中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2014年
11期
2059-2064
,共6页
刘娜%孙康钦%白莲琴%李腾腾%董红燕%袁宝强
劉娜%孫康欽%白蓮琴%李騰騰%董紅燕%袁寶彊
류나%손강흠%백련금%리등등%동홍연%원보강
银杏苦内酯%谷氨酸%缺氧诱导因子1,α亚基%神经干细胞%PI3K/Akt信号通路
銀杏苦內酯%穀氨痠%缺氧誘導因子1,α亞基%神經榦細胞%PI3K/Akt信號通路
은행고내지%곡안산%결양유도인자1,α아기%신경간세포%PI3K/Akt신호통로
GINKGOLIDE%Glutamic acid%Hypoxia-inducible factor 1,alpha subunit%Neural stem cells%PI3K/Akt signaling pathway
目的：探讨银杏内酯B（GKB）对谷氨酸损伤神经干细胞（NSCs）内的磷脂酰肌醇-3激酶（PI3K）、磷酸化丝氨酸-苏氨酸蛋白激酶（p-Akt）、低氧诱导因子-1α（HIF-1α）活性的影响，以进一步明确银杏内酯B神经保护作用的分子机制。方法体外培养新生大鼠海马神经干细胞，并建立体外谷氨酸神经干细胞损伤模型。将其分为：（1）对照组；（2）GKB组（终浓度40 mg/L）；（3） wortmannin＋GKB组；（4）wortmannin组（终浓度10 nmol/L）。Western blot 印迹法检测各组NSCs中 PI3K、p-Akt、HIF-1α蛋白水平的表达。结果从新生大鼠海马区分离培养出具有自我更新、增殖的神经球，并在细胞球中可以检测到NSCs表面标记物巢蛋白（nestin）及5-溴-2-脱氧尿嘧啶（BrdU）的表达。100～400μmol/L谷氨酸作用NSCs 30 min，培养基中乳酸脱氢酶含量随着谷氨酸浓度的提高而明显增高[分别为（26.94±4.75）U/L，100μmol/L GLU；（38.22±5.86）U/L，200μmol/L GLU；（45.64±5.35）U/L，400μmol/L GLU]。经wortmannin处理后PI3K、p-Akt、HIF-1α蛋白活性较对照组明显下降，差异有统计学意义（P＜0.05）。wortmannin＋GKB组和wortmannin组比较，PI3K、p-Akt、HIF-1α表达明显上调。施加GKB后，PI3K、p-Akt、HIF-1α蛋白表达均较对照组增加，差异有统计学意义（P＜0.05）。结论银杏内酯B处理诱导损伤神经干细胞HIF-1α蛋白的表达可能与PI3K/Akt信号通路的激活有关。
目的：探討銀杏內酯B（GKB）對穀氨痠損傷神經榦細胞（NSCs）內的燐脂酰肌醇-3激酶（PI3K）、燐痠化絲氨痠-囌氨痠蛋白激酶（p-Akt）、低氧誘導因子-1α（HIF-1α）活性的影響，以進一步明確銀杏內酯B神經保護作用的分子機製。方法體外培養新生大鼠海馬神經榦細胞，併建立體外穀氨痠神經榦細胞損傷模型。將其分為：（1）對照組；（2）GKB組（終濃度40 mg/L）；（3） wortmannin＋GKB組；（4）wortmannin組（終濃度10 nmol/L）。Western blot 印跡法檢測各組NSCs中 PI3K、p-Akt、HIF-1α蛋白水平的錶達。結果從新生大鼠海馬區分離培養齣具有自我更新、增殖的神經毬，併在細胞毬中可以檢測到NSCs錶麵標記物巢蛋白（nestin）及5-溴-2-脫氧尿嘧啶（BrdU）的錶達。100～400μmol/L穀氨痠作用NSCs 30 min，培養基中乳痠脫氫酶含量隨著穀氨痠濃度的提高而明顯增高[分彆為（26.94±4.75）U/L，100μmol/L GLU；（38.22±5.86）U/L，200μmol/L GLU；（45.64±5.35）U/L，400μmol/L GLU]。經wortmannin處理後PI3K、p-Akt、HIF-1α蛋白活性較對照組明顯下降，差異有統計學意義（P＜0.05）。wortmannin＋GKB組和wortmannin組比較，PI3K、p-Akt、HIF-1α錶達明顯上調。施加GKB後，PI3K、p-Akt、HIF-1α蛋白錶達均較對照組增加，差異有統計學意義（P＜0.05）。結論銀杏內酯B處理誘導損傷神經榦細胞HIF-1α蛋白的錶達可能與PI3K/Akt信號通路的激活有關。
목적：탐토은행내지B（GKB）대곡안산손상신경간세포（NSCs）내적린지선기순-3격매（PI3K）、린산화사안산-소안산단백격매（p-Akt）、저양유도인자-1α（HIF-1α）활성적영향，이진일보명학은행내지B신경보호작용적분자궤제。방법체외배양신생대서해마신경간세포，병건입체외곡안산신경간세포손상모형。장기분위：（1）대조조；（2）GKB조（종농도40 mg/L）；（3） wortmannin＋GKB조；（4）wortmannin조（종농도10 nmol/L）。Western blot 인적법검측각조NSCs중 PI3K、p-Akt、HIF-1α단백수평적표체。결과종신생대서해마구분리배양출구유자아경신、증식적신경구，병재세포구중가이검측도NSCs표면표기물소단백（nestin）급5-추-2-탈양뇨밀정（BrdU）적표체。