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目的观察精原干细胞在中药生精制剂中的生长特性,为探讨精原干细胞的多向分化能力提供实验依据。方法取5～7d龄昆明系雄性小白鼠,脱颈椎处死后,无菌取出睾丸,进行精原干细胞分离、培养与鉴定。取培养3d的精原干细胞,分为对照组和实验组。对照组用DMEM/F12完全培养液(含10%FBS、4mmoi/ L谷氨酰胺、0.12%NaHCO_3、0.5mmol/L丙酮酸钠、15mmol/LHEPES、50U/mL青霉素和50μg/mL链霉素)培养,实验组用条件培养液(DMEM/F12完全培养液中添加中药生精制剂生精胶囊浓缩液)培养。在倒置显微镜下观察细胞的形态变化,酶联吸收OD值,细胞DNA倍体分析。结果实验组精原干细胞在中药生精制剂条件培养液中较对照组细胞生长快,条件培养3～7d后见细胞生长迅速,呈集落样增长,细胞立体感增强,但未见明显细胞间桥;诱导培养15d见细胞增殖达高峰,增殖细胞的胞质间桥仍然不明显。酶联吸收OD值两组之间有显著性差异(P<0.01)。细胞DNA倍体分析,实验组DNA四倍体含量与对照组比较,两组之间有显著性差异(P<0.01)。结论小鼠精原干细胞在中药生精制剂培养条件下能较快速生长与增殖,在增殖过程中表现出与正常体内细胞增殖的某些特性,这为进一步研究精原干细胞多向分化潜能提供实验参考,也为中药制剂治疗少、弱精子症提供理论依据。
Objective To observe the growth characteristics of spermatogonial stem cells in spermicidal agents of Chinese traditional medicine and provide experimental evidence for exploring the multidirectional differentiation ability of spermatogonial stem cells. Methods Male Kunming mice aged 5 to 7 days were sacrificed after removing the cervical spine. The testis was isolated and cultured, and the spermatogonial stem cells were isolated, cultured and identified. Spermatogonial stem cells were cultured for 3 days and divided into control group and experimental group. The control group was treated with DMEM / F12 complete medium (containing 10% FBS, 4 mmoi / L glutamine, 0.12% NaHCO 3, 0.5 mmol / L sodium pyruvate, 15 mmol / LHEPES, 50U / mL penicillin and 50 μg / The experimental group was cultured with conditioned medium (DMEM / F12 complete medium plus Chinese medicinal sperm preparation concentrate capsule concentrate). Observed under inverted microscope cell morphology, enzyme-linked absorption OD value, cell DNA ploidy analysis. Results Spermatogonial stem cells grew faster in the culture medium of spermatogenic agent of experimental group than that of the control group. After 3 ~ 7 days of culture, the cells grew rapidly, showed colony-like growth and increased the stereophism, but no obvious intercellular space was observed After cultured for 15 days, the cell proliferation peaked and the cytoplasm of proliferating cells was still not obvious. There was a significant difference between two groups (P <0.01). Cell DNA ploidy analysis, the experimental group DNA tetraploid content compared with the control group, there was a significant difference between the two groups (P <0.01). Conclusion The mouse spermatogonial stem cells can grow faster and proliferate under the condition of spermicidal agent of Chinese traditional medicine, and exhibit certain characteristics of proliferation in normal cells in vivo, which provides experimental evidence for further research on the multipotential differentiation ability of spermatogonial stem cells Reference, but also for the treatment of traditional Chinese medicine less, asthenospermia provide a theoretical basis.