骨髓源性EPCs对脊髓源性NSCs增殖分化的影响  

The effects of bone marrow-derived endothelial progenitor cells on the proliferation and differentiation of spinal cord-derived neural stem cells

作　　者：张硕[1] 杜怡斌[2] 杜公文 张辉[2] 余涛[2] 方家刘 高维陆[2] 尹宗生[1] 

机构地区：[1]安徽医科大学第四附属医院骨科,合肥230022 [2]安徽医科大学第一附属医院骨科,合肥230022

出　　处：《安徽医科大学学报》2015年第1期20-24,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81171173);安徽省自然科学基金(编号:11040606Q25)

摘　　要：目的观察骨髓源性内皮祖细胞(EPCs)对脊髓源性神经干细胞(NSCs)增殖分化的影响。方法通过密度梯度离心法获取骨髓血单个核细胞,以EBM-2进行诱导培养EPCs并进行免疫细胞化学染色鉴定,成熟的方法获取及鉴定SD大鼠的脊髓NSCs,1×105/ml第3代NSCs置于Transwell小室下层与1×105/ml上层原代EPCs进行体外1∶1共培养,以单纯第3代的NSCs培养为对照,培养7 d,双盲法分别计数各组在相差显微镜下神经球形成的数目,并用目镜测微尺测量神经球的平均直径,通过5%血清诱导培养NSCs7 d后,行β-微管蛋白-Ⅲ免疫荧光染色,Hoechst细胞核染色后在显微镜下计算神经元/细胞总数得出百分率。结果骨髓源性EPCs与脊髓源性NSCs共培养组神经球平均数目为(22.27±3.85)个,平均直径为(61.70±7.21)μm,诱导培养后分化为神经元的平均百分率为(46.10±3.70)%,与对照组比较差异均有统计学意义(P<0.01)。结论骨髓源性EPCs能促进脊髓源性NSCs增殖及其向神经元分化。Objective To investigate the effects of bone marrow-derived endothelial progenitor cells（ EPCs） on the proliferation and differentiation of spinal cord-derived neural stem cells（ NSCs） . Methods Bone marrow mononu-clear cells were isolated by density gradient centrifugation methods and EPCs were cultured by EBM-2 basal medi-um, identified by fluorescent immunocytochemistry. Spinal cord-derived NSCs were isolated, cultured and identi-fied by the mature methods. 1 × 105/ml tertiary NSCs were plated on the base of culture wells, the upper transwell compartment was seeded with 1 × 10^5/ml primary EPCs, EPCs and NSCs （1 ∶ 1） were co-cultured in vitro, set the untreated tertiary NSCs as a control group. 7 days after co-culture, the number and diameter of neurospheres were calculated and measured with the double blind method. After that, NSCs were maintained for 7 days in DMEM/F12＋ 5 % serum medium, and immunochemically stained with neuronal specific marker β-tubulin-Ⅲ, ratio of posi-tive cells to total cells was calculated. Results The average number of neurospheres in co-culture group was （22. 27 ± 3. 85） and the average diameter was（61. 70 ± 7. 21）μm. The percentage of neurons immunostaining was （46. 10 ±3. 70）%, differences are statistical significance compared with the control group （P〈0. 01）. Conclu-sion Bone marrow-derived EPCs promote the proliferation of spinal cord-derived NSCs and induce the differentia-tion of spinal cord-derived neural stem cells into neurons.

关 键 词：NSCS EPCS 增殖 分化 微环境 脊髓损伤 

分 类 号：R681.54[医药卫生—骨科学]