13-顺维甲酸体外诱导神经母细胞瘤干细胞分化的体外实验

The experimental research of using 13-cis-retinoic acid to induce differentiation of stem cells of neuroblastoma in vitro

目的体外观察并鉴定13-顺维甲酸诱导神经母细胞瘤(NB)干细胞的分化作用,为临床用维甲酸治疗NB微小残留病灶提供实验依据。方法 取14例伴骨髓转移的Ⅳ期NB患儿的骨髓液标本,分离出NB细胞,将原代肿瘤细胞接种于无血清干细胞培养基中悬浮培养;在含5μmol·L^(-1)13-顺维甲酸的分化培养基中培养神经球细胞,观察细胞的形态变化;RT-PCR法检测神经球细胞诱导前、诱导3和9 d Oct-4表达水平的改变;利用细胞免疫荧光技术比较诱导前及诱导9d神经球细胞nestin表达的差异。结果 14例骨髓标本中,4例分离到原代NB细胞。无血清培养基中培养4～6d后,可见原代悬浮神经球形成,传代后成球细胞能再次分裂增殖为神经球。神经球细胞在血清培养基中呈贴壁生长,呈三角形或星形,添加5μmol·L^(-1)13-顺维甲酸后,细胞生长速度降低,形态发生明显改变。RT-PCR法检测结果显示,13-顺维甲酸诱导9d后,神经球细胞Oct-4表达水平逐渐降低;细胞免疫荧光显示诱导9d后神经球细胞nestin表达减弱。结论 13-顺维甲酸能有效诱导NB干细胞分化。

Objective To observe and identify that 13-cis-retinoic acid can induce differentiation of stem cells of neuroblastoma, to provide experimental evidence for clinical application of 13-cis-retinoic acid for the treatment of neuroblastoma. Methods Bone marrow aspirates were obtained from children with neuroblastoma at stage IV, tumor cells were collected and cultivated in the serum-free stem cell medium containing DMEM-F12, 20 μmol · L^-1 EGF, 20μmol · L^-1 bFGF and 2% B27;A mesh sieve with 400 holes was used to leach the medium which contained neurospheres, collected the neurospheres which could not get through the mesh sieve and then reaked up the clusters of neurospheres using mechanical method to produce the suspension of single cell neurospheres. A single cell neurosphere was placed into DMEM/F12 differentiation medium with 10% FBS to cultivate, added 13-cis-retinoic acid into differentiation medium to make the final density of 5μmol · L^-1 and then observed morphological changes of the cells after adding 13-cis-retinoic acid into differentiation medium one day 0, day 3 and day 9, respectively. The RNAs of neuroblastoma neurospheres were extracted with the cases after 0 day, 3 days and 9 days induction, respectively, then they were reverse transcripted to obtain cDNA. RT-PCR method was used to detect the change of expression level of stem cell Oct-4 gene during the process of induction. Neurosphere cells without induction or with induction by 5 μmol · L^-1 13-cis-retinoic acid were added respectively into DMEM/F12 differentiation medium with 1% FBS. Adding these two groups of cells into sterile coverslip treated by Poly-D-Lysine, then the cell immunofluorescence technology was used to determine the difference of neuro-progenitor cell marker-nestin after cells adhesion to the coverslip. Results Primary suspended neurospheres could be observed after cultivation in the serum-free stem cell medium for 4 - 6 days. Neurosphere cells cultivated in the serum medium began to grow and adhere to the coverslip. The growing neurosphere cells were in the shape of star or triangle, the cell processes were blunt and short. After adding 5 μmol · L^-1 13-cis-retinoic acid into the medium, the growing speed of cells slowed down, morphogenesis of the ceils changed greatly, cell processes increased in number and it＇s shape became longer then connected into network. The result of RT-PCR revealed that the uninduced NB neurosphere cells could express Oct-4, the ability to express Oct-4 of the NB neurosphere cells induced by 13-cis-retinoic acid decreased after 3 days and under the same condition the ability to express Oct-4 almost disappeared after 9 days. It was evident that the ability to express Oct-4 of the NB neurosphere cells induced by 13-cis-retinoic acid was decreased as time elapsed. Cell immunofluorescence indicated that the NB neurosphere cells without induction could express nestin, the ability to express nestin of the NB neurosphere cells induced by 13-cis-retinoic acid was weaken greatly after 9 days. Conclusions 13-cis-retinoic acid could induce differentiation of stem cells of neuroblastoma efficiently in vitro.