大鼠分化期神经干细胞过氧化氢氧化耐受差异的实验研究

Studies on the Variance of Oxidative Tolerance against Hydrogen Peroxide in Differentiated Neural Stem Cells of Rat

目的探讨大鼠分化期神经干细胞(differentiated neural stem cell,DNSC)对过氧化氢(H2O2)氧化耐受(oxidative tolerance,OT)的差异。方法取SD胎鼠培养DNSC,用浓度为0.05mmol/L、0.10mmol/L、0.25mmol/L,0.50mmol/L和1.0mmol/L的H2O2进行损伤干预,观察2,4,8,12和24h后细胞损伤情况。同时观察用H2O2(0.5mmol/L)损伤2h后,DNSC用Hoechst33342和碘化丙啶(PI)染色的结果。观察热休克蛋白(Hsp)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)的表达情况。用透射电镜(TEM)检测DNSC损伤前后超微结构变化。结果当H2O2浓度达到0.25mmol/L时,DNSC开始死亡。给予0.5mmol/LH2O2损伤2h后,位于神经球周边的DNSC死亡并脱落,存活的细胞位于神经球中心。近中心区域Hsp70免疫组化阳性细胞数目明显上升,而Hsp27、CAT和SOD无明显阳性表达。TEM检测结果显示,存活细胞均为比较原始的细胞;距离中心越远,细胞分化程度越高,中心区域细胞具有原始细胞特点。结论随着DNSC的发育,其抗H2O2损伤的能力下降。相对原始的DNSC抗H2O2的OT能力要高于分化较好的DNSC。经H2O2损伤后存活的DNSC仍然具有增殖和细胞分化能力。

Objective To evaluate the variance of oxidative tolerance（OT） against hydrogen peroxide （ H2O2 ） in differentiated neural stem cells （DNSC）. Methods The DNSC of SD rat obtained after 8 days＇ differentiation, were cultured in media with H2O2 （0.5 mmol/L） for 2 hours, followed by observation of their variance of oxidative tolerance. The survial was cultured for another 21 days, then observed the capability of proliferation and differentiation. The viability was assessed with fluorescent dye including Hoechst 33342 and PI. The antioxidant stress test included the expression of heat shock proteins （Hsps）, catalase（CAT） and superoxide dismutase（SOD）. The cellular types were determined by its markers, such as nestin, proliferating cell nuclear antigen （ PCNA）, MAP-2 and tubulin-β, GFAP, Gal-C. The transmission electron microscope（TEM） was used to study the uhrastructure of DNSC. Results After H2O2 attacks, DNSC at the periphery died and fell off from the cover glass, while those located at the central part survived and kept on differentiation and demonstrated positive staining by nestin（ PCNA）, MAP-2, tubulin-β, GFAP, and Gal-C, with no significant difference from the control group（P 〉0.05）. TEM demonstrated the DNSC at the periphery with a relatively mature organelle and those at the central part with immature characteristics. Conclusion The relatively immature DNSC has a higher resistance against H2O2 than those relatively well- differentiated DNSC. DNSC surviving from H2O2 attack still keep the capability proliferate and differentiate.