不同供者脐带间充质干细胞生物学特性的比较研究-制药工程专业论文.docxVIP

中文摘要 脐带间充质干细胞(UC-MSCs)具有自我更新、增殖和分化的潜能，在造血恢 复、修复损伤或病变的组织器官、免疫抑制治疗等方面具有重要临床应用前景。 为了获得稳定质量的 UC-MSCs，研究不同供者 UC-MSCs 生物学特性的差异是 十分必要的。为此，我们对脐带的存放时间和 UC-MSCs 的培养进行研究，通过 观察不同供者来源的 UC-MSCs 的细胞形态、增殖能力、多向分化能力、表面分 子标志物和细胞因子的表达量，比较其生物学特性的差异。用 UC-MSCs 培养上 清，比较不同供者来源 UC-MSCs 促造血因子的分泌能力。 采集后的新鲜脐带存放时间不宜超过 48h。脐带存放时间 48~72h 时，分离 UC-MSCs 的成功率下降为 80%，细胞数减少为 3.83×104/cm、原代培养时间延 长为 10.8d。DMEM/F12 是 UC-MSCs 的最适培养基，原代培养时间为 8.24d，获 得的细胞数达 3.84×106。20~31 岁的不同供者的 UC-MSCs 体外培养均呈纺锤形、 贴壁生长，在诱导培养基中均能分化为脂肪细胞和成骨细胞，表达表面分子标志 CD73、CD90 和 CD105，不分泌 M-CSF 和 EPO。UC-MSCs 培养上清均能促进 脐带血 CD34+细胞产生 CFU-G、CFU-GM 和 CFU-M，但不产生 CFU-E 和 CFU-GMEM。20~23 岁供者 UC-MSCs 的增殖能力强于 28~31 岁供者。20~23 岁 生男孩的供者 UC-MSCs 细胞因子 G-CSF、M-CSF、GM-CSF、IL-6、SOD2、 TGF-β2、CDK6、JAG1 的表达量较高，培养上清中促造血细胞因子 GM-CSF、 G-CSF 和 IL-6 的含量较高，支持脐带血 CD34+细胞形成较多集落。 关键词：间充质干细胞；脐带；分离；鉴定；细胞因子 ABSTRACT Human umbilical cord mesenchymal stem cells (UC-MSCs) have the capacity of self-renewal, proliferation and multilineage differentiation. UC-MSCs accelerate hematopoietic recovery, repair the damaged tissues and organs, and function immunosuppress. UC-MSCs have important and promising values of clinical application. In order to obtain the quality stable UC-MSCs, the biology of UC-MSCs derived from the various donors should be characterized. We studied storage of human umbilical cord and the culture of UC-MSCs. The biologic characteristics of UC-MSCs derived from the various donors were compared by morphology, proliferation, multi-differentiation, and the expression of cluster of differentiation (CD) and cytokines. The culture supernatant of UC-MSCs was tested to promote hematopoietic clonization. The human umbilical cord should storage less than 48h before UC-MSCs were isolated. When storagd within 48 to 72h, the isolation rate of UC-MSCs dropped down to 80%, and the isolated number of UC-MSCs decreased to 3.83×104/cm, the primary culture last 10.8d. On the suitable DMEM/F12, the primary culture of UC-MSCs was 8.24d