Accutase消化传代对人胚胎纹状体来源神经干细胞凋亡的影响  

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

作　　者：李婷[1,2] 王小莉[2] 宋娟[2] 李晨[1] 张翠英[1] 赵杰[3] 王金胜[4] 

机构地区：[1]长治医学院生理学教研室,山西长治046000 [2]中南大学湘雅医学院病理生理学系,长沙410008 [3]中南大学湘雅医院神经外科,长沙410008 [4]长治医学院病理学教研室,山西长治046000

出　　处：《中南大学学报（医学版）》2015年第5期471-478,共8页Journal of Central South University ：Medical Science

基　　金：山西省中美国际合作项目(2013081054);长治医学院创新团队项目(CX201408)~~

摘　　要：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(Td T-mediated d UTP 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。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 after 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.

关 键 词：神经干细胞 神经球 accutase TUNEL 凋亡 活性caspase-3 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]