人脐带血中内皮克隆形成细胞的分离和培养

目的:脐带血来源的内皮克隆形成细胞具有高度增殖、自我更新和血管生成的能力,是再生医学和母胎医学领域研究的热点。本研究旨在提出一种有效的、可靠的人脐带血中内皮克隆形成细胞分离和培养方法。方法:取抗凝脐血,采用密度梯度离心法分离单核细胞并进行贴壁培养,在细胞克隆形成但未融合成片时,进行单克隆的挑取再培养,最后对所得细胞进行表型和功能的鉴定。结果:所获细胞增殖能力强,形态单一,呈鹅卵石样,连续传代8次,未见细胞性状改变。接种到基质胶上,细胞可连接为管状、网状结构。能摄取培养液中的低密度脂蛋白并结合荆豆凝集素,细胞免疫荧光显示其表达典型的内皮细胞标志性分子CD31、e NOS、VE-Cadherin、v WF。结论:通过本研究的方法,我们从脐带血中获得足够数量和高纯度的内皮克隆形成细胞,这为进一步的实验研究提供了可靠的基础。

Isolation and Primary Culture of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

ABSTRACT Objective: Umbilical cord blood-derived endothelial colony forming cells(ECFCs) have high proliferation potential, self-renewal and vessel forming capacity. It is well studied in the field of regenerative and maternal-fetal medicine. This study aims to propose an effective and reliable method for ECFCs isolation and culture from umbilical cord blood. Methods: Heparin is used as antico- agulant, density gradient centrifugation and adherent culture is used for ECFCs isolation. When clones are formed but not fused, mono- clonal is selected and passaged. Finally, the obtained cells are subjected to phenotypic and functional characterization. Results: The ob- tained cells exhibit high proliferation ability , presented as cobblestone-like appearance and can form tubular structure. They can intake ac-LDL, combine Ulex-lectin and express the typical molecular markers of endothelial cells(namely CD31, eNOS, VE-Cadherin, vWF). Conclusion: By this efficient isolation method, sufficient and high-quality of ECFCs from cord blood can be obtained, providing a reli- able basis for further experimental research.