腹主动脉移植后小鼠外周血内皮祖细胞数量的动态变化及意义

目的 观察腹主动脉移植后小鼠移植物动脉硬化的病变过程及外周血内皮祖细胞(EPC)数量的动态变化,探讨EPC与动脉内膜损伤和修复的相互关系.方法 以C57BL/6小鼠和Balb/c小鼠为供、受者,建立腹主动脉原位移植后移植性模型.术后3 d、2周、4周、6周,观察移植动脉病理改变,并采用计算机图像分析系统分析移植血管内膜增生情况.使用流式细胞仪监测术后外周血中EPC数量的变化.结果 术后3 d,移植动脉内皮细胞损伤,并伴有明显的炎症细胞浸润.术后2周,即可观察到移植动脉新生内膜形成,存在急性排斥反应;术后4周、6周内膜厚度逐渐增生,移植动脉管腔明显狭窄.术后早期外周血中EPC的数量增多,3 d时达到高峰,此后迅速减少,术后14和28 d时,显著低于术前水平(P＜0.05).结论 通过同种小鼠腹主动脉原位移植可成功复制出移植物动脉硬化的病变特点;外周血中EPC的数量与移植动脉内膜损伤的修复密切相关,可能成为移植物动脉硬化的发病指标和干预靶点.

Abstract：

Objective To investigate changes in the number of endothelial progenitor cells (EPC) from peripheral blood and pathological feature in the development of transplant arteriosclerosis in mouse abdominal aortic allografts, and discuss their correlations. Methods A segment of abdominal aorta was transplanted orthotopically from C57BL/6 to Balb/c mice. The grafts were harvested at 3rd day, 2nd week, 4th week and 6th week after the operation and studied by light and electronic microscopy. Regional changes in the lumen and intima were measured with computer imaging analysis system. EPC from peripheral blood were quantified by flow cytometry. Results Endothelium injury and inflammatory cells infiltration were seen in the aortic allografts at 3rd day after transplantation.Neointimal lesions and acute rejection were observed as early as 2nd week after surgery. The lumen of allografts was significantly narrowed due to neointima hyperplasia and had progressed at 4th and 6th week postoperatively. The number of circulation EPC was increased from 1 st day after operation and reached the peak at 3rd day. Thereafter the number of EPC was decreased rapidly and significantly less at 14th and 28th day postoperation than that pre-operation. Conclusion Abdominal aortic transplantation from C57BL/6 to Balb/c mice presents typical pathological feature of transplant arteriosclerosis. The number of EPC from peripheral blood is related to the process of injured endothelial repair and neointima formation of aortic grafts. EPC count may be considered a novel biological marker and therapeutic intervention for transplant arteriosclerosis.     

腹主动脉移植后小鼠外周血内皮祖细胞数量的动态变化及意义

目的 观察腹主动脉移植后小鼠移植物动脉硬化的病变过程及外周血内皮祖细胞(EPC)数量的动态变化,探讨EPC与动脉内膜损伤和修复的相互关系.方法 以C57BL/6小鼠和Balb/c小鼠为供、受者,建立腹主动脉原位移植后移植性模型.术后3 d、2周、4周、6周,观察移植动脉病理改变,并采用计算机图像分析系统分析移植血管内膜增生情况.使用流式细胞仪监测术后外周血中EPC数量的变化.结果 术后3 d,移植动脉内皮细胞损伤,并伴有明显的炎症细胞浸润.术后2周,即可观察到移植动脉新生内膜形成,存在急性排斥反应;术后4周、6周内膜厚度逐渐增生,移植动脉管腔明显狭窄.术后早期外周血中EPC的数量增多,3 d时达到高峰,此后迅速减少,术后14和28 d时,显著低于术前水平(P＜0.05).结论 通过同种小鼠腹主动脉原位移植可成功复制出移植物动脉硬化的病变特点;外周血中EPC的数量与移植动脉内膜损伤的修复密切相关,可能成为移植物动脉硬化的发病指标和干预靶点.     

