Accutase 消化传代对人胚胎纹状体来源神经干细胞凋亡的影响

目的：观察人类胚胎纹状体来源神经干细胞(neural stem cells，NSCs)经消化酶accutase消化传代后是否 发生了大量细胞凋亡。方法：提取人13~18周自然流产胚胎脑组织纹状体的NSCs，体外形成神经球并培养至3~5 代。经消化酶accutase消化后传代，运用活性caspase-3染色、TUNEL(TdT-mediated dUTP nick end labeling)染色等方 法对多聚甲醛固定的NSCs进行凋亡检测，同时采用Annexin V和Hoechst33342/PI等联合染色方法对活细胞进行凋亡 鉴定，以确定NSCs在体外传代后的凋亡情况。结果：在传代后检测的3个时间点(1，24，72 h)中TUNEL染色和活 性caspase-3染色均高度重合。传代后1 h，TUNEL和活性caspase-3染色所检测到的凋亡率为20%~25%，传代后24 h增 高为75%~80%(P<0.01)，而传代后72 h，由于存活下来的细胞开始增殖，所以整体凋亡率降为60%~70%，明显低于 24 h(P<0.01)。结论：由人类纹状体来源NSCs形成的神经球，经accutase消化传代后24 h内即发生大量细胞凋亡。对于 体外培养的NSCs，抑制其凋亡可能是促使其自我更新和增殖，最终起到提高细胞扩增能力的有效手段。

Effect of accutase dissociation and passage on the apoptosis of human striatum derived neural stem cells

Objective: To explore the status of apoptosis in human striatum derived neural stem cells (NSCs) aft er accutase dissociation and passage. Methods: The NSCs were isolated from fetuses obtained through spontaneous abortion at 13- 18 weeks of pregnancy, which formed neurospheres in vitro. At passages of 3-5, the neurospheres were disassociated into single cell by accutase digestion and then passaged. At 1, 24 and 72 h aft er passage, the apoptosis of NSCs was measured by several methods, including active caspase-3 or TUNEL staining for fixed cells, Annexin V, Hoechst or PI staining for live cells. Results: At all of the 3 time points, the staining of TUNEL and active caspase-3 overlapped perfectly. The apoptosis rate of NSCs increased significantly from 20%-25% at 1 h to 75%-80% at 24 h after passage (P<0.01). At 72 h, the apoptosis rate was significantly decreased as compared to that at 24 h time point because of the self-renewal and proliferation of survived NSCs (P<0.01). Conclusion: Many cells in the neurospheres formed by human striatum-derived NSCs underwent apoptosis soon after accutase disassociation. For NSCs cultured in vitro, anti-apoptosis treatments might be a good method to increase the self-renewal and the proliferation of NSCs.