人精原干细胞分离方法的改进.docVIP

人精原干细胞分离方法的改进该题目修改后不象论著题目，更像技术方法类文献，请作者斟酌 该题目修改后不象论著题目，更像技术方法类文献，请作者斟酌 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30 d。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] A Improvement of the method to isolate human Spermatogonial stem cells Liu Shixue, Tang Wei，Xiong Tao （Department of urology，First Affiliated Hospital of Chongqing Medical University，400016） [Abstract] Objective Two-step method and two enzymes isolates spermatogonial stem cells in human testis. 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 density gradient centrifugation coupled with differentiated adherence centrifugal purification, were confirmed to be human spermatogonial stem cells as assayed by immunocytochemistry against OCT-4. The purity of Oct-4 positive cells was 86.7% as detected by flow cytometry. Oct-4 positive cells can stable growth 30 days on the feeder layer of the sertoli cells. Conclusion The Oct-4 positiv