CAJ | 학술논문

目的研究神经干细胞对老化小胶质细胞存活及c-Jun氨基末端激酶(JNK)磷酸化蛋白(p-JNK)表达的调控作用. 方法原代培养的小胶质细胞和神经干细胞分别取自12～18个月龄的ICR小鼠及孕12.5 d的ICR小鼠,特异性标记物IB4染色、免疫荧光检测nestin的表达进行鉴定.利用插入式共培养系统将传代后稳定的小胶质细胞与神经干细胞(1∶4)共培养,噻唑蓝(MTT)法检测共培养前后细胞增殖能力的变化；用20 ng/mL.JNK抑制剂Sp600125预处理小胶质细胞4h,再加入神经干细胞共培养3d,Western blotting检测共培养前后小胶质细胞p-JNK的相对表达量的变化. 结果原代培养的小胶质细胞呈贴壁生长,折光性强,特异性标记物IB4染色显示细胞纯度在80％以上.神经干细胞nestin染色呈阳性；共培养组细胞的增殖能力显著高于单纯小胶质细胞和神经干细胞,差异有统计学意义(P＜0.05)；共培养的小胶质细胞+神经干细胞组小胶质细胞的p-JNK的相对表达量高于小胶质细胞组,Sp600125共培养组p-JNK的相对表达量高于单纯Sp600125刺激组,差异均有统计学意义(P＜0.05). 结论神经干细胞可以通过激活JNK信号通路促进老化小胶质细胞的存活.
목적 연구신경간세포대노화소효질세포존활급c-Jun안기말단격매(JNK)린산화단백(p-JNK)표체적조공작용. 방법 원대배양적소효질세포화신경간세포분별취자12～18개월령적ICR소서급잉12.5 d적ICR소서,특이성표기물IB4염색、면역형광검측nestin적표체진행감정.이용삽입식공배양계통장전대후은정적소효질세포여신경간세포(1∶4)공배양,새서람(MTT)법검측공배양전후세포증식능력적변화；용20 ng/mL.JNK억제제Sp600125예처리소효질세포4h,재가입신경간세포공배양3d,Western blotting검측공배양전후소효질세포p-JNK적상대표체량적변화. 결과 원대배양적소효질세포정첩벽생장,절광성강,특이성표기물IB4염색현시세포순도재80％이상.신경간세포nestin염색정양성；공배양조세포적증식능력현저고우단순소효질세포화신경간세포,차이유통계학의의(P＜0.05)；공배양적소효질세포+신경간세포조소효질세포적p-JNK적상대표체량고우소효질세포조,Sp600125공배양조p-JNK적상대표체량고우단순Sp600125자격조,차이균유통계학의의(P＜0.05). 결론 신경간세포가이통과격활JNK신호통로촉진노화소효질세포적존활.
Objective To investigate the regulation of neural stem cells (NSCs) on the viability and stress-activated kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling of aging microglia cells.Methods Primary microglia cells were isolated from 12-18 months old ICR mouse and NSCs were isolated from 12.5 days pregnancy ICR mouse.Staining test with Isolectin-B4,the specific marker for microglia,was performed and NSCs were verified by expression of nestin with immunofluorescence.Four groups were chosen in our experiment,including microglia cells group,co-cultured group,Sp600125 stimulated group and Sp600125-stimulation co-cultured group; in the Sp600125 stimulated group,microglia cells were pretreated with 20 ng/mL SP600125,a specific inhibitor of JNK,for four h; in the co-cultured group,microglia and NSC cells (1:4) were co-cultured using a Millicell Hanging Cell Culture Insert plates; and Sp600125-stimulation co-cultured group was also pretreated with 20 ng/mL SP600125 for four h firstly,and then,NSCs were added to co-culture with microglia cells for 3 d.MTT assay was performed to analyze the proliferation ability; Western blotting was used to detect the protein expression level of phosphorylated JNK signaling.Results After culturing for 2 weeks,primary microglia cells had a good adherence ability and strong refractivity.Staining test with Isolectin-B4 showed that the purity reached 80％.Neural stem cells grew like suspended spheres and nestin-positive.As compared with microglia cells group and stem cells group,co-cultured group had a significantly higher proliferation ability in MTT assay (P＜0.05).The phosphorylated JNK level in the co-cultured group was significantly up-regulated as compared with that in the microglia cells group (P＜0.05);Sp600125-stimulation co-cultured group had obviously higher phosphorylated JNK level than that in the Sp600125-stimulated group (P＜0.05).Conclusion NSCs might promote the survival of aging microglia cells through activation of JNK signaling pathway.