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目的:探讨支持细胞对体外培养精原干细胞的作用途径。方法:选用7 d龄雄性昆明种小鼠,两步酶消化法获得睾丸组织细胞悬液,差异时间贴壁法分离精原干细胞和支持细胞,免疫荧光法和油红O染色法分别对其进行生物学鉴定,流式细胞仪对精原干细胞进行纯度分析。按培养条件的不同将实验分为3组:精原干细胞与支持细胞共培养组(A组)、条件培养基组(B组)、常规培养基组(C组)。其中条件培养基按单纯支持细胞培养清液∶双倍浓缩的DMEM/F12∶胎牛血清=4.5∶4.5∶1的比列配置;常规培养基即含体积分数10%胎牛血清的DMEM/F12。台盼蓝法测定各组贴壁率,四甲基偶氮唑盐(MTT)法测定各组精原干细胞的吸光度并绘制增殖曲线。倒置显微镜下观察各组精原干细胞增殖特点及集落形成情况。比较各组精原干细胞24 h贴壁率、增殖曲线及存活时间的不同。结果:A组精原干细胞24 h贴壁率大于B组及C组(P<0.05),而B、C组间无差异(P>0.05);A组精原干细胞接种起即稳定增殖,于7~10 d形成稳定集落并维持约30 d的特性。B组和C组均表现为精原干细胞经短暂的增殖后呈现快速减少的趋势,培养1周后,精原干细胞数目明显减少。结论:支持细胞对体外精原干细胞的作用是依靠两者之间的直接联系和支持细胞的旁分泌2种途径;而仅依靠支持细胞的旁分泌作用不能促进精原干细胞的贴壁和增殖。
Objective: To explore the role of supportive cells in culturing spermatogonial stem cells in vitro. Methods: Seven-day-old male Kunming mice were used to obtain testicular cell suspension by two-step enzymatic digestion. Spermatogonial stem cells and supporting cells were isolated by differential time-adherence method. Immunofluorescence and oil red O staining were performed on them respectively Biological identification, purity analysis of spermatogonial stem cells by flow cytometry. The experiment was divided into three groups according to different culture conditions: co-culture of spermatogonial stem cells and supporting cells (group A), conditioned medium group (group B) and conventional medium group (group C). The conditioned medium was prepared according to the ratio of purely cultured cell culture supernatant: twice concentrated DMEM / F12: fetal bovine serum = 4.5: 4.5: 1; conventional medium, DMEM / F12 containing 10% fetal bovine serum . The adherent rate of each group was determined by trypan blue method. The absorbency of spermatogonial stem cells in each group was determined by MTT assay and the proliferation curve was drawn. Under inverted microscope, the proliferation characteristics and colony formation of spermatogonial stem cells in each group were observed. The adherent rate, proliferation curve and survival time of spermatogonial stem cells in each group were compared. Results: The adherent rate of spermatogonial stem cells in group A was significantly higher than those in group B and C (P <0.05), but not in group B and C (P> 0.05) From 7 to 10 days, stable colonies formed and maintained for about 30 d. In group B and group C, spermatogonial stem cells showed a tendency of rapid decrease after transient proliferation, and the number of spermatogonial stem cells decreased significantly after one week of culture. CONCLUSION: The effect of supporting cells on spermatogonial stem cells in vitro depends on the direct connection between them and the paracrine way of supporting cells. Paracrine effect of supporting cells can not promote the adherence and proliferation of spermatogonial stem cells.