CAJ | 학술논문

背景:内皮祖细胞不仅参与胚胎血管生成,也参与出生后血管发生和血管内膜损伤后修复,对治疗缺血性疾病意义重大,但目前对内皮祖细胞的分离、培养、鉴定还存在争议.目的:体外分离、培养大鼠骨髓与外周血来源的内皮祖细胞,并比较其生物学特性.方法:密度梯度离心法分离 SD 大鼠骨髓和外周血单个核细胞,接种于纤维连接蛋白铺被的培养瓶中贴壁培养,用加入血管内皮生长因子、碱性成纤维细胞生长因子及表皮生长因子的完全培养基诱导培养,对获得的贴壁细胞进行细胞形态学,免疫细胞化学染色,流式细胞仪,透射电镜,以及 Dil-acLDL、FITC-UEA-1双荧光染色法检测.结果与结论:骨髓来源的内皮祖细胞数量多,集落状生长,增殖能力强;外周血来源的内皮祖细胞数量较少,散在生长,消化后能贴壁但不能传代.两种不同来源的内皮祖细胞免疫细胞化学检测贴壁细胞CD133、CD34、Flk-1、Ⅷ因子在不同时段呈阳性表达;激光共聚焦显微镜观察,Dil-acLDL、FITC-UEA-1均为双染.透射电镜检查外周血来源的内皮祖细胞发现 W-P 小体.提示大鼠骨髓和外周血均能分离培养出内皮祖细胞,但前者是早期内皮祖细胞,后者为晚期内皮祖细胞,两者生物学特性各不相同.
배경:내피조세포불부삼여배태혈관생성,야삼여출생후혈관발생화혈관내막손상후수복,대치료결혈성질병의의중대,단목전대내피조세포적분리、배양、감정환존재쟁의.목적:체외분리、배양대서골수여외주혈래원적내피조세포,병비교기생물학특성.방법:밀도제도리심법분리 SD 대서골수화외주혈단개핵세포,접충우섬유련접단백포피적배양병중첩벽배양,용가입혈관내피생장인자、감성성섬유세포생장인자급표피생장인자적완전배양기유도배양,대획득적첩벽세포진행세포형태학,면역세포화학염색,류식세포의,투사전경,이급 Dil-acLDL、FITC-UEA-1쌍형광염색법검측.결과여결론:골수래원적내피조세포수량다,집락상생장,증식능력강;외주혈래원적내피조세포수량교소,산재생장,소화후능첩벽단불능전대.량충불동래원적내피조세포면역세포화학검측첩벽세포CD133、CD34、Flk-1、Ⅷ인자재불동시단정양성표체;격광공취초현미경관찰,Dil-acLDL、FITC-UEA-1균위쌍염.투사전경검사외주혈래원적내피조세포발현 W-P 소체.제시대서골수화외주혈균능분리배양출내피조세포,단전자시조기내피조세포,후자위만기내피조세포,량자생물학특성각불상동.
@@@@BACKGROUND: Endothelial progenitor cells (EPCs) not only participate in embryonic angiogenesis, but also participate in postnatal angiogenesis and vascular intima damage repair, exhibiting a great significance for the treatment of ischemic diseases. Nevertheless, the isolation, culture and identification of endothelial progenitor cells remain controversial. OBJECTIVE: To isolate and culture endothelial progenitor cells from peripheral blood and bone marrow in vitro, and to compare their biological characteristics.METHODS: SD rat bone marrow and peripheral blood mononuclear cells were isolated by density gradient centrifugation, inoculated on fibrinin-coated culture flask and cultured with complete medium containing vascular endothelial growth factor, basic fibroblast growth, and epidermal growth factor. Cel morphplogy, immunocytochemical staining, flow cytometry and transmission electron microscopy as wel as Dil-acLDL, FITC-UEA-1 double fluorescence staining were performed on the harvested adherent cells. RESULTS AND CONCLUSION: A large number of endothelial progenitor cells were harvested from bone marrow, and they were grown in a colony-like manner and show strong proliferative capacity. A smal number of endothelial progenitor cells were harvested from peripheral blood. They were grown in a sparse manner and could adhere to flask wal but could not be passaged. Immunocytochemical staining showed that adherent cells from two sources were positive for CD133, CD34, Flk-1 and VII factors at different time periods. Laser confocal microscopy showed that adherent cells from two sources can stain Dil-acLDL and FITC-UEA-1. Transmission electron microscopy showed that W-P body was found in the peripheral blood endothelial progenitor cells. These results indicate that endothelial progenitor cells can be cultured from both bone marrow and peripheral blood, but early-stage endothelial progenitor cells can be cultured from bone marrow and late-stage endothelial progenitor cells can be cultured from peripheral blood. These two kinds of cells show different biological characteristics.