CAJ | 학술논문

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.
목적 건립간단쾌속획득Leydig세포적분리순화신방법.방법 분계단응용저농도효원매,채용차속소화법분리순화소서고환내적Leydig세포.장불동계단획득적세포분별배양,관찰기원대생장정황.채용면역형광염색,관찰배양적Leydig세포내적표지성매--3β간기류고순탈경매(3β-HSD)、담고순측련렬해매(CYP11A1)화17α간화매(CYP17A1)적표체정황.결과 사용저농도효원매단시간중복삼차소화,이제이차소화획득적Leydig세포순도최고69.6%±4.16%,증식능력최강.배양7 d후순도증고지90%.원대배양적각개계단균가검측도고동합성매보적표체.결론 응용저농도효원매차속소화법가이획득대량고활력적Leydig세포,원대배양효과이상,세포공능보존완호.
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.