低氧对人骨髓间充质干细胞趋化因子受体CXCR4和CX_3CR1表达的影响

Effects of hypoxia on chemokine receptor CXCR4 and CX_3CR1 expression in human bone marrow mesenchymal stem cells

背景：移植的骨髓间充质干细胞能向损伤病灶部位定向迁移，进而发挥治疗作用，但关于其向病灶定向迁移的具体机制还不十分清楚。目的：观察低氧对人骨髓间充质干细胞趋化因子受体CXCR4和CX3CIR1表达的影响。设计、时间和地点：细胞学体外观察，于2008-02／2009-02在解放军第三军医大学新桥医院中心实验室进行。材料：骨髓标本取自解放军第三军医大学附属新桥医院血液科收治的15-40岁正常或原发病未累及骨髓患者。方法：穿刺采集骨髓，密度梯度离心结合贴壁培养法分离纯化入骨髓间充质干细胞。取生长良好的第3代细胞接种于25cm。培养瓶中，培养至70％-80％融合后，置于37℃、体积分数分别为3％O2、5％CO2，92％N2的饱和湿度孵育箱内培养48h，以常氧培养作为对照。主要观察指标：相差显微镜下观察细胞形态，流式细胞仪检测细胞表面标记物，Realtime荧光定量PCR法检测CXCR4和CX3CR1 mRNA的表达，免疫细胞化学和Westernblot法检测CXCR4和CX3CR1蛋白的表达。结果：体外分离纯化的人骨髓间充质干细胞呈成纤维细胞样，培养12-14d达90％汇合，呈极性排列，集落呈漩涡状：CD105和CD29阳性率分别为99．38％和99．13％，而CD14，CD45均呈阴性表达。在体积分数为3％的02培养条件下，人骨髓间充质干细胞CXCR4和CX3CR1mRNA的表达分别是常规培养条件下的2．130倍和2．361倍，CXCR4和CX3CR1蛋白的表达分别是常规培养条件下的1．69倍和1．93倍，CXCR4和CX3CR1主要表达于人骨髓间充质干细胞的胞膜和胞浆。结论：体积分数为3％的02低氧条件能够促进人骨髓间充质干细胞趋化因子受体CXCR4和CX3CR1mRNA及蛋白的表达，这可能是体内移植的骨髓间充质干细胞向损伤病灶定向迁移的机制之一。

BACKGROUND： Transplanted bone marrow mesenchymal stem cells （BMSCs） migrate to the injured regions and exert their therapeutic effects. The specific mechanisms involved in their directional migration to lesions remain unclear. OBJECTIVE： To investigate the chemokine receptor CXCR4 and CX3CR1 expression of human BMSCs in hypoxia culture. DESIGN, TIME AND SETrlNG： The cytology in vitro study was performed at the Central Laboratory, Xingqiao Hospital, Third Military Medical University of Chinese PLA from February 2008 to February 2009. MATERIALS： Cells harvested from the iliac heparinized bone marrow were obtained by iliac crest aspiration from healthy adult volunteers, aged 15 to 40 years old, at the Department of Hematology, Xinqiao Hospital, Third Military Medical University of Chinese PLA. METHODS： Bone marrow was obtained by puncture. Human BMSCs were harvested by combination of density and gradient centrifugation and different adherent method. Cells at passage 3 were incubated in a 25 cm2 flask. When 70% 80% confluence was found, cells were incubated at 37 ℃ and saturated humidity in an incubator containing 3% 02, 5% CO2, 92% N2 for 48 hours and those incubated under normal oxygen as controls. MAIN OUTCOME MEASURES： Morphology was examined by phase contrast microscopy. Cell surface markers were tested by flow cytometer. The CXCR4 and CX3CR1 mRNA expression were detected by real-time PCR. The CXCR4 and CX3CR1 protein expression were determined by Western blot assay. RESULTS： All of the cells had a fibroblast-iike morphology cultured in vitro and reached 90% confluence at 12 14 days, with the presence of polarity arrangement and whirlpool-shape. Cells were uniformly positive for CD105 （99.38%） and CD29 （99.13%）, but negative for CD14 and CD45. Exposure of BMSCs to 3% 02 increased expression of the CXCR4 mRNA and CX3CR1 mRNA, which were respectively 2.130 times and 2.361 times of normal culture; expression of the CXCR4 protein and CX3CR1 protein was respectively 1.69 times and 1.93 times of normal culture. CXCR4 and CX3CR1 mainly expressed in membrane and cytoplasm of human BMSCs. CONCLUSION： Hypoxia （3% O2） can upregulate the expression of CXCR4 and CX3CR1 in human BMSCs, which might be one of the machenisms underlying the migration of BMSCs.