绿荧光蛋白标记人骨髓源性神经干细胞的体外实验研究

In Vitro experimental study on marking neural stem cells derived from human bone marrow with green fluorescence protein

目的:探讨重组腺相关病毒包装绿荧光蛋白(rAAV-GFP)标记人骨髓源性神经干细胞(BM.NSCs)的可行性,为进一步行标记细胞移植实验研究奠定基础。方法:以成人自愿者的骨髓为研究对象,梯度密度离心法分离获取骨髓基质细胞并用“Cytokine·神经干细胞培养基”诱导分化为神经干细胞,分化的细胞以免疫细胞化学鉴定。对接种生长的BM.NSCs(1×105～6/mL)以无血清培养基冲洗并重悬细胞,依最佳病毒感染指数(MOI)为105的标准计算、加入包装了GFP的rAAV病毒颗粒,连续培养12h(37℃,50mL/LCO2)以感染细胞;补加100g/L胎牛血清后,于神经干细胞培养基中继续细胞培养24h(37℃,50mL/LCO2)以上,荧光显微镜(OlympusIX70,Japan)追踪观察rAAV-GFP标记的BM.NSCs。结果:BM.NSCs于标记后1周内见不到GFP阳性表达;在标记10d后才出现有GFP阳性表达情况(平均每个200倍镜下视野可见到5～10个GFP阳性细胞),所标记的细胞在荧光显微镜下呈亮绿色;随着培养时间延长,BM.NSCs表达GFP也逐渐增强、阳性细胞数量逐渐增多,并可于1个月时最明显(标有GFP阳性的细胞可达BM.NSCs总数的60%),追踪观察到到2.5个月时亦未见衰减。结论:rAAV-GFP在人骨髓源性神经干细胞中的表达较迟,但呈渐强趋势;rAAV-GFP标记人骨髓源性神经干细胞具有可行性。

AIM:To explore the feasibility of marking neural stem cells derived from human bone marrow (BM.NSCs) with green fluorescence protein packed by recombinant ade no associated virus ( rAAV-GFP). METHODS:Bone marrow stroma cells (BMSCs) were derived from adult volunteers wi th density gradient centrifugation and cultured in Cytokine·NSCs medium to be i nduced into neural stem cells. All of the differentiated cells were identified b y immunocytochemistry.The inoculated BM.NSCs, with the density of 1×105-6/mL, were washed with serum-free medium, and then suspended in the same medium. Afte r the rAAVs-GFP were added into the cells-suspension upon the best multiplicit y of infection (MOI, 105), the BM.NSCs were cultured at 37 ℃and 50 mL/L CO2 for 12 hours, and then supplied with fetal calf serum (FCS) at 100 g/L of final con centration for continuous culture in NSCs-medium for over 24 hours (37 ℃, 50 m L/L CO2). All of BM.NSCs marked by rAAVs-GFP were traced by fluorescence micros copy (Olympus IX70,Japan). RESULTS:Within 1 week, no any cell was positively marked by GFP. At 10 days af ter marked, however, the GFP positive expression appeared in BM. NSCs(average 5-10 cells with positive GFP expression per vision field of 200×microscope), w hich showed light green under the fluorescence microscopy. It enhanced gradually along with the prolonged culturing time, and showed the strongest positive-GFP at 1 month after marked, with about 60%of GFP-positive cells in the total BM. NSCs. The GFP didn't attenuate even until 2.5 months following the mark.