低温冻存对胚胎大鼠神经干细胞生物学特性的影响

Effects of cryopreservation on biological characteristics of neural stem cells in fetal rats

目的 研究不同冻存液对胚胎大鼠神经干细胞的保护作用及低温冻存对神经干细胞增殖及分化潜能的影响。方法 分别应用冻存液Ⅰ和冻存液Ⅱ对源于胚胎大鼠的神经干细胞球进行冷冻,于复苏后进行传代培养,并鉴定其增殖和分化能力。结果 复苏后的神经干细胞可以多次传代,冻存神经干细胞的克隆增殖率为(36.80±3.81)％,未冻存者为(38.15±4.80)％,两者相比差异无显著性(P>0.05)。冻存液Ⅰ和冻存液Ⅱ保存的神经干细胞分化为神经元的比例分别为(7.61±0.74)％和(12.76±2.53)％(P>0.05);分化为少突胶质细胞的比例分别为(0.90±0.50)％和(2.18±0.33)％(P>0.05);分化为星形细胞的比例为(47.67±2.10)％和(35.38±3.14)％(P<0.05)。结论 冻存的胚胎大鼠神经干细胞复苏后仍具有活跃的增殖和分化潜能,含10％血清的冻存液可以促使神经干细胞向星形细胞方向转化。

Objective To study the efficiency of different cryopreservation protecting reagents on neural stem cells and try to find out whether or not these neural stem cells can keep capacity of selfrepro- duction and multipotentialty. Methods Cryopreservation solution Ⅰand Ⅱ were applied for the cryop- reservation of neural stem cells cultured from E13 Wistar rat. The thawed neural stem cells were cultured and identified for the selfreproduction and multipotentialty by immunocytochemistry. Results Thawed neural stem cells could proliferate actively and maintained the ability of selfreproduction and passage re- peatedly. The clonal proliferative rate of cryopreserved and noncryopreserved neural stem cells was (36. 80 ±3. 81)% and (38. 15±4. 80)% respectively (P >0. 05 ). The rate of differentiation into neu- ron in cryopreservation Ⅰand Ⅱ was (7. 61±0. 74)% and (12. 76±2. 53)% respectively (P >o. o5); The rate of differentiation into oligodendrocyte was (0. 90 ± 0. 50)% and (2. 18±0. 33)% respectively (P >0. 05). Whereas the rate of differentiation into astrocyte in cryopreservation Ⅰ(47. 67±2. 10)% was higher than that of cryopreservation Ⅱ (35. 38±3. 14)% (P < 0. 05 ). Conclusion Embryonic neural stem cells can preserved in cryopreservation. Thawed neural stem cells keep the ability of reproduc- tion and differentiation. The 10% dimethyl sulfoxide in the cryopreservation solution is enough to keep the vitality of neural stem cells and the 10% serum in the cryopreservation solution can derive the neural stem cells to differentiation into astrocyte.