骨髓间充质干细胞对骨髓CD34~+细胞增殖影响的体外研究

研究骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSC)对骨髓CD34+细胞增殖的影响,为骨髓CD34+细胞体外扩增的深入研究提供依据。用MACS进行骨髓CD34+细胞的分选;用流式细胞术进行CD34+细胞纯度鉴定;用密度梯度离心法分离引产胎儿骨髓单个核细胞,结合贴壁法进行BMMSC的体外扩增培养;用流式细胞术进行BMMSC表面标志鉴定;用Transwell培养板培养CD34+细胞与BMMSC;用自动细胞计数仪计数有活性的CD34+细胞数量;用MTT比色法检测骨髓CD34+细胞增殖活性。流式细胞术检测结果显示分选所得的CD34+细胞纯度达到90%以上;流式细胞术检测显示传至第3代继续培养72h后的BMMSC高表达CD44、CD29,而HLA-DR、CD45、CD34表达阴性,说明所培养的BMMSC纯度很高;Transwell培养板培养CD34+细胞与BMMSC,在倒置显微镜下观察以及自动细胞计数仪计数发现,实验组有活性的CD34+细胞数量高于对照组(P<0.05);采用MTT比色法检测骨髓CD34+细胞的增殖活性,实验组高于对照组(P<0.05)。以上结果说明BMMSC有促进骨髓CD34+细胞增殖的作用,这种作用可能与其分泌的细胞因子有关。

Effect of bone marrow mesenchymal stem cells on the proliferation of bone marrow CD34~+ cells in vitro

To investigate the affection imposed on the marrow CD34+ cells by bone marrow mesenchymal stem cells(BMMSC),VarioMACS was used to sorting bone marrow CD34+ cells,and then the purity of CD34+ cell was tested by FCM.Marrow mononuclear cells from abortion fetal bone marrow were isolated,and BMMSC were extracted from the marrow mononuclear cells,and cell cultivation was processed with combination of the explant-attached method.The CD34+cells and BMMSC were cultivated in Transwell chambers and the living CD34+ were counted.The FCM assay showed high purity of the sorted CD34+ cells(≥90%).BMMSC highly expressed cell surface marker CD44,CD29,but negatively expressed HLA-DR CD45 and CD34.The amount of the active CD34+ cells in the test group was significantly higher than that in the control group(P<0.05),and the marrow CD34+ cells proliferation activity was significantly higer in the test group compared with the normal group(P<0.05).These results demonstrate the BMMSC promotes proliferation of marrow CD34+ cells.