腹主动脉移植后小鼠外周血内皮祖细胞数量的动态变化及意义

目的 观察腹主动脉移植后小鼠移植物动脉硬化的病变过程及外周血内皮祖细胞(EPC)数量的动态变化,探讨EPC与动脉内膜损伤和修复的相互关系.方法 以C57BL/6小鼠和Balb/c小鼠为供、受者,建立腹主动脉原位移植后移植性模型.术后3 d、2周、4周、6周,观察移植动脉病理改变,并采用计算机图像分析系统分析移植血管内膜增生情况.使用流式细胞仪监测术后外周血中EPC数量的变化.结果 术后3 d,移植动脉内皮细胞损伤,并伴有明显的炎症细胞浸润.术后2周,即可观察到移植动脉新生内膜形成,存在急性排斥反应;术后4周、6周内膜厚度逐渐增生,移植动脉管腔明显狭窄.术后早期外周血中EPC的数量增多,3 d时达到高峰,此后迅速减少,术后14和28 d时,显著低于术前水平(P＜0.05).结论 通过同种小鼠腹主动脉原位移植可成功复制出移植物动脉硬化的病变特点;外周血中EPC的数量与移植动脉内膜损伤的修复密切相关,可能成为移植物动脉硬化的发病指标和干预靶点.     

内皮祖细胞移植治疗应用及作用机制

背景 最近的研究表明内皮祖细胞(endothelial progenitor cells,EPCs)在维持正常内皮功能和修复损伤血管内皮发挥重要的作用.目的 分析和总结了近年来EPCs移植治疗应用的现状,同时探讨了EPCs可能的作用机制.内容 描述了EPCs的生物学特性;总结了EPCs移植治疗的应用现状;探讨了EPCs...     

人外周血内皮祖细胞培养及特性

目的 探讨人外周血来源的内皮祖细胞(EPCs)体外诱导培养和扩增的方法，以期证实人外周血是临床治疗缺血性疾病一个理想的内皮祖细胞来源。方法 采集正常人外周血单个核细胞进行体外培养，应用免疫组织化学和免疫荧光技术进行内皮细胞表面标记物检测，应用细胞荧光化学检测进行培养细胞的内皮细胞功能检测。结果 人外周血单个核细胞(MNCs)经体外培养，表达内皮细胞的特异性抗原，包括Ⅷ因子(VWF)、FLK-1、CD31和CD34，并显示出能内吞乙酰低密度脂蛋白(acLDL)，结合UEA-1等内皮细胞的特性，提示这些培养细胞具有内皮细胞的表面蛋白特性和具有内皮细胞的功能。结论 人外周血中存在具有分化增殖能力的EPCs，可体外扩增和收集。     

HSCT联合不同剂量内皮祖细胞输注对小鼠造血重建的影响

目的 探讨异基因造血干细胞移植联合不同剂量内皮祖细胞(EPC)输注对移植后造血重建的影响.方法 以C57BL/6小鼠为供鼠,Balb/c小鼠为受鼠,进行HSCT,输注骨髓单个核细胞数量为5×106个/只.仅进行HSCT者为单纯骨髓细胞移植组;行HSCT的同时经尾静脉输注供者骨髓单个核细胞诱导培养的EPC的受鼠为EPC联合移植组,EPC的输注量分别为5×104、1×105、5× 105和1×106个/只.另设正常对照组和致死量照射组.观察小鼠的存活率、造血重建情况及骨髓微环境的变化.结果 各EPC联合移植组小鼠存活时间长于单纯骨髓移植组,5×105 EPC联合移植组至观察结束时存活率为100％,高于其他各组(P＜0.05).移植后10和15 d,5×105 EPC联合移植组外周血白细胞数量高于其他组(P＜0.05).移植后15 d,5×105 EPC联合移植组外周血血小板数量高于其他组(P＜0.05).5×105 EPC联合移植组造血组织增生程度也好于其他组.5×105 EPC联合移植组骨髓内HSC比例为(1.06±0.03)％,高于其他各组(P＜0.05).结论 小鼠异基因骨髓移植中联合输注5×105 EPC能够有效促进造血重建,提高小鼠存活率.     

血管内皮祖细胞在人工血管移植后内皮化中的作用机制

血管内皮祖细胞具有高度的分化增殖能力,可以定向分化为成熟内皮细胞,参与体内内皮损伤后的修复和血管再生。研究表明血管内皮祖细胞在人工血管移植后内皮化中也起重要作用,现就近年来的研究进展综述如下。     

