小鼠肿瘤坏死因子α增强造血干／祖细胞归巢效率的机制研究

Study on the mechanism of enhancing homing efficiency of human hematopoietic stem/progenitor cells into bone marrow after manipulation with tumor necrosis factor α in xenotransplanted BALB/c mouse model

目的探讨肿瘤坏死因子（TNF）α在调节造血干／祖细胞（HS／PC）归巢中的增效作用及其机制。方法将荧光染料CFSE标记的脐血CD34^＋细胞移植入接受照射（对照组）或联合TNFα预处理（实验组）的BALB／c受鼠。移植后20h，采用流式细胞术（FACS）检测脐血CD34^＋细胞在BALB／c受鼠中的分布（外周血、肝脏、肺脏）与归巢（骨髓、脾脏）特征，计算其相应的归巢效率；酶联免疫吸附实验（ELISA）检测BALB／c受鼠血清基质衍生因子（SDF）-1α水平；FACS检测TNFα预处理前、后脐血CD34^＋细胞表面CXCR4表达水平的变化；免疫组化法检测BALB／c受鼠骨髓及脾脏组织切片中SDF-1α的表达水平。结果脐血CD34^＋细胞主要归巢于BALB／c受鼠骨髓和脾脏；经TNFα预处理的实验组BALB／c受鼠骨髓归巢率[（0．65±0．13）％]显著高于对照组[（0．30±0．09）％，P〈0．01]；但TNFα预处理同时显著抑制脐血CD34^＋细胞归巢于BALB／c受鼠脾脏（P〈0．01）；不同剂量的TNFα预处理不影响受鼠血清SDF-1α水平；新鲜分离的脐血CD34^＋细胞与不同浓度TNFα共孵育18h后，其表面CXCR4表达水平并无明显变化；但TNFα预处理受鼠骨髓龛组成细胞SDF—1α表达增高，且脾脏红髓区小梁动脉和小梁静脉内皮细胞SDF-1d表达增高。结论TNFα通过提升骨髓龛SDF-1α浓度梯度促进HS／PC归巢于骨髓，这一发现将有助于为临床提供可行性HS／PC归巢增效剂。

Objective To investigate the role of tumor necrosis factor （TNF） α on the homing efficiency of hematopoietic stem/progenitor cells （HS/PC） into bone marrow and its mechanism. Methods CFSE-labeled umbilical cord blood （UCB） CD34^＋ cells were transplanted into irradiated （control group） or combined with TNFαrepared （experimental group） BALB/c recipient mice. The distribution in peripheral blood, liver, lung and homing characteristics in bone marrow and spleen of UCB CD34^＋ cells, in BALB/c recipient mice were determined 20 hours after xenotransplantation by flow cytometry （FACS） and their homing efficiency was calculated. ELISA was used to measure serum SDF-1α level. CXCR4 expression levels of on UCB CD34^＋ cells were assessed by FACS pre-/post-manipulation with TNFα. SDF-1α expression level in bone marrow and spleen was tested by immunohistochemistry. Results UCB CD34^＋ cells mainly homed into recipient mice bone marrow and spleen ; The homing efficiency in experimental group bone marrow [ （0.65 ± 0.13） % ] was significantly higher than that in control ones [ （ 0.30 ± 0.09 ） %, P 〈 0.01 ] , whereas the homing efficiency in experimental group spleen was dramatically lower than that in control ones （P 〈0.01 ） ; Treatment with TNFα did not affect recipient serum SDF-1α level; After 18 hours co-cultured with TNFα, the CXCR4e expression level on UCB CD34^＋ cells was similar to that on fresh ones ; TNFα treatment induced significantly higher SDF-1α expression on osteoblastic and stromal cells in bone marrow, and reversed spleen SDF-1α gradient that was originally favorable for CD34^＋ cells homing. Conclusion TNFα enhances the homing efficiency of HS/PC via up-regulating SDF-1α gradient in bone marrow, and might be an useful enhancer for HS/PC homing in clinical practice.