人精原干细胞的分、培养及鉴定.docVIP

人精原干细胞的分离、培养及鉴定 刘世学1 唐伟* 熊涛2 （重庆医科大学附属第一医院泌尿外科，400016） 基金项目：重庆市卫生局重点项目（No. 2009-2-80） This study was supported by Key project research grant from Research Development Foundation of Health Bureau of ChongQing (No. 2009-2-80) 【摘要】目的：分离培养人睾丸精原干细胞请更具体些，如该方法也是文章讨论重点，也可采用：采用……方法分离……，为体外稳定培养精原干细胞系提供新的实验方法。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；运用细胞免疫荧光术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证实是精原干细胞结果不能用结论性语言，而需要描述形态观察结果或数据支持，流式细胞技术检测所获精原干细胞细胞纯度为86.7%，能够在支持细胞饲养层上形成稳定的精原干细胞集落同上。结论：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结论首先应对应结果，即培养分离的是什么细胞，然后再对技术进行结。。 请更具体些，如该方法也是文章讨论重点，也可采用：采用……方法分离…… 结果不能用结论性语言，而需要描述形态观察结果或数据支持 同上 结论首先应对应结果，即培养分离的是什么细胞，然后再对技术进行结。 【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] Isolation,culture,and identification of human Spermatogonial stem cells Liu Shi-xue, Tang Wei， Xiong Tao （Department of urology，First Affiliated Hospital of Chongqing Medical University，400016） Abstract Objective: To establish an in vitro experimental method for stable culturing of human spermatogonial stem cells by isolating and screening stem spermatogonium. Methods: The isolation and culture of spermatogonial stem cells were performed using enzymatic digestion of type I collagenase and trypsin, Percoll density gradient centrifugation, and differentiated wall-adherence method. Cytoimmunity fluorescence technology was employed for identification of stem spermatogonium and flow cytoimmunity fluorescence technology was employed for screening of OCT-4 labeled positive cells. The purity of the human spermatogonial stem cells was determined and a co-culture system for spermatogonial stem cells and Sertoli cells was established. Results: The viability of the cells was 91.07% in the suspension of human spermatogonial stem cells digested in a two-step enzymatic process. The cells obtained after Percoll de