不同方法分离的成人睾丸支持细胞与胰岛共移植的效果对比

目的比较成人睾丸支持（Sertoli）细胞不同分离方法的效果，建立成人Sertoli细胞简便高效的分离方法。方法将质量相等的睾丸组织按照不同分离方法随机分为3组：A组采用胰蛋白酶、DNA酶、胶原酶和透明质酸酶一步消化法；B组采用胰蛋白酶和DNA酶第一步消化，胶原酶和透明质酸酶第二步消化；C组采用胰蛋白酶和DNA酶第一步消化，透明质酸酶第二步消化，胶原酶第三步消化；D组为对照组。采用形态学观察和免疫组化鉴定Sertoli细胞；MTT法和流式细胞仪法测定3组Sertoli细胞的活性和纯度；应用生存分析方法比较3组Sertoli细胞与胰岛共移植至糖尿病鼠的效果。结果分离获得的细胞经形态学和免疫组化鉴定，具有Sertoli细胞的特征，A、B、C三组Sertoli细胞的纯度分别为（85．17±1．8）％、（92．33±2．5）％和（93．12±2．6）％，B组和C组的Sertoli细胞纯度显著高于A组（t=7．35，t=7．95，P=0．00，P=0．00）。B组Sertoli细胞活性于培养14d时达到峰值，此后缓慢下降。B组Sertoli细胞活性显著高于A组和C组（t=4．02，t=2．77，P=0．00，P=0．01），且B组Sertoli细胞与胰岛共移植术后胰岛移植物存活时间显著高于A、c、D组（F=165．548，P=0．000）。结论采用两步消化的方法能够获得纯度和活性较高的Sertoli细胞，其与胰岛共移植能够显著延长移植物存活。

Comparison of different isolation methods for adult Sertoli cells and co-transplantation with islet

Objective To compare the efficiency of different isolation methods for adult Sertoli cells. Methods Testis tissues were equally divided into three parts and treated with different isolation methods for Sertoli cells. Trypsin, DNase, collagenase and hyaluronidase were used for one-step digestion in method A. In method B testis tissues were first digested with trypsin and DNase, then with collagenase and hyaluronidase. In method C, trypsin, DNase were first used, followed by hyaluronidase, and then by collagenase. Sertoli cells were identified by morphology and immunohistochemistry method. The viability and purity of Sertoli cells were evaluated by MTT and flow cytometry （FCM） method. The effects of Sertoli cell co-transplantation on islet grafts were compared among three groups. Results Typical Sertoli cells were identified by morphology and immunohistochemistry method. Viability of Sertoli cells was highest with method B （t = 7.35, t = 7.95, P = 0.00, P = 0.00）. The purity of Sertoli cells detected by FCM was lower with method A （t = 4.02, t = 2.77, P = 0.00, P = 0.01）.Islets were co-transplanted with Sertoli cells isolated with three methods to renal capsule of diabetic mouse. Islets with Sertoli cells isolated by method B had the longest survival time （F = 165.548, P = 0.000）. Conclusion Sertoli cells obtained by two-step digestion method have the highest purity and viability and significantly prolong the survival of islet graft by co- transplantation.