神经生长因子诱导后的神经元样PC12细胞与成肌干细胞共培养的实验研究

Co-culture of myoblasts with differentiated PC12 cells induced by nerve growth factor

目的探讨神经元样PC12细胞对C3H小鼠成肌干细胞C2C12增殖与分化的影响。方法利用Transwell建立PC12细胞和C2C12细胞共培养体系，实验分为3组：空白对照组：C2C12细胞单独培养；实验组：C2C12细胞与已分化的PC12细胞共培养；阴性对照组：C2C12细胞与未分化的PC12细胞共培养。免疫荧光染色鉴定神经生长因子（NGF）对PC12细胞的促分化效应；流式细胞仪检测共培养条件下C2C12细胞的增殖情况；逆转录聚合酶链式反应（RT—PCR）和实时荧光定量核酸扩增（qPCR）检测与PC12细胞共培养3、7d后C2C12细胞生肌素、结蛋白基因的表达。结果体外条件下，NGF诱导PC12细胞呈现神经元样特征：具有细长的细胞突起，并阳性表达微管相关蛋白-2。流式细胞检测证实：已经分化的PC12细胞抑制C2C12细胞的增殖。实验组DNA合成前期的C2C12细胞所占百分比（77．2％±0．4％）较空白对照组（71．0％±0．6％）和阴性对照组（70。8％±0．8％）高，反应增殖活力的增殖指数（22．8％±0．4％）较空白对照组（29．0％±0．6％）和阴性对照组（29．2％±0．8％）低，差异均有统计学意义（P〈0．05）。实验组C2C12细胞生肌素、结蛋白基因的表达高于同一培养时间其他组，差异均有统计学意义（P〈0．05）。结论神经化的PC12细胞抑制C2C12细胞的增殖，但促进其分化。

Objective To explore the effect of neuron-like cells on proliferatinn and differentiation of C2C12 cells. Methods A co-culture system of PC12 cells and C2C12 cells was established using Transwell. They were allocated into 3 groups at random. In tbe blank control group, merely C2C12 cells were cultured： in the experimental group, tbe C2C12 cells were co-cultured with differentiated PC12 cells; in the negative control group, tbe C2C 12 cells were co-cultured with undifferentiated PC 12 cells. The effects of nerve growtb factor （NGF） on differentiation of PC12 cells were identified by the immunofluorescence method. Proliferation of C2C12 ceils was determined by flow cytumetry. The expressions of Myngenin anti Desmin in the C2C12 cells co-cultured with PC12 cells at 3 and 7 days were detected by RT-PCR and qPCR. Results The PC12 cells showed neurun-like appearance with neurite structure due to the effects of NGF and were MAP-2 positive. The differentiated PC12 cells inhibited the proliferation of C2C12 cells. The percentage of C2C12 cells at the DNA G1 period in the experimental group （77.2%±0.4% ） was significantly higher than that in the blank control group （71.0% ±0.6% ） and in the negative control group （70.8% ±0.8% ） （ P 〈 0.05）. The proliferatiun index （Prl） in the experimental group （22.8% ± 0. 4% ） was significantly lower than that in the blank control group （29.0% ±0.6% ） and in the negative control group （29.2% ±0.8% ） （ P 〈 0.05） . The expressions of Myogenin and Desmin in the C2C12 cells in the experimental group were significantly bigher than those in the other 2 groups at the same time point. Conclusion The differentiated PC12 cells may inhibit the proliferation of C2C12 cells but activate their differentiation.