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[目的]研究兔前软骨干细胞PSCs,(precartilagious stem cells)的分离、培养方法及增殖、表型特征,为细胞移植治疗椎间盘退变,提供合适的种子细胞。[方法]取胎兔骨骺周围软骨膜(LaCroix环)中的细胞进行原代培养,然后采用免疫磁珠分离系统分选纯化PSCs。体外培养,传代,冻存复苏,绘制生长曲线,观察细胞特性。分别采用免疫组化、免疫荧光、RT-PCR等方法鉴定纯化后的PSCs。[结果]免疫磁珠分离可获得纯度较高的兔PSCs,培养后成活率高,细胞状态良好。细胞冻存复苏后,细胞增殖速度、细胞形态及表面标志物无明显变化。免疫组化、免疫荧光、RT-PCR等方法鉴定后,细胞都有明显的PSCs表面特异性标记物的表达。[结论]PSCs存在于LaCroix环中,免疫磁珠分离法得到的PSCs,体外培养条件下,细胞增殖较快,生物学特性稳定。
[Objective] To study the isolation, culture, proliferation and phenotypic characteristics of precartilagious stem cells (PSCs) from precancerous rabbit stem cells and to provide suitable seed cells for the treatment of degenerative disc degeneration. [Methods] The cells in the peritoneal epiphyseal peritoneal membrane (LaCroix ring) were primary cultured, then the purified PSCs were sorted by immunomagnetic bead separation system. In vitro culture, passage, cryopreservation recovery, draw the growth curve, observe the cell characteristics. Purified PSCs were identified by immunohistochemistry, immunofluorescence and RT-PCR respectively. [Result] The rabbit PSCs with high purity could be obtained by immunomagnetic bead separation. The cultured rabbit PSCs had high survival rate and good cell state. Cell cryopreservation and recovery, cell proliferation rate, cell morphology and surface markers no significant change. Immunohistochemistry, immunofluorescence, RT-PCR and other methods identified cells were significantly PSCs surface-specific markers. [Conclusion] The PSCs were present in LaCroix rings. The PSCs obtained by immunomagnetic bead separation could proliferate rapidly and have stable biological characteristics in vitro.