促红细胞生成素与甲基强的松龙对星形胶质细胞的作用研究

摘要

Objective To study the effect of methylprednisolone(MPSS) combined with erythropoietin(EPO) to in vitro cultured primary astrocytes(AST). Methods The cell suspension was prepared by using the cerebrum of neonatal rats within 3 d of birth,cultured by the DMEM culture solution containing 15% fetal calf serum,gradually passaged after shaking for purifying astrocytes. After simulating nulli-nutrition model for 3 h by phosphate buffer solution(PBS) for substituting medium,MPSS 10 μmol/L,EPO 10 U/L and MPSS 10 U/L plus EPO 10 U/L were immediately added and the continuous culture for 3 d was performed. Astrocytes were identified by the immunofluorescent technique;the proliferation activity and apoptosis of astrocytes were detected by MTT;the expression level of GFAP mRNA was detected by PCR. Results Astrocytes were passaged to the third generation after shaking,the astrocytes purity reached more than 95% by identification;part of astrocytes became round in morphology and even died after nulli-nutrition for 3 h;after 3 d of applying MPSS,EPO and MPSS plus EPO in the normal group and the damage group,both cytoactive and expression level of GFAP mRNA in the damage group were significantly increased ( P ﹤ 0. 05 ) ,moreover compare with the single application of MPSS or EPO,the cytoactive and GFAP mRNA expression level in the combined application of MPSS and EPO were significantly increased( P ﹤ 0. 05),but no obvious change was found in the normal group( P ﹥ 0. 05). Conclusion The combined application of proper dose of MPSS and EPO has obviously protection effect on nulli-nutrition damaged astrocytes,moreover has significant difference compared with the single appli-cation of MPSS or EPO,but has no obvious protective effect on normal astrocytes.%目的：研究甲基强的松龙（MPSS）与促红细胞生成素（EPO）联合应用对体外培养的原代星形胶质细胞的作用。方法采用出生3 d内SD新生鼠的大脑制成细胞悬浊液后，以含15%胎牛血清的DMEM培养液培养，摇床后逐渐传代、纯化星形胶质细胞，磷酸盐缓冲液（PBS）代替培养基模拟缺营养模型3 h后即刻分别加入MPSS 10μmol/L，EPO 10 U/L，MPSS 10μmol/L﹢EPO 10 U/L，分别继续培养3 d，采用免疫荧光技术鉴定星形胶质细胞，MTT法检测细胞的增殖活性和凋亡，聚合酶链式反应（PCR）技术测定细胞胶质纤维酸性蛋白( GFAP ) mRNA表达水平。结果星形胶质细胞摇床后传至第3代，经鉴定细胞纯度达95%以上；缺营养3 h后部分细胞形态变圆甚至死亡漂浮于正常细胞表面；正常组和损伤组分别加入MPSS，EPO，MPSS﹢EPO 3 d后，损伤组细胞活性与GFAP mRNA表达水平均明显升高（ P﹤0.05），且MPSS和EPO联合应用与单用MPSS、单用EPO相比，细胞活性与GFAP mRNA表达水平明显升高（ P﹤0.05），但正常组无明显变化（ P﹥0.05）。结论适当剂量的MPSS与EPO联合应用对缺营养损伤星形胶质细胞有显著的保护作用，且与单用MPSS、单用EPO相比有显著性差异，但对正常星形胶质细胞无显著的保护作用。