CAJ | 학술논문

背景:内皮祖细胞因其分离与培养的方法各不相同,在实验中难以重复.目的:探讨大量获取骨髓源性内皮祖细胞分离与培养的方法.方法:通过密度梯度离心法从4周龄 SD 大鼠骨髓中分离单个核细胞,使用 EGM-2 MV 培养基进行诱导培养,采用形态学特征观察、摄取 Dil-Ac-LDL 与结合 FITC-UEA-1实验、免疫荧光化学鉴定其表面抗原 CD133与 VEGFR2等方法对其进行鉴定,并通过管腔形成实验观察形成管腔的能力.结果与结论:①形态学观察:分离的骨髓单个核细胞经诱导培养后,在生长的早期(8 d 左右)、晚期(15 d左右)其细胞形态有一定差异,早期以纺锤形、三角形、圆形细胞多见,晚期以圆形、短梭形细胞多见.②摄取 Dil-Ac-LDL 与结合 FITC-UEA-1实验:显示8,21 d 的细胞均为阳性.③免疫荧光化学染色:8 d的细胞表达 CD133、VEGFR2.④管腔形成实验:在 Matrigel 基质上15 h 左右能够生成血管样结构.结果表明:利用密度梯度离心法分离大鼠骨髓单个核细胞后以 EGM-2 MV 进行诱导培养,经过鉴定证明获得的细胞符合内皮祖细胞的特征.这种方法能够简单、快速、可靠、大量地获取内皮祖细胞.
배경:내피조세포인기분리여배양적방법각불상동,재실험중난이중복.목적:탐토대량획취골수원성내피조세포분리여배양적방법.방법:통과밀도제도리심법종4주령 SD 대서골수중분리단개핵세포,사용 EGM-2 MV 배양기진행유도배양,채용형태학특정관찰、섭취 Dil-Ac-LDL 여결합 FITC-UEA-1실험、면역형광화학감정기표면항원 CD133여 VEGFR2등방법대기진행감정,병통과관강형성실험관찰형성관강적능력.결과여결론:①형태학관찰:분리적골수단개핵세포경유도배양후,재생장적조기(8 d 좌우)、만기(15 d좌우)기세포형태유일정차이,조기이방추형、삼각형、원형세포다견,만기이원형、단사형세포다견.②섭취 Dil-Ac-LDL 여결합 FITC-UEA-1실험:현시8,21 d 적세포균위양성.③면역형광화학염색:8 d적세포표체 CD133、VEGFR2.④관강형성실험:재 Matrigel 기질상15 h 좌우능구생성혈관양결구.결과표명:이용밀도제도리심법분리대서골수단개핵세포후이 EGM-2 MV 진행유도배양,경과감정증명획득적세포부합내피조세포적특정.저충방법능구간단、쾌속、가고、대량지획취내피조세포.
@@@@BACKGROUND: Endothelial progenitor cel s have a very broad application prospect, but the isolation and culture methods differ greatly and therefore are hard to repeat. OBJECTIVE: To investigate the isolation and culture methods of bone marrow-derived endothelial progenitor cel s. METHODS: Bone marrow mononuclear cel s were isolated from 4-week-old Sprague-Dawley rats by density gradient centrifugation methods and cultured by EGM-2 MV medium. Then the cel s were identified by morphological observation, FITC-UEA-1 binding and Dil-Ac-LDL uptake assay, and fluorescent immunocytochemistry for detection CD133 and VEGFR2 expression. In addition, angiogenic tube formation was determined by Matrigel tube formation assays. RESULTS AND CONCLUSION: (1) Morphology: After induced culture, the isolated bone marrow mononuclear cel s exhibited a spindle-shaped, triangular, and round appearance in the early stage (about the 8th day) and round and short spindle-shaped appearance in the late stage (about the 15th day). (2) FITC-UEA-1 binding and Dil-Ac-LDL uptake assay: Cel s were positive on days 8 and 21. (3) Fluorescent immunocytochemistry: on day 8, cel s expressed CD133 and VEGFR2. (4) Matrigel tube formation assays: Capil ary-like structures formed at 15 hours on Matrigel. These findings suggest that rat bone marrow mononuclear cel s isolated by density gradient centrifugation method and cultured by EGM-2MV medium correspond to the characteristics of endothelial progenitor cel s. This method is simple, quick, and reliable to harvest enough endothelial progenitor cel s.