:P2级。①实验材料:6～8月龄雄性新西兰大白兔由解放军第二军医大学实验动物中心提供.体质量(2.5±0.5)kg,清洁级,实验过程中对动物处置符合动物伦理学标准。②实验方法:抽取成年兔骨髓分离制备骨髓单个核细胞,体外扩增法培养出骨髓来源内皮祖细胞.在培养第7天通过Ac-Dil-LDL、FITC-BS-1双染色及流式细胞仪检测CD34、CD133、FIK-1表达进行细胞鉴定,应用DAPI荧光染料体外标记培养7 d的骨髓来源内皮祖细胞、将成年新西兰大白兔23只随机分为细胞移植组(n=13)和对照组(n=10)进行自体左颈外静脉、颈总动脉移植术,在建模后第3天将DAPI标记的骨髓来源内皮祖细胞悬液经耳缘静脉植入细胞移植组动物体内,埘照组植入等量的PBS液③实验评估:细胞移植后4周.观察兔静脉移植段血管性及内膜厚度。结果:23只免均进入结果分析,无脱落:①通过体外扩增法培养的骨髓来源内皮组细胞,培养至7 d可见AC-Dil-LDI及FITC-BS-1双染色阳性细胞并表达CD34、CD133及FIK-1。②在呈绿荧光染色的血管内皮中可见有部分不均匀的散发蓝色荧光(DAPI标记细胞的细胞核)。③细胞移植组兔静脉血管内皮有DAPI标记的细胞.且内膜厚度明显低于对照组(P<0.05)结论:自体骨髓来源的内皮祖细胞移植可以促进损伤部位血管内皮的修复.并抑制内膜的过度增生。

AIM： Studies had verified that endothelial progenitor cells from bone marrow could repair the injured vascular endothelium and inhibit the intimal hyperplasia. Thus, whether the endothelial progenitor cells from bone marrow can repair the venous vascular endothelium and inhibit the intimal hyperplasia after autologous vein transplantation？ This article investigates the effects of autologous bone marrow derived endothelial progenitor cell transplantation on reendothelialization and intimal hyperplasia of vein grafts after autologous vein transplantation. METHODS： Experiments were performed at the Laboratory of Department of Internal Medicine of Hospital Affiliated to Beihua University from January to August 2007. The laboratory was in grade P2. ①Clean male New Zealand rabbits aged 6-8 months weighting （2.5±0.5） kg were provided by Experimental Animal Center of Second Military Medical University of Chinese PLA. Animal intervention met the animal ethical standard. ②Bone marrow was collected from adult rabbits to harvest mononuclear cells. Endothelial progenitor cells from bone marrow were incubated by in vitro amplification method. The ingestion of Ac-DiI-LDL and FITC-BS-1 was evaluated; The expression of cell marker CD133, CD34, FLK-1 was assessed using flow cytometry. Endothelial progenitor cells were stained with DAPI. Twenty-three adult rabbits were divided into a transplantation group （n =13） and a control group （n =10）. These rabbits received autologous left external jugular vein and common carotid artery transplantation. Three days later, rabbits in the cell transplantation group were transplanted with autologous endothelial progenitor cells via auricular veins. Rabbits in the control group were treated with the same volume of PBS. ③Four weeks after the transplantation, the grafted veins and the thickness of vein grafts were measured. RESULTS： Twenty-three rabbits were involved in the result analysis. ①Endothelial progenitor cells from bone marrow were positive for AC-DiI-LDI and FITC-BS-1 as well as CD34, CD133 and FIK-1 7 days after the transplantation. Uneven blue fluorescence （nuclei labeled by DAPI） partially appeared in endothelium showing green fluorescence. ② DAPI labeled cells appeared in the rabbit vein in the cell transplantation group, and the endometrium was thinner than the control group （P 〈 0.05）. CONCLUSION： Autologous endothelial progenitor cell transplantation may repair the injured vascular endothelium and inhibit the intimal hyperplasia.