血管内皮祖细胞促进游离移植脂肪颗粒组织存活的实验

目的探讨血管内皮祖细胞（endothelial progenitor cell，EPC）移植促进游离移植的脂肪颗粒组织的血管新生，提高移植脂肪颗粒组织存活率。方法体外分离、培养人脐血中EPCs，然后与来自人体的脂肪颗粒组织混合移植于裸鼠背部。结果脐血中分离培养的EPCs表达血管内皮细胞生长因子受体（KDR）及细胞表面标记CD34、CD133，EPCs与脂肪颗粒组织混合移植到裸鼠3个月后，EPCs整合到缺血部位新生血管中，与对照组的脂肪颗粒组织存活率分别为（89．3±6．8）％、（42．2±2．5）％（P〈0．05），而且实验组与对照组脂肪颗粒组织周边区毛细血管密度差异有统计学意义（P〈0．05）。术后3个月2组脂肪颗粒组织周边区的EPCs密度分别为（95．2±10．5）个／mm^2、0个／mm^2（P％O．05）。结论体外培养的脐血EPCs移植体内可促进游离移植的脂肪颗粒组织的血管新生，提高存活率。     

联合内皮祖细胞移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用

目的 研究联合内皮祖细胞(EPC)移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用.方法 将C57BL/6小鼠分为4组,每组10只.(1)正常对照组:小鼠不做任何处理,仅作为正常对照;(2)单纯照射组:单次给予全身照射(TBI)预处理,不进行骨髓移植;(3)单纯移植组:给予单纯照射组相同的TBI预处理,TBI后4 h内经小鼠尾静脉输注C57BL/6小鼠骨髓单个核细胞5×106/只;(4)联合移植组,小鼠的处理方式与单纯移植组相同,仅在骨髓移植的同时经尾静脉输注C57BL/6小鼠EPC 5×105/只.TBI后第2、4、7、14、21天,检测各组小鼠肝脏重量的变化情况,并于TBI后第4、7、14、21天对各组小鼠肝脏进行组织病理学检查.结果 单纯照射组、单纯移植组和联合移植组小鼠肝脏重量均于TBI后第2天开始明显增加,于第14天达到高峰,峰值分别为正常对照组的(1.65±0.15)倍(P＜0.05)、(1.61±0.06)倍(P＜0.05)和(1.11±0.4(0)倍(P＜0.05);以后均呈下降趋势,第21天时单纯照射组和单纯移植组肝脏重量仍明显高于正常对照组(P＜0.05),但联合移植组小鼠肝脏重量已完全恢复正常.组织病理学检查显示,单纯照射组小鼠肝窦内皮损伤明显,肝细胞水肿及严重的炎症细胞浸润,第7天时肝细胞水肿、坏死较前明显加重,几乎无存活的肝血窦内皮细胞;第14天时单纯移植组小鼠肝窦内皮损伤较前有所减轻,但到第21天时仍未恢复正常;联合移植组小鼠第7天时肝窦内皮及肝细胞水肿、坏死程度均较轻,到第14天时已基本恢复正常.结论 造血干细胞移植前的预处理会造成受者肝脏内皮损伤,且此损伤持续存在;移植时联合输注EPC能修复肝窦内皮的损伤.

Abstract：

Objective To study the repair function of united endothelial progenitor cells (EPC)transplantation on injured liver endothelium by bone marrow transplantation (BMT) conditioning.Methods C57BL/6 mice were divided into four groups randomly: normal control group, without any treatment; irradiation alone group, administered a total body irradiation(TBI) pretreatment, without BMT; (3) BMT alone group: C57BL/6 mice were infused with bone marrow mononuclearcells (MNC) 5 × 106/only through caudal vein not more than 4 h after the same TBI pretreatment as the irradiation alone group; united transplantation group: receiving the same way as the BMT alone group, but C57BL/6 mice were infused with EPC 5 × 105/only at the same time. Two, 4, 7, 14, and 21 days after the TBI, the changes of the liver weight were observed regularly. The histopathological examination of liver was done at the 4th, 7th, 14th, and 21st day after the TBI. Results In irradiation alone group, BMT alone group and united transplantation group the liver weight began to increase significantly on the day 2 and peaked at 14th day after the TBI, and the peaks were respectively (1.65±0. 15) times (P＜0. 05), (1.61 ±0.06) times (P＜0.05), and (1.11 ±0.40)times (P＜0. 05) of those in normal control group. At the day 14, the liver weight in irradiation alone group, BMT alone group and united transplantation group began to decrease, and on the day 21 the liver weight in united transplantation group had been completely restored to normal level, however the liver weight in irradiation alone group and BMT alone group were still significantly heavier than that in normal control group (P＜0. 05). Liver histopathological examination revealed that there were obvious sinusoidal endothelial cells (SEC) injury, hepatocyte edema and severe inflammatory cell infiltration in irradiation alone group, and on the day 7 the hepatocyte edema and necrosis were significantly worse than before, and almost no alive SEC were found. On the day 14 the injury of SEC in BMT alone group was lighter than before, but on the day 21 the injury had not returned to normal. On the day 7 the injury of SEC, hepatocyte edema and necrosis were alleviated in united transplantation group as compared with irradiation alone group and BMT alone group, and on the day 14 the injury had returned to normal basically. Conclusion The transplantation conditioning could damage recipient liver endothelium and the injury would persist, and united EPC infusion could repair the injured SEC following BMT.     

