论文部分内容阅读
目的探讨钙拮抗剂对铁诱导的原代神经干细胞损伤是否具有保护作用。方法以大鼠胚胎(E17.5)海马神经干细胞(hNSC)为细胞模型。首先鉴定hNSC的神经干细胞特征,确认其表达L型钙通道Cav1.2基因mRNA及相关蛋白;然后分别给予细胞如下处理:1对照;2加铁(0.9mmol/L铁剂);3加钙拮抗剂(氟桂利嗪0.1μmol/L或尼莫地平10nmol/L);4钙拮抗剂+铁,观察钙拮抗剂对铁诱导的hNSC细胞活性及凋亡的影响;最后观察铁超负荷对细胞外信号调节激酶(ERK)磷酸化的影响及丝裂原活化蛋白激酶(MAPK)/ERK激酶(MEK)抑制剂U0126对铁诱导细胞活性的影响。免疫组化法鉴定细胞神经干细胞特征;逆转录聚合酶链反应(RT-PCR)法检测Cav1.2基因mRNA表达,免疫组化法及流式细胞仪法检测蛋白表达;XTT比色法检测细胞活性;Annexin V/碘化丙啶(PI)流式细胞仪检测细胞凋亡;抗磷酸化-ERK1/2流式细胞仪检测ERK磷酸化水平。结果 hNSC呈现典型神经干细胞特征,表达Cav1.2基因mRNA(158bp)及相关蛋白;钙拮抗剂可以显著增加临床相关浓度(0.9mmol/L)铁超负荷诱导的hNSC的细胞活性(尼莫地平,P<0.01),以及减少细胞凋亡;临床相关浓度铁超负荷可以诱导hNSC ERK磷酸化,MEK抑制剂U0126可显著增加铁损伤hNSC的细胞活性(P<0.01)。结论钙拮抗剂改善铁诱导的大鼠胚胎hNSC凋亡,可能通过抑制ERK磷酸化过程。
Objective To investigate whether calcium antagonists have a protective effect on iron-induced primary neural stem cell injury. Methods Rat embryonic (E17.5) hippocampal neural stem cells (hNSC) were used as the cell model. Firstly, the characteristics of neural stem cells in hNSC were identified and the expression of Cav1.2 mRNA and related protein in L-type calcium channels was confirmed. Then the cells were treated with 1 control, 2 iron (0.9 mmol / L iron), 3 calcium antagonist (Flunarizine 0.1μmol / L or nimodipine 10nmol / L); 4 calcium antagonist + iron, to observe the effect of calcium antagonists on iron-induced hNSC cell activity and apoptosis; and finally observe the effects of iron overload on cells ERK phosphorylation and the effect of mitogen-activated protein kinase (MAPK) / ERK kinase (MEK) inhibitor U0126 on iron-induced cell activity. Immunocytochemistry was used to identify the characteristics of neural stem cells. The expression of Cav1.2 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of Cav1.2 mRNA was detected by immunohistochemistry and flow cytometry. The apoptosis of cells was detected by Annexin V / propidium iodide (PI) flow cytometry. The phosphorylation of ERK was detected by anti-phospho-ERK1 / 2 flow cytometry. Results The expression of Cav1.2 mRNA (158 bp) and its related proteins in hNSCs was typical of hNSCs. Calcium antagonists could significantly increase the cell viability of hNSCs induced by iron overload (nimodipine, P <0.01), and decreased the apoptosis of cells. The iron overload of clinically relevant concentrations could induce the ERK phosphorylation of hNSC, and MEK inhibitor U0126 could significantly increase the cell viability of hNSCs with iron injury (P <0.01). Conclusion Calcium antagonists can improve the iron-induced apoptosis of rat hNSC embryos by inhibiting the phosphorylation of ERK.