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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;采用细胞免疫荧光法进行人精原干细胞的鉴定;流式细胞法对Oct-4标记阳性的细胞进行筛选,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、简单易行、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饲养层上形成稳定集落。
Objective To isolate and culture human testicular spermatogonial stem cells by two steps of two enzymes. Methods Human spermatogonial stem cells were isolated and cultured using collagenase Ⅰ and trypsin digestion, Percoll density gradient centrifugation and differential adherence method. The expression of human spermatogonial stem cells was detected by immunofluorescence method. Flow cytometry The Oct-4 positive cells were screened and the purity of spermatogonial stem cells was tested simultaneously to establish spermatogonial stem cells and supportive cells co-culture system. Results The viability of human testicular cell suspension digested by two enzymes was 91.07%. Percoll density gradient centrifugation and differential adherent centrifugation purified cells were detected by Oct-4 immunofluorescence. Flow cytometry The percentage of positive cells was 86.7%. The positive cells could grow stably for 30 days on the supportive feeder layer. Conclusion The positive cells of Oct-4 expression are spermatogonial stem cells. Type I collagenase and trypsin two-step enzymatic digestion is an economical, simple and easy method for the isolation and culture of human spermatogonial stem cells with less pollution to cells. The human spermatogonial stem cells isolated by this method can be cultured in supportive cells Formation of stable colonies on the layer.