Differentiation and proliferation of endothelial progenitor cells from canine peripheral blood mononuclear cells

BACKGROUND: The isolation, differentiation, and expansion of endothelial progenitor cells (EPCs) from peripheral blood have potential applicability in areas of therapeutic neovascularization, vascular repair, and tissue engineering. The purpose of the current study was to elucidate a simple method of isolation and differentiation of EPCs by defining the endothelial morphology, surface marker expression, and proliferative capacity of EPC outgrowth from canine peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: PBMCs were isolated from fresh canine blood and cultured in fibronectin-coated plates in which EPCs were identified from cell morphology and outgrowth characteristics. Cell surface markers were determined with flow cytometry analysis to identify differentiation of cultured and subcultured colonies. A hematologic counter with phase contrast microscopy was used to study cell growth curves of EPCs as compared with mature human coronary artery endothelial cells. RESULTS: During the first week of canine PBMC culture, cells were morphologically round and varied in size, but in the course of the second and third week of culture, the cells, respectively, became spindle-shaped and displayed an endothelium-like cobblestone morphology with outgrowth. CD34 was significantly decreased at 21 days as compared with 7 days culture (36.04% to 21.37%), whereas vWF (from 77.26% to 96.37%) and eNOS (from 0% to 14.97%) were significantly increased. VEGFR-2 was slightly increased, and P1H12 (CD146) was unchanged. Subcultured canine EPCs displayed a higher proliferation rate as compared to mature human coronary artery endothelial cells in the same culture conditions. CONCLUSIONS: These data demonstrate that canine EPCs can be isolated and cultured from the canine PBMC fraction. These outgrowth cells displayed characteristics of endothelial morphology with endothelial cell-specific surface markers. Furthermore, it was revealed that canine EPCs have a greater growth potential as compared to mature endothelial cells. This study suggests that PBMCs could be used as a source of EPCs for potential applications in tissue engineering and vascular therapy.     

鼠外周血内皮祖细胞体外培养鉴定与诱导分化

目的探讨鼠外周血来源的血管内皮祖细胞（EPCs）体外培养鉴定及诱导分化的方法。方法从鼠外周血中分离获取EPCs,进行体外培养扩增,并在培养液中添加VEGF及bFGF,采用免疫组织化学技术和流式细胞仪进行EPCs的细胞表面标志物检测。结果培养3-4d,可观察到梭形贴壁细胞,14d左右贴壁细胞呈条索状结构,贴壁细胞是表达血管内皮细胞的特异性标志。结论鼠外周血中含有EPCs,在一定条件下,可分化成血管内皮细胞。     

鼠外周血内皮祖细胞体外培养鉴定与诱导分化

目的探讨鼠外周血来源的血管内皮祖细胞(EPCs)体外培养鉴定及诱导分化的方法。方法从鼠外周血中分离获取EPCs,进行体外培养扩增,并在培养液中添加VEGF及bFGF,采用免疫组织化学技术和流式细胞仪进行EPCs的细胞表面标志物检测。结果培养3~4d,可观察到梭形贴壁细胞,14d左右贴壁细胞呈条索状结构,贴壁细胞是表达血管内皮细胞的特异性标志。结论鼠外周血中含有EPCs,在一定条件下,可分化成血管内皮细胞。     

红细胞生成素对慢性肾衰竭大鼠外周血内皮祖细胞的影响

目的 探讨红细胞生成素（EPO）对慢性肾衰竭大鼠外周血内皮祖细胞（EPC）数量和功能的影响。 方法 采用分阶段5/6肾切除制备大鼠慢性肾衰竭模型。实验动物按数字随机表法分为4组（均n = 7）：假手术组、慢性肾衰竭组（模型组）、30 U/kg EPO干预组（小剂量组）和50 U/kg EPO干预组（大剂量组）。大鼠皮下注射EPO 6周后,取其外周血分离培养EPC,并检测EPC数量及其增殖、黏附和形成血管结构的能力。 结果 与假手术组比较,慢性肾衰竭大鼠外周血EPC数量及其增殖、黏附与形成血管结构的能力均显著下降（均P < 0.05）。应用EPO治疗能显著增加慢性肾衰竭大鼠外周血EPC数量（P < 0.05）,改善外周血EPC增殖、黏附及形成血管结构的能力（均P < 0.05）,并且呈剂量依赖性。 结论 EPO可改善慢性肾衰竭大鼠外周血EPC的数量和功能。     

