Accutase和胰蛋白酶消化传代对人胚纹状体来源神经干细胞凋亡的影响

目的探讨Accutase和胰蛋白酶消化神经干细胞(NSCs)后对其凋亡过程的影响。方法来自人类12~16周自然流产胚胎纹状体组织,分离并体外培养NSCs,将体外培养的第3~5代神经球,分别用胰蛋白酶、Accutase消化,消化过程中仅振荡打散,避免巴斯德吸管反复吹打。将神经球打散成的单细胞悬液接种。用Annexin V/碘化丙啶染色、Hoechst 33342活细胞染色镜下观察等方法,在培养传代后2和24 h时间点检测神经干细胞凋亡率。结果两种酶消化后立即用台盼蓝染色判断细胞活性,Accutase消化后活细胞比例为(91.65±4.43)%,胰蛋白酶仅有(83.10±6.76)%,二者比较差异有统计学意义(P<0.05)。体外培养的第3~5代人胚纹状体NSCs形成的神经球传代后2 h,Accutase和胰蛋白酶传代的细胞凋亡率差异有统计学意义(P<0.01),而到24 h时间点,Accutase消化的细胞凋亡率经Annexin V/碘化丙啶、Hoechst 33342活细胞染色两种方法检测已经达到50%,而胰蛋白酶消化后的细胞凋亡率在此时间点维持在15%~20%。Accutase消化后的NSCs生长4 d后,克隆形成率和神经球直径分别为(7.83±1.32)%和(43.5±9.76)μm,均显著低于胰蛋白酶传代组的(19.22±3.66)%和(58.4±18.73)μm(P<0.01)。结论虽然台盼蓝染色显示Accutase消化神经球后的细胞存活比胰蛋白酶更多,但消化后24 h内,Accutase消化的细胞凋亡率显著升高,最终神经球新克隆形成率低,直径更小。胰蛋白酶消化振荡打散神经球后的凋亡率较Accutase具有明显优势;神经球消化后台盼蓝即时染色的结果不足以预示后期NSCs凋亡率。

Effect of Accutase or Trypsin Dissociation on the Apoptosis of Human Striatum-derived Neural Stem Cells

Objective To observe the apoptosis of neural stem cells(NSCs)at differential time points after the dissociation of neurospheres by Accutase or trypsin.Methods The NSCs were isolated from striatum of human fetals that suffered abortion at 12-16 weeks of pregnancy.The 3^rd-5^th passages of NSCs were digested by Accutase or trypsin.Only vortexing was applied,and the triturating by Pasteur pipette was avoided to attenuate the injury to the cells during the dissociation.The single cells were then stained by Annexin V/propidium iodide and Hoechst 33342.The apoptosis rates 2 and 24 hours after passaging were evaluated.Results The trypan blue staining confirmed that immediately after the dissociation,the viability of cells digested by trypsin was(83.10±6.76)%,which was significantly lower than that digested by Accutase,which was(91.65±4.43)%(P＜0.05).The apoptosis of the NSCs digested by Accutase was higher than that digested by trypsin at both 2 and 24 hours after passaging(P＜0.01).Four days after the passaging,both the new clone formation rate and diameter of new spheres after trypsin digestion were significantly higher than those after Accutase digestion(P＜0.01).Conclusions Although the viability of NSCs immediately after the disassociation by trypsin is lower than that digested by Accutase,the apoptosis of NSCs subsequently caused by trypsin is lower than that caused by Accutase.Trypan blue test immediately after the disassociation can not be used as an indicator in estimating the apoptosis of NSCs during the expanding.