摘要
目的:为克服类胚体和视黄酸诱导体系的不足,实验使用胎鼠脑源性神经干细胞条件培养液定向诱导分化胚胎干细胞,观察是否可以获得大量具有高度增殖和进一步分化能力的神经干/祖细胞。方法:实验于2005-06/2006-09在中山大学干细胞与组织工程中心实验室完成。①取孕12.5d的C57BL/6J小鼠胚胎脑组织,放入含有神经干细胞培养液(含B27、肝素5mg/L、碱性成纤维细胞生长因子20μg/L、表皮生长因子20μg/L、100U/mL青霉素、0.1g/L链霉素的DMEM/F12)的离心管内,接种后37℃、体积分数为0.05的CO2培养箱中饱和湿度培养。3～5d后可见团状细胞球出现,即为原代的神经干细胞。取P5～10代神经干细胞消化成单细胞悬液,以5×104/cm2密度接种于神经干细胞培养液中,4～5d离心后收集上清液,即为胎鼠脑源性神经干细胞条件培养液。②P30代SC1002鼠胚胎干细胞消化成单个细胞后,移入上述制备的条件培养液中,5d后消化细胞,再重新接种于条件培养液中培养5d。常规消化后接种于神经干细胞培养液中,培养4～5d,即得到胚胎干细胞来源的神经干/祖细胞。对照组用神经干细胞培养液代替条件培养液。③所得细胞进行特异性抗原免疫细胞化学染色,并计数免疫染色阳性细胞率。RT-PCR检测标志性基因的表达。结果:①胚胎干细胞分化为神经干/祖细胞的比例:当消化成单个细胞的胚胎干细胞接种到神经干细胞条件培养液后,24h内聚集成细胞球并悬浮生长。免疫细胞化学检测少部分细胞表达nestin,在随后的培养过程中nestin阳性细胞逐渐增多。第4天细胞球周围的细胞几乎全部呈nestin阳性。当消化后重新接种于神经干细胞条件培养液中,可见部分细胞贴壁生长,10d后再常规消化接种于神经干细胞培养液中,呈典型的双极外形,nestin及RC2检测均呈阳性。RT-PCR检测显示Oct-4不再表达,而sox2,sox3,nestin,fabp7及bHLH转录因子均有显著表达。另一方面,当单个胚胎干细胞直接培养在神经干细胞培养液中,大量细胞死亡,仅10%存活,存活细胞表达Oct-4和nestin。②胚胎干细胞来源的神经干/祖细胞向神经元和胶质细胞的分化:经过10～12d的诱导分化,微管相关蛋白2和胶质纤维酸性蛋白细胞阳性率分别为(27.6±9.2)%和(29.7±11.6)%。结论:不需要共培养或添加诱导因子,应用胎鼠脑源性神经干细胞条件培养液成功地将胚胎干细胞定向诱导分化为大量高纯度的神经前体细胞,并进一步得到神经元和神经胶质细胞。
AIM： To conquer the deficiency of the embryonic stem cells （ESCs） and retinoic acid induction into neural cells, neural stem cell conditioned medium （NSC-CM） is employed to induced ESCs expect to generate neural stem/progenitor cells （NSCs/NPCS） of proliferating highly and differentiating further. METHODS： The experiment was performed at Center of Stem Cells and Tissue Engineering, Sun Yat-sen University from June 2005 to September 2006. ①C57BL/6J mouse fetuses on embryonic day 12.5 were isolated from their mother under deep anesthesia and placed into NSC medium containing B27, 5 mg/L heparin, both epidermal growth factor （EGF） and basic fibroblast growth factor （bFGF） at 20 μg/L, 100 U/mL Penicillin and 0.1 g/L streptomycin and DMEM/F-12 nutrient. The dissociated cells were incubated at 37℃ and CO2 condition of 0.05 volume fraction. The NSCs grew as free-floating clusters or neurospheres after 3-5 day culture. NSC-CM was prepared by trypsinizing NSCs at passage 5-10 to a single cell and seeded at 5×10^4 cells/cm^2 in NSC medium. NSC-CM was collected after 4-5 day cultures. ②After dissociated into single cells, SC1002 mouse ESCs at P30 were cultured in NSC-CM. After 5 days, cells were trypsinized and cultured in NSC-CM to differentiate for another 5 days. The resulting cells were trypsinized and transferred to NSC medium to expand for 4-5 days. NSC medium were instead of NSC-CM to work as control. ③The resulting cells were analyzed with specific antigen immunocytochemical staining and the rate of positive cells was calculated. RT-PCR was used to detect the expression of specific gene. RESULTS： ①Ratio of differentiation of ESCs into NSCs/NPCs： Single ESCs developed into a number of floating clusters called cell spheres when cultured in the NSC-CM in 24 hours. Results of immunocytochemical method found that parts of the cells in the cell spheres expressed nestin, after plating and the nestin positive cells increased gradually in subsequent culture period. Cells round the spheres were almost nestin positive at day 4. When the resulting cell spheres were dissociated and cultured in fresh NSC-CM, some cells attached. After 10 days the resulting cells were reseeded in NSC medium, bipolar cells of neural precursor morphology appeared and were identified as neural precursors by staining with nestin and RC2. RT-PCR analysis showed that all ESCs-derived NSCs/NPCs expressed the neural stem/precursor marker genes sox2, sox3, nestin, fabp7 and the basic helix-loop helix （bHLH） but oct-4. On the other hand, when single ESCs were cultured in NSC medium, there was no cell spheres formation. About 10% of cells survived and attached. A minority of survived cells expressed nestin and oct-4. ② Differentiation of neuron and glial cells from ESCs-derived NSCs/NPCs： After cultured in differentiation medium for 10-12 days, cells differentiated into neuronal and glial morphology. The positive rates of microtubule-associated protein 2 （MAP2） and glial fibrillary acidic protein （GFAP）-positive cells were respectively （27.6±9.2）% and （29.7±11.6）%. CONCLUSION： NSC-CM is a highly potent reagent for inducing the development of mouse ESCs into NSCs/NPCs thence into neurons and gila without any reagents and co culture.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第28期5489-5493,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助(30100188
30571891)~~
