低浓度胶原酶差速消化法分离纯化小鼠Leydig细胞的研究  

A novel method of isolating Leydig cells from mice testes using low concentration of collagenase digestions and differential centrifugation

作　　者：钟量[1] 孙杰[2] 陈凡凡[3] 朱英坚[1] 刘国华[1] 

机构地区：[1]上海市第一人民医院泌尿外科,200080 [2]上海儿童医学中心 [3]上海组织工程研究与开发中心

出　　处：《中华小儿外科杂志》2010年第9期694-697,共4页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金（编号：30700878）

摘　　要：目的 建立简单快速获得Leydig细胞的分离纯化新方法.方法 分阶段应用低浓度胶原酶,采用差速消化法分离纯化小鼠睾丸内的Leydig细胞.将不同阶段获得的细胞分别培养,观察其原代生长情况.采用免疫荧光染色,观察培养的Leydig细胞内的标志性酶--3β羟基类固醇脱氢酶（3β-HSD）、胆固醇侧链裂解酶（CYP11A1）和17α羟化酶（CYP17A1）的表达情况.结果 使用低浓度胶原酶短时间重复三次消化,以第二次消化获得的Leydig细胞纯度最高69.6%±4.16%,增殖能力最强.培养7 d后纯度增高至90%.原代培养的各个阶段均可检测到睾酮合成酶谱的表达.结论 应用低浓度胶原酶差速消化法可以获得大量高活力的Leydig细胞,原代培养效果理想,细胞功能保存完好.Objective To establish a novel method of isolating and purifying Leydig cells from mice testes.Methods Testes of postpuberal mice were harvested and digested in low concentration of collagenase for 15 minutes.The digestion was repeated for 3 times.Cells were harvested,resuspended and cultured.Leydig cell were identified by its specific markers such as 3β-hydroxysteroid dehydrogenase （3β-HSD）,cholesterol side-chain cleavage enzyme （CYP11A1）,17α-hydroxylase （CYP17A1）.Results Leydig cells harvested after 2 times digestion had the highest purity （69.6% ± 4.16%）and proliferative capacity.The 90% purity achieved after 7 days＇ culture.Positive staining of steroidogenic enzymes appeared all the time in primary culture.Conclusions Leydig cells can be isolated and purified by low concentration of collagenase digestions and differential centrifugation.

关 键 词：莱迪希细胞 细胞分离 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]