人精原干细胞体外分离、纯化及培养体系的初步研究
发布时间：2019-06-07 13:58
【摘要】：目的 采用两种酶两步法分离纯化人睾丸精原干细胞,探索精原干细胞体外培养方法,在体外建立稳定的精原干细胞培养体系。 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。
[Abstract]:Objective to isolate and purify human testicular spermatogonial stem cells by two enzyme two-step method, to explore the culture method of spermatogonial stem cells in vitro, and to establish a stable spermatogonial stem cell culture system in vitro. Methods Human spermatogonia stem cells were isolated and cultured by type I collagenase and trypsin digestion, Percoll density gradient centrifugation and differential adherent method, and the spermatogonia stem cells were identified by cellular immunofluorescence assay. Oct-4 positive cells were screened by flow cytometry, and the purity of spermatogonia stem cells was detected at the same time, and the co-culture system of spermatogonia stem cells and Sertoli cells was established. Results the living cell rate of human testicular cell suspension digested by two-step digestion was 21.07%. The expression of human testicular cell suspension purified by Percoll density gradient centrifugation combined with differential adherent centrifugation was detected by Oct-4 cell immunofluorescence assay. The proportion of positive cells detected by flow cytometry was 86.7%. The positive cells could grow stably on the feeder layer of Sertoli cells for 30 days. Conclusion the positive cells of Oct-4 expression are spermatogonial stem cells. Type I collagenase and trypsin two-step enzyme digestion is an economical, simple and easy method to isolate and culture human spermatogonial stem cells with less cell pollution. Human spermatogonia stem cells isolated by this method can form stable colonies on the supporting cell layer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2494848
[Abstract]:Objective to isolate and purify human testicular spermatogonial stem cells by two enzyme two-step method, to explore the culture method of spermatogonial stem cells in vitro, and to establish a stable spermatogonial stem cell culture system in vitro. Methods Human spermatogonia stem cells were isolated and cultured by type I collagenase and trypsin digestion, Percoll density gradient centrifugation and differential adherent method, and the spermatogonia stem cells were identified by cellular immunofluorescence assay. Oct-4 positive cells were screened by flow cytometry, and the purity of spermatogonia stem cells was detected at the same time, and the co-culture system of spermatogonia stem cells and Sertoli cells was established. Results the living cell rate of human testicular cell suspension digested by two-step digestion was 21.07%. The expression of human testicular cell suspension purified by Percoll density gradient centrifugation combined with differential adherent centrifugation was detected by Oct-4 cell immunofluorescence assay. The proportion of positive cells detected by flow cytometry was 86.7%. The positive cells could grow stably on the feeder layer of Sertoli cells for 30 days. Conclusion the positive cells of Oct-4 expression are spermatogonial stem cells. Type I collagenase and trypsin two-step enzyme digestion is an economical, simple and easy method to isolate and culture human spermatogonial stem cells with less cell pollution. Human spermatogonia stem cells isolated by this method can form stable colonies on the supporting cell layer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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相关期刊论文 前3条
1 毕罡,李彦锋,李黔生,靳风烁,张勇,徐序广,靳文生,霍文谦;人睾丸精原细胞的分离和纯化[J];第三军医大学学报;2005年11期
2 采克俊;张易祥;丁志丽;丁海雷;张念慈;刘莉;;精原干细胞的分离纯化方法[J];上海畜牧兽医通讯;2008年01期
3 胡红梅;徐富翠;李伟;吴绍华;;精原干细胞在成骨培养条件下的生物学特征[J];中国组织工程研究与临床康复;2007年33期
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