大鼠骨髓单个核细胞诱导扩增为内皮祖细胞的细胞分离方法、接种数目、培养瓶包被条件  

Cell isolation method,seeding number,and coating conditions for inducing and amplifying rat bone marrow mononuclear cells into endothelial progenitor cells

作　　者：孙白羽 陈静依 姜志超[4] SUN Baiyu;CHEN Jingyi;JIANG Zhichao(School of Stomatology,Cheeloo College of Medicine,Shandong University,Jinan China;不详)

机构地区：[1]山东大学齐鲁医学院口腔医学院·口腔医院,济南250012 [2]山东省口腔组织再生重点实验室 [3]山东省口腔生物材料与组织再生工程实验室 [4]山东大学齐鲁医院急诊外科

出　　处：《山东医药》2024年第21期44-48,共5页Shandong Medical Journal

摘　　要：目的筛选大鼠骨髓单个核细胞(BMMNCs)诱导扩增为内皮祖细胞(BM-EPCs)的细胞分离方法、接种数目、培养瓶包被条件,构建一个高效、高产量、高纯度的骨髓来源BM-EPCs分离培养诱导扩增方法。方法取2周龄雄性SD大鼠,脱颈处死后分离大鼠双侧胫骨和股骨,收集骨髓细胞悬液。配制30%、50%、60%和70%浓度的Percoll细胞分离液,通过Percoll密度梯度离心法分离出大鼠BMMNCs种子细胞,并计算活细胞比例。将获得的BMMNCs分为1×10^(5)、5×10^(5)、1×10^(6)、2.5×10^(6)、5×10^(6)、1×10^(7)六个组别,分别接种于25 cm^(2)无菌培养瓶中,培养7 d后镜下观察各组细胞集落形成数目,并计算每10^(6)细胞的集落形成数。运用Graphpad prism9.5软件进行Logistic拟合曲线,根据相关系数R^(2)确定相关性,根据其P值将有统计学差异的接种数目纳入范围,随后使用R语言编程定义计算函数,根据已知种子细胞总数及相关性函数限制下,通过迭代寻找最佳的BMMNCs细胞接种数目。分别配制20、50、100 nmol/L浓度的人纤连蛋白(FN)溶液,以不添加FN的空白溶液为对照,分别包被空白培养瓶2、6、12、24 h,将收集的48 h未贴壁BMMNCs接种于FN包被的各培养瓶中,静置培养3 d后计算各组集落形成数目,确定FN包被的最佳浓度与时间。接种48 h未贴壁BMMNCs于25 cm^(2)培养瓶底,使用EGM-2完全培养基定向诱导,于显微镜下观察集落形成及诱导扩增进程。取培养14 d的BM-EPCs,分别采用双阳性染色法和流式细胞术鉴定BM-EPCs的纯度。结果使用Percoll分离法可把BMMNCs细胞清晰的分为5层,其中30%与50%Percoll细胞分离层之间为BMMNCs活细胞比率最高。BMMNCs的最优接种数目为2.5×10^(6)个。以50 nmol/L的FN溶液包被24 h或以100 nmol/L的FN溶液包被6 h皆可有效促进细胞集落形成。细胞接种7 d后获得形态良好的铺路石样细胞并建立生长优势,表明BMMNCs已经诱导成为形态良好的BM-EPCsObjective To select the cell isolation method,seeding number,and coating conditions for culture flasks to induce and amplify rat bone marrow mononuclear cells(BMMNCs)into endothelial progenitor cells(BM-EPCs),aiming to establish an efficient,high-yield,and high-purity method for the separation,culture,induction,and amplification of BMEPCs derived from bone marrow.Methods Two-week-old male SD rats were used and euthanized by cervical dislocation,then we isolated the tibia and femur bilaterally to collect bone marrow cell suspensions.We prepared Percoll cell separation solutions at concentrations of 30%,50%,60%,and 70%,and separated rat BMMNCs seed cells using the Percoll density gradient centrifugation method,and calculated the ratio of viable cells.The obtained BMMNCs were divided into six groups(1×10^(5),5×10^(5),1×10^(6),2.5×10^(6),5×10^(6),and 1×10^(7) cells)and were inoculated in the 25 cm^(2)sterile culture flasks.After 7 days of culture,we observed the number of cell colonies formed in each group under a microscope and calculated the number of colonies formed per 10^(6) cells.Logistic fitting curves were plotted using GraphPad Prism 9.5 software to determine correla‑tion based on the R^(2) coefficient,and the P-values were used to include statistically significant seeding numbers.We then used R language to define calculation functions and iteratively found the optimal BMMNCs seeding number based on the known total number of seed cells and the correlation function limitations.Fibronectin(FN)solutions at concentrations of 20 nmol/L,50 nmol/L,and 100 nmol/L were prepared,and culture flasks were coated for 2,6,12,and 24 h with blank solu‑tions without FN as controls.We inoculated BMMNCs that did not adhere after 48 h into FN-coated culture flasks and cul‑tured them for 3 days to calculate the number of colonies formed in each group,and determined the optimal FN concentration and coating time.BMMNCs that did not adhere after 48 h were inoculated in the 25 cm^(2)culture flasks,and were induced with EG

关 键 词：内皮祖细胞 骨髓来源内皮祖细胞 单个核细胞 骨髓单个核细胞 Percoll密度梯度离心法 人纤连蛋白 骨组织工程 细胞分离方法 细胞培养方法 

分 类 号：R782.1[医药卫生—口腔医学]