N-钙黏素融合蛋白对P19细胞神经分化的影响

The effect of N-cad-Fc chimera protein on neuronal differentiation of P19 cells

目的探讨细胞间黏附分子对胚胎癌P19细胞体外定向神经细胞分化的作用。方法通过单层培养技术，采用视黄酸为诱导剂诱导P19细胞神经细胞分化，在浓度分别为0．1％明胶、2．5mg／L和10mg／L的N-钙黏素融合蛋白（N-cad-Fc）包被的培养板上进行培养，观察细胞形态的变化情况，用神经细胞特异标志蛋白β-Ⅲ型微管蛋白（Tuj-1）抗体进行免疫荧光染色，反转录聚合酶链式反应（RT-PCR）检测干细胞标志蛋白（oct3／4）、神经前体细胞标志蛋白巢蛋白（nestin）、神经元素1（ngn1）、微管相关蛋白（map2）和神经胶质细胞的特异性标志蛋白（gfap）的表达情况。结果使用5×10^-7mol／L的视黄酸成功诱导了P19细胞神经细胞分化，0．1％明胶组、2．5mg／L N—cad—Fc组和10mg／L N-cad-Fc组分化为神经元细胞的百分率分别为7．8％、28．6％和26．8％，在包被有N-cad-Fc的培养板上培养，神经标志性蛋白ngn1和map2的表达明显提前、增强，干细胞标志蛋白oct3／4表达提前减弱，未见神经胶质细胞的特异性标志蛋白gfap表达。结论视黄酸可以诱导P19细胞向神经细胞分化，细胞间黏附分子对分化有促进作用。

Objective To observe the effect of intercellular adhesion molecule on neuronal differentiation of P19 cells in vitro. Methods Monolayer P19 cells were cultured on the plates coated with 0.1% gelatin, 2.5 mg/L and 10 mg/L N-cad-Fe （a fusion protein of the N-eadherin extracellular domain and the IgG Fc domain）, and then 5 × 10^-7 mol/L retinoic acid （RA） was used to induce neuronal differentiation of P19 cells. Neuron specific class Ⅲ β tubulin （Tuj-1） antibody was examined by immunofluorescence staining. The protein expressions of oct3/4 in stem cells, nestin in precursor cells, ngnl and map2 in neurons and gfap in glial cells were detected by reverse transcription-polymerase chain reaction （RT PCR）. Results RA at 5 × 10^-7 mol/L could induce P19 cells to differentiate into neurons. The average differentiation rates on 0.1% gelatin, 2.5 mg/L and 10 mg/L N-cad-Fc were 7.8%, 28.6% and 26.8%, respectively. Cells cultured on N cadFc coated plates dramatically up-regulated the expressions of neural specific proteins ngnl and map2. The expression of stem cell specific protein oct3/4 was decreased ahead of schedule and the expression of glial cell specific protein gfap could not be found by RT-PCR. Conclusions RA can induce P19 cells to differentiate into neuronal cells. Intercellular adhesion molecule has promotional effect on cell differentiation.