人精原干细胞分离,培养及鉴定

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1、人精原干细胞分离、培养及鉴定刘世学，唐伟，熊涛（400016重庆，重庆医科大学附属第一医院泌尿外科）[摘要]目的采用两种酶两步法分离培养人睾丸精原干细胞。方法先后应用I型胶原酶和胰蛋白酶消化法，Percoll密度梯度离心，差异贴壁法，进行人精原干细胞的分离及培养；细胞免疫荧光法行人精原干细胞的鉴定；流式细胞免疫荧光法对Oct-4标记阳性的细胞行筛选，并同时检测所获精原干细胞的纯度，建立精原干细胞和支持细胞共培养体系。结果两种酶两步法消化的人睾丸细胞悬液活细胞率为91.07%，Percoll密度梯度离心结合差异贴壁离心纯化后的细胞经Oct-4细胞免疫荧光法检测表达阳性，流式细胞技术

2、检测阳性细胞所占比例为86.7%，该阳性细胞能够在支持细胞饲养层上稳定生长30d。结论：所获Oct-4表达阳性细胞为精原干细胞。I型胶原酶和胰蛋白酶两步酶消化法是一种经济的、简单易行的、对细胞污染损伤少的分离培养人精原干细胞的方法，利用此方法分离的人精原干细胞能够在支持细胞层上形成稳定集落。【关键词】精原干细胞；分离；培养；鉴定[中图法分类号][文献标志码]AIsolation,culture,andidentificationofhumanSpermatogonialstemcellsLiuShixue,TangWei，XiongTao（Departmentofurology，

3、FirstAffiliatedHospitalofChongqingMedicalUniversity，400016）[Abstract]ObjectiveTwo-stepmethodandtwoenzymesisolatesspermatogonialstemcellsinhumantestis.MethodsTheisolationandcultureofspermatogonialstemcellswereperformedusingenzymaticdigestionoftypeIcollagenaseandtrypsin,Percolldensitygradientce

4、ntrifugation,anddifferentiatedwall-adherencemethod.CytoimmunityfluorescencetechnologywasemployedforidentificationofstemspermatogoniumandflowcytoimmunityfluorescencetechnologywasemployedforscreeningofOct-4labeledpositivecells.Thepurityofthehumanspermatogonialstemcellswasdeterminedandaco-cultur

5、esystemforspermatogonialstemcellsandSertolicellswasestablished.ResultsTheviabilityofthecellswas91.07%inthesuspensionofhumanspermatogonialstemcellsdigestedinatwo-stepenzymaticprocess.ThecellsobtainedafterPercolldensitygradientcentrifugationcoupledwithdifferentiatedadherencecentrifugalpurificat

6、ion,wereconfirmedtobehumanspermatogonialstemcellsasassayedbyimmunocytochemistryagainstOct-4.ThepurityofOct-4positivecellswas86.7%asdetectedbyflowcytometry.Oct-4positivecellscanstablegrowth30daysonthefeederlayerofthesertolicells.ConclusionTheOct-4positivecellsisspermatogonialstemcells.Thetwo-s

7、tepenzymedigestion(bytypeIcollagenaseandtrypsin)methodiseconomical,simple,andfeasibleforisolatingandculturingofhumanspermatogonialstemcells.Withlittlecontaminationandlesscelldamage,thismethodfacilitatesisolatedhumanspermatogonialstemcellstofo