骨髓基质细胞表面CD40分子配基化促进IL-6和Flt3配体的释放及其作用

目的：分析人骨髓基质细胞（BMSC）CD40分子及其配体（CD40L）的表达，观察BMSC经CD40激发型单抗（5C11）激发后，其培养上清中IL－3、IL－6、Flt3配体（FL）和干细胞因子（SCF）含量变化及对纯化脐血CD34^ 细胞的作用。方法：新鲜分离人骨髓单个核细胞，经体外培养获得BMSC。间接免疫荧光标记，流式细胞仪分析细胞表面CD40和CD40L的表达，并与5C11（20μg/ml）共同孵育，分别于24，48和72h取上清液，ELISA法测定IL－3、IL－6、FL和SCF含量；在与5C11共同培养24h后，收取培养上清，与纯化的脐血CD34^ 细胞共同孵育，1周后进行细胞计数、细胞集落培养，并用钙磷脂结合蛋白（Annexin V)标记，流式细胞仪分析细胞凋亡。结果：发现BMSC表达CD40分子，经5C11激发后，BMSC IL－6和FL分泌增加，而对IL－3及SCF含量无影响。BMSC经5C11激发培养后支持脐血CD34^ 细胞的增殖，培养7d后其细胞数和集落数显著多于对照组，且细胞凋亡率低，二者差异有显著性（P＜0．01）。结论：BMSC CD40分子配基化可促进IL－6和FL的分泌，并支持CD34^ 细胞的增殖，CD40／CD40L可能通过调节细胞因子的释放参与造血调控。

CD40 ligandization promotes IL-6 and Flt3 ligand production of bone marrow stromal cells

OBJECTIVE: To analyze the expression of the CD(40) and its ligand (CD(40)L) on bone marrow stromal cells (BMSC), and investigate interleukin-3 (IL-3), IL-6, Flt3 ligand (FL) and stem cell factor (SCF) production of BMSC after stimulated with CD(40) agonistic monoclonal antibody (5C11) and its role in the regulation of hematopoiesis. METHODS: BMSC were freshly isolated from adult bone marrow. The expression of CD(40) on these cells was determined with flow cytometry, the concentrations of IL-3, IL-6, FL and SCF in the supernatant at 24, 48 and 72 hours after BMSC cultured with 5C11 at a dose of 20 micro g/ml were determined by the ELISA assay. After BMSC incubated with 5C11 for 24 hours, the supernatant was collected and cultured with purified cord blood CD(34)(+) cells for 7 days under 37 degrees C in a fully humidified atmosphere supplement with 5% CO(2). Colony formation assay and Annexin V assay were employed to determine the proliferation and apoptosis of the CD(34)(+) cells. RESULTS: BMSC expressed CD(40) and the production of IL-6 and FL increased after stimulated with 5C11 while IL-3 and SCF had no change. The supernatant collected from the stimulated BMSC promoted proliferation of CD(34)(+) cells, increased the CFU-GM yields and had anti-apoptosis effects. CONCLUSION: CD(40) ligandization on BMSC increased the production of IL-6 and FL and promoted the proliferation of CD(34)(+) cells. The couple CD(40)/CD(40)L may be involved in the control of hematopoiesis via modulation of the cytokine network in the bone marrow.