钙拮抗剂改善铁诱导的大鼠胚胎海马神经干细胞凋亡  

Calcium channel blockers alleviated rat embryomic hippocampal neural stem cells apoptosis induced by iron

作　　者：陈文雄 黄珍妮[2] 杨默[3] 吴武田[4] 苏焕兴[5] 陈志峰[2] 

机构地区：[1]广州市妇女儿童医疗中心脑病中心神经内科,广东广州510623 [2]香港大学李嘉诚医学院儿童与青少年系 [3]南方医科大学南方医院儿科 [4]香港大学李嘉诚医学院解剖学系 [5]澳门大学中华医药研究院中药质量研究国家重点实验室

出　　处：《中华高血压杂志》2016年第9期850-856,共7页Chinese Journal of Hypertension

基　　金：香港大学CRCG-HKU研究基金(10207982-08296-21100-323)

摘　　要：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 explore the protective potential of calcium channel blockers on iron-injured primary neural stem cells. Methods Rat embryonic （E17.5） hippocampal neural stem cells （hNSC} served as a cell model. Im- munostaining method was used to identify the characteristics of hNSC, while reverse transcription-polymerase chain reaction （RT-PCR）, immunostaing and flow cytometry methods was respectively adopted to confirm the expression of mRNA and protein of L type calcium channel Cav 1.2 gene of hNSC. XTT colorimetric method was used to de tect the cell viability. The Annexin V/propidium iodide（H） was used to measure apoptotic cells, while anti-phos pho-extracellular regulated protein kinase（ERK）1/2 was adopted to detect ERK phosphorylation, both via flow cy- tometry. The hNSC were divided into the control, iron-overload （0.9 mmol/L）, calcium channel blockers （flunari- zine 0.1 μmol/L or nimodipine 10 nmol/L） or mitogen-activated protein kinase（MAPK）/ERK kinase（MEK） inhibi- tor （U0126）, and calcium channel blockers or U0126 plus iron-overload groups, to explore the effects of calcium channel blockers or MEK inhibitor on celt viability as well as apoptosis of iron-injured hNSC. Results The hNSC showed typical characteristics of neural stem cells, and expressed mRNA （ 158 bp） as well as relevant protein of Cav1. 2 gene of L-type calcium channel. Calcium channel blockers could alleviate clinically relevant doses of iron （0.9 mmol/L） induced apoptosis, and increased cell viability of hNSC （nimodipine P〈0.01）. Clinically relevant doses of iron induce ERK activation of hNSC, and MEK inhibitor （U0126） could significantly increase cell viability of hNSC （P〈0.01） in clinically relevant dose of iron-overloaded. Conclusion Calcium channel blockers could po- tentially protect iron-induced neurotoxicity in hNSC, probably by inhibiting ERK activation.

关 键 词：铁超负荷 神经干细胞 钙拮抗剂 神经退行性障碍 出血性脑卒中 细胞外信号调节激酶 

分 类 号：R741[医药卫生—神经病学与精神病学]