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 proteolytic mechanism of amyloid precursor protein （APP） and to explore amyloidbeta （Aβ） generation in living neurons. Methods DNA fragments were amplified by PCR or synthesized. The four fragments, CFP, 54bp, YFP and C99 were ligated into pcDNA3.0 vector to construct the recombinant plasmids pcDNA3.0-CFP-54bp- YFP and pcDNA3.0-CFP-54bp-YFP-C99. The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99. The expression of fusion gene was examined under a multiphoton laser scanning microscope. Fluorescence resonance energy transfer （FRET） was used to measure the β cleavage and γ cleavage of APE Aβ generation was confirmed by immunocytochemistry and multiphoton laser scanning microscopy. Cell viability was tested by MTT assay at different time points. Results （1） The double restriction endonuclease digestion and sequencing analysis confirmed the authenticity of the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp- YFP-C99. （2） Blue and yellow fluorescences were detected in the transfected cells. （3） FRET occurred in pcDNA3.0-CFP- 54bp-YFP-transfected cells but not in pcDNA3.0-CFP-54bp-YFP-C99-transfected cells. （4） Aβ was produced in the pcDNA3.0- CFP-54bp-YFP-C99 transfected cells. （5） Aβ-deposition was widespread in the cell. （6） Cell viability decreased along with the intracellular Aβ deposition. Conclusion C99 is important for the APP β cleavage. Aβ may be generated and deposited in cells at the early stage of Alzheimer＇s disease. Intracellular Aβ accumulation brings deleterious effects on cells.