大鼠外周血内皮前体细胞体外程序化培养的研究

目的探讨从大鼠外周血中获取内皮前细胞的方法，为缺血性心脏病细胞移植治疗提供新的移植材料。方法首先应用密度梯度离心法分取大鼠外周血中的单个核细胞，然后以IMEM、胎牛血清、马血清、VEGF、bFGF等混合而成的培养基，进行体外培养。最后将此方法收获的细胞经一系列免疫荧光染色，并检测其结合UEA-1、摄取acLDL的能力。结果细胞CD31、CD34、Flk-1和vWF免疫荧光染色阳性，并能结合UEA-1、摄取acLDL。同时结合细胞的形态学变化特点，证实得到的细胞为内皮前细胞。结论通过一定的体外分离、培养途径，可以从大鼠外周血获得较为纯化的内皮前细胞。     

急性胰腺炎时骨髓和循环内皮祖细胞的变化研究

目的 探讨急性胰腺炎时骨髓及循环内皮祖细胞(EPCs)的数量变化及机制.方法 将BALB/c小鼠随机分为对照组和腹腔注射雨蛙素组,腹腔注射后12 h、24 h、48 h,用流式细胞仪测定骨髓和循环中EPCs数量,分别采用ELISA法和Western blot检测循环中VEGF、TNF-α、ET-1含量及胰腺VEGF表达,TUNEL法原位检测胰腺血管内皮细胞凋亡.结果 腹腔内雨蛙素注射后骨髓和循环中EPCs的数量显著增加(P<0.05)并伴有血浆高水平的VEGF、TNF-α、ET-1(P<0.05),骨髓和循环中EPCs数量与血浆VEGF水平呈线性相关(r分别为0.77和0.67);雨蛙素注射后12 h胰腺VEGF表达明显增加;雨蛙素注射后24 h胰腺血管内皮细胞凋亡明显增加.结论 急性胰腺炎可导致胰腺局部血管内皮损伤,并可动员骨髓内EPCs进入循环.     

外周血来源间充质干细胞/内皮祖细胞复合细胞膜片的构建及其生物学特性初步研究

目的从外周血中分离获取外周血来源间充质干细胞(peripheral blood mesenchymal stem cells,PBMSC)和外周血内皮祖细胞(peripheral blood endothelial progenitor cells,PBEPC),构建复合细胞膜片并初步研究其生物学特性。方法抽取健康成年新西兰大白兔外周血,采用密度梯度离心法分离获取PBMSC和PBEPC,分别行形态学观察及鉴定。取第3代PBMSC和PBEPC以1∶1比例进行成膜诱导形成复合细胞膜片,取单纯第3代PBMSC同法制备单一细胞膜片。取两种细胞膜片行HE染色,观察细胞膜片内细胞分布情况;分别对两种细胞膜片行成骨诱导,收集诱导1、5、10 d的上清液,利用ELISA法检测两种细胞膜片上清液内ALP、骨钙素(osteocalcin,OCN)和VEGF的表达情况。结果PBMSC细胞形态单一,呈纺锤形或多角形,具备良好的成骨、成脂分化能力;PBEPC细胞形态单一,呈铺路石样,成管试验阳性。两种细胞膜片均呈白色半透明膜状物。HE染色示,与单一细胞膜片组相比,复合细胞膜片组膜片更厚,具备更多细胞层数及更高的细胞密度。ELISA法检测示,随诱导时间延长,两组细胞膜片上清液中ALP、OCN和VEGF表达量均有所增加。复合细胞膜片组诱导培养5、10 d OCN表达量显著高于单一细胞膜片组,10 d ALP表达量显著高于单一细胞膜片组,1、5、10 d VEGF表达量均显著高于单一细胞膜片组,差异均有统计学意义(P<0.05);其余各时间点两组间各蛋白表达量比较差异均无统计学意义(P>0.05)。结论PBMSC具备稳定的MSCs分化能力,因其取材微创具备良好的应用前景。通过与PBEPC共培养、成膜诱导等手段构建复合细胞膜片,为组织缺损的修复提供了一种新的思路与探索。