人脂肪源性血管外膜细胞对CD34^(+)造血干/祖细胞体外支持的实验研究  

Experimental Study on the Supporting Effect of Human Adipose-Derived Pericyte/Perivascular Cells on CD34^(+) Hematopoietic Stem/Progenitor Cells in Vitro

作　　者：周芮 杨婷婷 张磊[1] 许飞[3] 郑波 ZHOU Rui;YANG Tingting;ZHANG Lei;XU Fei;ZHENG Bo(Ningxia Medical University,Yinchuan 750004,China;Human Stem Cell Institute of Ningxia,Yinchuan 750004,China;Department of Hematology,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏人类干细胞研究所,银川750004 [3]宁夏医科大学总医院血液内科,银川750004

出　　处：《宁夏医科大学学报》2021年第5期453-460,473,共9页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81860028)。

摘　　要：目的建立以人脂肪源性血管外膜细胞(human adipose-derived pericyte/perivascular cells,hAD-PCs)为基质层与人脐血(umbilical cord blood,UCB)CD34^(+)造血干/祖细胞(hematopoietic stem/progenitor cells,HSPCs)共培养体系,评估hAD-PCs对UCB CD34^(+)HSPCs的体外支持效力。方法实验分3组,实验组(hAD-PCs组)以hAD-PCs为基质细胞与UCB CD34^(+)HSPCs共培养;阳性对照组(BMSCs组)以骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)为基质细胞与UCB CD34^(+)HSPCs共培养;空白组(无基质细胞组)以UCB CD34^(+)HSPCs单独培养。在共培养的1、2、4周,倒置显微镜下观察UCB CD34^(+)HSPCs细胞形态;应用台盼蓝拒染法计算细胞数量;多参数流式细胞术分析血细胞标记CD45、CD34、CD33、CD19、CD10和CD14的表达;镜下观察UCB CD34^(+)HSPCs培养后集落形成能力;通过酶联免疫吸附法(ELISA)检测造血因子分泌情况。结果UCB CD34^(+)HSPCs细胞增殖结果显示:hAD-PCs组及BMSCs组共培养的UCB CD34^(+)HSPCs细胞数在第1周略有增长,无基质细胞组细胞数降低,组间比较差异无统计学意义(P>0.05);第2周时hAD-PCs组和BMSCs组细胞数继续增长达到高峰,无基质细胞组细胞数持续降低,hAD-PCs组、BMSCs组细胞数量均高于无基质细胞组(P均<0.05);第4周时hAD-PCs组和BMSCs组细胞数略有下降,无基质细胞组细胞全部死亡。hAD-PCs组和BMSCs组细胞计数各时间点差异无统计学意义(P均>0.05)。血细胞标记表达情况:hAD-PCs组和BMSCs组的全血细胞标记CD45^(+)、造血祖细胞标记CD34^(+)CD33^(-)、淋巴细胞标记CD10^(+)/19^(+)和髓系细胞标记CD14^(+)表达在1、2、4周差异均无统计学意义(P均>0.05)。UCB CD34^(+)HSPCs集落形成结果显示:hAD-PCs组和BMSCs组在1、2、4周形成的集落数量差异均无统计学意义(P均>0.05)。细胞因子分泌情况:hAD-PCs组和BMSCs组IL-3、IL-6、TPO、IFN-γ及TNF-α分泌水平差异均无统计学意义(P均>0.05);hAD-PCs组的SCF在1周时�Objective To establish human adipose-derived pericyte/perivascular cells(hAD-PCs)as a trophoblast and human umbilical cord blood(UCB)CD34^(+)hematopoietic stem/progenitor cells(HSPCs)co-culture system,to explore and evaluate whether hAD-PCs have a supporting effect on UCB CD34^(+)HSPCs in vitro.Methods The experiment was set to 3 groups.The experimental group(hAD-PCs group)was co-cultured with UCB CD34^(+)HSPCs with hAD-PCs as stromal cells;the positive control group(BMSCs group)was co-cultured with UCB CD34^(+)HSPCs with BMSCs as stromal cells;the group(without stromal cells group)had no stromal cells,and UCB CD34^(+)HSPCs were cultured alone as blank group.The changes of cell morphology were observed under inverted microscope at week 1,2 and 4 after co-culture.Trypan blue rejection method was used to calculate the number of cells.The expression of hematopoietic markers CD45,CD34,CD33,CD19,CD10 and CD14 were analyzed by multi-parameter flow cytometry,and the colony forming ability of UCB CD34^(+)HSPCs was observed under a microscope,and hematopoietic factors were detected by enzyme-linked immunoassay adsorption assay(ELISA).Results The UCB CD34^(+)HSPCs proliferation was analyzed.The number of viable cells in the hAD-PCs group and BMSCs group increased slightly in the first week,while the number of cells in the blank group decreased,and there was no statistical difference(P>0.05).In the second week,the number of cells in the hAD-PCs group and BMSCs group continued to increase and reached a peak,while the number of cells in the without bland group continued to decrease.The number of cells in the hAD-PCs group and the BMSCs group was higher than that in the without stromal cells group,and the difference was statistically significant(P all<0.05).In the 4 th week,the number of cells in the hAD-PCs group and the BMSCs group decreased slightly,and all cells in the without stromal cells group died.hAD-PCs group and BMSCs group had no significant difference in cell count at each time point(P all>0.05).Cytometric cell co

关 键 词：间充质干细胞 血管外膜细胞 共培养 造血支持 

分 类 号：R457.7[医药卫生—治疗学] R394.2[医药卫生—临床医学]