中国医学科学院学报ACTAACADEMIAEMEDICINAESINICAEVol.37No.2185基金项目:国家自然科学基金青年基金(81100057)和长治医学院创新团队项目(CX201408)SupportedbytheNationalNaturalSciencesYouthFounda-tionofChina(81100057)andChangzhiMedicalCollegeInnovationTeamProject(CX201408)·论著·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李婷1,2,李晨1,张翠英1,赵杰31长治医学院生理学教研室,山西长治0460002中南大学湘雅医学院病理生理学系,长沙4100083中南大学湘雅医院神经外科,长沙410008通信作者:张翠英电话:0355-3151440,电子邮件:zhangcy157@sohu.com;赵杰电话:0731-84327039,电子邮件:steelzj@126.com摘要: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AnnexinV/碘化丙啶染色、Hoechst33342活细胞染色镜下观察等方法,在培养传代后2和24h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2h,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24h时间点,Accutase消化的细胞凋亡率经AnnexinV/碘化丙啶、Hoechst33342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4d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24h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。关键词:神经干细胞;神经球;Accutase;胰蛋白酶;凋亡中图分类号:R392.2+8文献标志码:A文章编号:1000-503X(2015)02-0185-10DOI:10.3881/j.issn.1000-503X.2015.02.009EffectofAccutaseorTrypsinDissociationontheApoptosisofHumanStriatum-derivedNeuralStemCellsLITing1,2,LIChen1,ZHANGCui-ying1,ZHAOJie31DepartmentofPhysiology,ChangzhiMedicalCollege,Changzhi,Shanxi046000,China2DepartmentofPathophysiology,XiangyaSchoolofMedicine,CentralSouthUniversity,Changsha410008,China3DepartmentofNeurosurgery,XiangyaHospital,CentralSouthUniversity,Changsha410008,ChinaCorrespondingauthor:ZHANGCui-yingTel:0355-3151440,E-mail:zhangcy157@sohu.com;ZHAOJieTel:0731-84327039,E-mail:steelzj@126.com中国医学科学院学报186April,2015ABSTRACT:ObjectiveToobservetheapoptosisofneuralstemcells(NSCs)atdifferentialtimepointsafterthedissociationofneurospheresbyAccutaseortrypsin.MethodsTheNSCswereisolatedfromstriatumofhumanfetalsthatsufferedabortionat12-16weeksofpregnancy.The3rd-5thpassagesofNSCsweredigestedbyAccutaseortrypsin.Onlyvortexingwasapplied,andthetrituratingbyPasteurpipettewasavoidedtoattenuatetheinjurytothecellsduringthedissociation.ThesinglecellswerethenstainedbyAnnexinV/propidiumiodideandHoechst33342.Theapoptosisrates2and24hoursafterpassagingwereevaluated.ResultsThetrypanbluestainingconfirmedthatimmediatelyafterthedissociation,theviabilityofcellsdigestedbytrypsinwas(83.10±6.76)%,whichwassignificantlylowerthanthatdigestedbyAccutase,whichwas(91.65±4.43)%(P<0.05).TheapoptosisoftheNSCsdigestedby...