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目的探讨突触蛋白-Ⅰ在体外诱导胚胎干细胞(ESC)向神经细胞分化过程中的作用,寻求这一过程的可调控点或调控切入点。方法采用“五步法”体外诱导 ESC 向神经细胞分化,于不同诱导阶段转染突触蛋白-Ⅰ反义寡核苷酸,观察转染后 ESC 的形态学、分化效率及其他神经特异性蛋白表达的变化。同时以突触蛋白-Ⅰ反义寡核苷酸对 PC12细胞诱导过程的影响作参照。结果胚胎干细胞分化的第3阶段反义链组的突起伸长速度较正常组和正义链组减慢,分化的神经前体细胞[nestin(+)]比例较正常组和正义链组明显减少(68.5%±4.2% vs 76.2%±5.1%和75.8%±4.9%,P<0.05)。第4阶段反义链组所扩增的神经前体细胞[nestin(+)]比例较正常组和正义链组明显减少(75.1%±4.7% vs 90.2%±4.3%和88.7%±4.5%,P<0.01)。第5阶段反义链组细胞之间的联系较正常组和正义链组减少,神经元样细胞[MAP2(+)]的比例较正常组和正义链组减少(30.7%±3.2% vs 41.2%±2.7%和40.5%±2.4%,P<0.05)。PC12细胞反义链组于诱导第1、4、7、10天细胞突起长度均较正常组和正义链组短,细胞分化率(0.33%±0.46%、9.78%±3.47%、45.3%±7.98%和34.2%±5.89%)显著低于正常组(1.81%±0.40%、45.13%±4.17%、90.26%±4.68%和84.66%±4.81%)和正义链组(P<0.01)。结论抑制突触蛋白-Ⅰ的表达可导致胚胎干细胞向神经细胞分化的进程滞后和神经分化效率降低,提示突触蛋白-Ⅰ在胚胎干细胞的体外神经分化过程中各阶段均起着重要的作用,可能参与了其中的调控机制。
Objective To investigate the role of synaptophysin-Ⅰ in inducing embryonic stem cells (ESCs) to differentiate into neural cells in vitro and to find a possible point of regulation or regulation of this process. Methods The “five-step method” was used to induce the differentiation of NSCs into neurons in vitro. Transfection of synuclein-I antisense oligonucleotides was performed at different induction stages. The morphology, differentiation efficiency and other neuro-specific Changes in sexual protein expression. At the same time, the effect of synapsin-I antisense oligonucleotide on the induction of PC12 cells was used as a reference. Results The rate of neurite outgrowth in the third stage of ES cell differentiation was slower than that in the normal group and the sense chain. The ratio of nestin (+) in differentiated neural progenitor cells was significantly lower than that in the normal group and the sense chain 68.5% ± 4.2% vs 76.2% ± 5.1% and 75.8% ± 4.9% respectively, P <0.05). The proportion of nestin (+) cells amplified by antisense group in stage 4 was significantly lower than that in normal group and sense chain (75.1% ± 4.7% vs 90.2% ± 4.3% and 88.7% ± 4.5%, respectively) P <0.01). Compared with normal group and sense chain group, the number of neuronal-like cells [MAP2 (+)] in the fifth phase of antisense group decreased as compared with the normal group and the sense chain group (30.7% ± 3.2% vs 41.2% ± 2.7% and 40.5% ± 2.4%, P <0.05). The length of protuberances on the 1st, 4th, 7th and 10th day in PC12 cells antisense group were shorter than those in normal group and sense strand group. The rates of cell differentiation (0.33% ± 0.46%, 9.78% ± 3.47%, 45.3% ± 7.98 % And 34.2% ± 5.89% respectively) were significantly lower than those in the normal control group (1.81% ± 0.40%, 45.13% ± 4.17%, 90.26% ± 4.68% and 84.66% ± 4.81% Conclusion Inhibition of the expression of synaptophysin-Ⅰ may lead to the process lag and the differentiation efficiency of embryonic stem cells differentiating into neurons, suggesting that synuclein-Ⅰ plays an important role in the process of embryonic stem cells in vitro differentiation. May be involved in the regulatory mechanism.