低氧有助于小鼠骨髓源间充质干细胞的定向分化

Hypoxia contributes to directed differentiation of mice bone marrow derived mesenchymal stem cells

目的本研究在测量小鼠体内股骨骨髓正常氧分压的基础上设定相应的体外低氧浓度(3%),研究低氧环境对小鼠骨髓源间充质干细胞向内皮细胞系定向分化功能的影响。方法用微电极测量正常小鼠股骨骨髓的氧分压,并以此为依据设定体外培养的低氧浓度。将小鼠骨髓源单个核细胞置于3%(低氧)和20%(常氧)氧浓度下用内皮细胞专用培养基(EGM-2)进行定向诱导分化培养,培养至第2代进行Dil-Ac-LDL摄取实验和FITC-UEA结合实验双染色鉴定。对3%和20%氧浓度下培养的内皮样细胞用MTT法进行增生能力的鉴定;用荧光染色法对其进行黏附能力的比较。结果小鼠股骨骨髓生理性氧分压浓度为(21.55±3.40)mm Hg(1 mm Hg=0.133 k Pa),相当于(2.8±0.45)%氧体积分数(约为3%)。在不同氧浓度下培养的细胞,DilAc-LDL摄取实验和FITC-UEA结合实验均为阳性。。MTT细胞增生实验显示低氧(3%)培养的细胞吸光度为0.31±0.05,常氧(20%)培养的细胞吸光度为0.16±0.01(P<0.01,n=5)。低氧培养的细胞,其黏附率为(8.72±2.95)%,常氧培养的内皮样细胞,其黏附率为(3.91±0.72)%(P<0.05,n=5)。结论 3%的氧浓度是骨髓生理状态下的氧浓度。在此浓度下,骨髓源间充质干细胞可被诱导分化为内皮样细胞。与常氧相比,低氧培养的内皮样细胞具有较高的增生和黏附能力。

Objective To monitor the intravital oxygen tension in mice femoral marrow, and assess effect of different oxygen tension on bone marrow derived mesenchymal stem cells （BM-MSCs）. Methods Oxygen tension of mice femoral bone marrow was monitored by micro electrode to determine hypoxic level for ex-vivo cell culture. Mice BM-MSCs were obtained by density gradient centrifugation and cultured under 3% oxygen （hypoxia） and 20% （normoxia） oxygen in endothelial growth media-2（EGM-2）. Dil-Ac-LDL and FITC-UEA absorbance assays were applied on 8 days culture of Passage 2. MTF proliferation and adhesion assays were performed on those endothelial-like cells under different oxygen condition. Results The oxygen tension in normal femoral bone marrow was （21.55 ± 3. 4） mmHg （1 mmHg = 0. 133 kPa）, which equaled to （2. 8 ±0. 45 ）% （approximately 3% ） oxygen in volume fraction. Mice BM-MSCs differentiated to endothelial-like cells in both 3% （hypoxia） and 20% （normoxia） oxygen. OA values for MTT proliferation assay was 0. 31 ±0. 05 and 0. 16 ±0. 01 in hypoxia and normoxia, respectively （P 〈0. 01, n =5） and adhesion rate of endothelial-like cells was （8.72 ±2. 95）% and （3.91 ±0. 72）%, respectively （P 〈0.05, n =5）. Conclusion Physiologic oxygen concentration in marrow is approximately 3%. At this level, directed differentiated endothelial-like cells present more proliferative and adhesive abilities.