CAJ | 학술논문

目的：观察选择性光刺激 NG2细胞对海马神经元内 Ca 2＋活性和电活动的影响，并初步探讨可能的作用机制。方法体外纯化培养新生 SD 大鼠 NG2细胞，通过脂质体转染的方法，在 NG2细胞中特异性表达光敏感通道（channelrhodopsin－2，ChR2）蛋白 DNA，并与体外培养7 d 的海马神经元共培养48 h，采用蓝光（470 nm、5 s、5 mW／cm2）选择性刺激表达 ChR2蛋白的 NG2细胞，利用 Ca2＋成像和电生理的方法记录 NG2细胞周围海马神经元中的 Ca 2＋浓度变化和电流改变情况，同时比较加入／未加入γ－氨基丁酸 A 型受体抑制剂情况下神经元的电流变化特点。结果当蓝光刺激表达 ChR2蛋白的 NG2细胞后，首先 NG2细胞内的 Ca 2＋信号增强，随后其周围海马神经元内的 Ca2＋升高约20％；Ca2＋缓慢下降后再次出现一个 Ca2＋峰，幅度较第1次小；NG2细胞周围海马神经元的电流频率增加，振幅增大。而当预先在细胞外液中加入50μmol／L γ－氨基丁酸 A 受体抑制剂后，海马神经元的电流频率和振幅较未加抑制剂组明显减小（P＜0．05）。结论选择性光刺激表达 ChR2蛋白的 NG2细胞可使其兴奋，且可能通过释放γ－氨基丁酸来调节神经元活动。
목적：관찰선택성광자격 NG2세포대해마신경원내 Ca 2＋활성화전활동적영향，병초보탐토가능적작용궤제。방법체외순화배양신생 SD 대서 NG2세포，통과지질체전염적방법，재 NG2세포중특이성표체광민감통도（channelrhodopsin－2，ChR2）단백 DNA，병여체외배양7 d 적해마신경원공배양48 h，채용람광（470 nm、5 s、5 mW／cm2）선택성자격표체 ChR2단백적 NG2세포，이용 Ca2＋성상화전생리적방법기록 NG2세포주위해마신경원중적 Ca 2＋농도변화화전류개변정황，동시비교가입／미가입γ－안기정산 A 형수체억제제정황하신경원적전류변화특점。결과당람광자격표체 ChR2단백적 NG2세포후，수선 NG2세포내적 Ca 2＋신호증강，수후기주위해마신경원내적 Ca2＋승고약20％；Ca2＋완만하강후재차출현일개 Ca2＋봉，폭도교제1차소；NG2세포주위해마신경원적전류빈솔증가，진폭증대。이당예선재세포외액중가입50μmol／L γ－안기정산 A 수체억제제후，해마신경원적전류빈솔화진폭교미가억제제조명현감소（P＜0．05）。결론선택성광자격표체 ChR2단백적 NG2세포가사기흥강，차가능통과석방γ－안기정산래조절신경원활동。
Objective It has been investigated that the change of Ca 2+and electrical activity in neurons of hippocampus influenced by NG2 cells which expressed channelrhodopsin-2 (ChR2) and were stimulated by blue light, and then discussed the probable mechanism.Methods We cul-tured NG2 cells from the cortex of neonatal rats, and transfected ChR2 DNA to NG2 cells through li-posomes, then co-cultured NG2 cells expressed ChR2 protein with hippocampal neurons for 48 hours.By way of calcium imaging and electrophysiological recording, we could observe the change of calcium concentration and current in hippocampal neurons around the NG2 cells expressed ChR2 which were stimulated by blue light (470 nm, 5 s,5 mW/cm 2 ).Meanwhile, we compared the change of current in hippocampal neuron pre-adding inhibitor in extracellular fluid with no inhibitor. Results In calcium imaging experiment, we found that there was an elevation of Ca 2+concentration in NG2 cells expressed ChR2 as soon as the blue-light stimulus, and then Ca 2+concentration in neu-rons around NG2 cells increased by 20%, following a second Ca 2+ peak which its amplitude was smaller than the first.In the electrophysiological experiment, we found that it was bigger of the fre-quency and amplitude of current in neurons around NG2 cells expressed ChR2 which were stimulated by blue light than those with no blue light.However, if we added γ-amino-butyric acid (GABA) A receptor inhibitor (bicuculin, 50 μmol/L) in extracellular fluid in advance, the frequency and am-plitude of current in neurons around NG2 cells expressed ChR2 which were stimulated by blue light (5 s, 5 mW/cm 2 )were significantly smaller than those with no inhibitor (P<0.05).Conclusion Selective stimulating NG2 cells expressed ChR2 protein with blue light is able to excite NG2 cells and may regulate neural activity by releasing GABA.