CD34+干细胞的分化及其在人工血管内皮化中的应用

目的:探讨外周血、骨髓和脐血中CD34 细胞作为人工血管内皮化的种子细胞来源问题.方法:采集犬外周血和骨髓及人脐血,经免疫磁珠分离出内皮细胞祖细胞,内皮细胞生长因子(VEGF)诱导分化为内皮细胞并扩增,光镜、扫描电镜和免疫细胞化学鉴定;将培养细胞种植于人工血管,扫描电镜观察.结果:经流式细胞仪测定,分离后的细胞中CD34 细胞:外周血(26.30±2.42)% 、骨髓(41.84±3.65)%、脐血(74.62±4.46)%,骨髓和脐血中CD34 细胞数量明显多于外周血,且增殖、分化能力强;CD34 细胞培养2周细胞长满瓶底并达到增殖高峰,细胞呈"铺路石"状排列.Ⅷ因子,CD31免疫细胞化学染色均为阳性,透射电镜细胞胞浆内可见W-P小体;扫描电镜下,内皮细胞平铺于人工血管表面,成单层排列,细胞排列无轴向性.结论:外周血、骨髓和脐血经免疫磁珠分离系统可分离出CD34 细胞,CD34 细胞经VEGF诱导可定向分化为内皮细胞.骨髓和脐血CD34 细胞可作为人工血管内皮化的种子细胞来源.

Induction of differentiation of CD34+ cells in vitro and application in endothelialization of blood vessel prostheses

OBJECTIVE: To induce differentiation of CD(34)+ cells from varying sources into epithelial cells for potential application of blood vessel prostheses. METHODS: CD(34)+ cells were isolated from canine peripheral blood and bone marrow or human umbilical cord blood by an immune magnetic cell sorting system. The isolated CD(34)+ cells were induced to differentiate into endothelial cells in a liquid culture with VEGF. Endothelial cells were evaluated by immunocytochemistry and transmission electron microscopy. CD(34)+ cells were seeded on PTFE prostheses, which were harvested and observed by electron microscopy. RESULTS: The average isolated CD(34)+ cells from peripheral blood, bone marrow and umbilical cord blood were (26.30+/-2.42)%,(41.84 +/-3.65)%and (74.62+/-4.46)%, respectively. The number of CD(34)+ cells increased with the culture duration and reached to the highest level at the 14th d. Immunostaining showed positive signal for CD31 in endothelial cells and positive for factor VIII in cell culture. Transmission electron microscopy found Weibel-Palade bodies in the cytoplasm. Scanning electron microscopy demonstrated a single- layer of flat cells in the innermost layer of PTFE prostheses, and the cells were not of tropism. CONCLUSION: CD(34)+ cells isolated from peripheral blood, bone marrow and umbilical cord blood can be induced into endothelial cells; bone marrow and umbilical cord blood can be used as the sources of seeding cells for endothelialization of prostheses.