大鼠精原细胞的分离纯化及免疫化学研究

Isolation,purification and immunochemical characteristics of spermatogonia of rat

目的探讨大鼠精原细胞的分离纯化方法及纯化后的细胞特性。方法应用复合酶消化法制备10d龄SD大鼠的睾丸单细胞悬液,采用差速贴壁结合非连续性Percoll密度梯度离心的方法纯化精原细胞。分别以c-kit和α6-整合素作为细胞标记,观察睾丸组织中的精原细胞与纯化后细胞的免疫化学特征。通过流式细胞仪分别检测纯化后细胞表达c-kit和α6-整合素的阳性率。结果在睾丸组织中精原细胞特异表达c-kit和α6-整合素。纯化后的单细胞中有c-kit和α6-整合素的阳性表达。流式细胞仪检测:以c-kit作为细胞标记,未纯化组的阳性表达率为1·59%±0·04%,纯化组为68·33%±2·45%,两组差异非常显著(P<0·01);以α6-整合素作为细胞标记,未纯化组的阳性表达率为2·38%±0·60%,纯化组为72·04%±3·65%,两组差异非常显著(P<0·01)。台盼蓝排斥试验表明纯化后细胞的活性率>95%。结论C-kit、α6-整合素可作为特定时期精原细胞的表面标志。采用复合酶消化、差速贴壁结合非连续性Percoll密度梯度离心的方法纯化精原细胞,能获得纯度和活性较高的精原细胞。

Objective To explore the approach of isolation and purification of spermatogonia and its immunochemical characteristics. Methods Compound enzymatic digestions were used to prepare germ cell suspensions of Sprague-Dawley rats aged 10 days, and velocity sedimentation and discontinuous Pereoll density gradient centrifugation were used to isolate and purify the spermatogonia. Using c-kit and α6-integrin multiclone antibodies as markers respectively, the immunochemical characteristics of the spermatogonia in the testicular tissue were observed and the c-kit and α6-integrin expression rates of the purified cells were detected by flow cytometry. Results The spermatogonia uniquely expressed c-kit and α6-Integrin in the testicular tissue. C-kit and α6-integrin were positively expressed in the purified cell suspensions. Using c-kit as the cell marker, the positive rate was 1.59% ±0.04% in the unpurified group, significantly lower than that of the purified group （68.33% ± 2. 45%, P 〈 0. 01 ）. Using α6-integrin as the cell marker, the positive rate of the unpurified group was 2. 38% ± 0. 60% , significantly lower than that of the purified group （72.04% ±3.65%, P 〈0.01 ）. Trypan blue staining showed that the cell viability of the purified cell suspensions was more than 95%. Conclusion c-kit and α6-integrin can be used as the molecular markers of spermatogonium at special stage. Spermatogonia with high purity and viability can be obtained via the steps including digestions with enzymes, velocity sedimentation and discontinuous pereoll density gradient centrifugation.