100～400μmol/L곡안산작용NSCs 30 min，배양기중유산탈경매함량수착곡안산농도적제고이명현증고[분별위（26.94±4.75）U/L，100μmol/L GLU；（38.22±5.86）U/L，200μmol/L GLU；（45.64±5.35）U/L，400μmol/L GLU]。경wortmannin처리후PI3K、p-Akt、HIF-1α단백활성교대조조명현하강，차이유통계학의의（P＜0.05）。wortmannin＋GKB조화wortmannin조비교，PI3K、p-Akt、HIF-1α표체명현상조。시가GKB후，PI3K、p-Akt、HIF-1α단백표체균교대조조증가，차이유통계학의의（P＜0.05）。결론은행내지B처리유도손상신경간세포HIF-1α단백적표체가능여PI3K/Akt신호통로적격활유관。
Objective To explore the effects of ginkgolide B (GKB) on the activity of the PI3K, phosphorylated Akt (p-Akt), hypoxia-inducible factor-1α (HIF-1α) in the cultured neuronal stem cells induced by glutamate, in order to provide the further evidence for the molecular mechanism of neuroprotection of ginkgolide B. Methods The neural stem cells from neonatal rat hippocampus were cultured and the model of injured NSCs by glutamate acid was built up. The cells were divided randomly into:(1) the control group;(2) GKB group (final concentration of 40 mg/L);(3) wortmannin+GKB group;(4) wortmannin group (final concentration of 10 nmol/L). Western blot was used to detect the protein levels of PI3K, p-Akt, HIF-1α in the 4 groups. Results The cells cultured from the hippocampus of neonatal rats grew with the self-renewal and proliferation of neural ball and the expression of nestin which was the surface marker of NSCs and the BrdU (5-bromo-2'-deoxyuridine) could be detected by immunocytochemical fluorescent staining. After the 100-400μmol/L range of glutamate dosage were used to intervene for 30 minutes, the lactate dehydrogenase in the neural stem cell damage model obviously improved with increased of the glutamate dosage, (26.94±4.75)U/L, 100μmol/L GLU;(38.22±5.86)U/L, 200 μmol/L GLU; (45.64±5.35)U/L, 400 μmol/L GLU. The expression of PI3K, p-Akt and HIF-1α was significantly decreased after treated by wortmannin, the difference was statistically significant (P<0.05). Wortmannin+GKB group compared with the control group, the expression of PI3K, p-Akt and HIF-1αhad no significant difference. The protein expression of PI3K, p-Akt and HIF-1αincreased compared with the control group after applying to GKB, the difference was statistically significant (P<0.05). Conclusion The expression of HIF-1αprotein in the cultured neural stem cells injured by glutamate induced by ginkgolide B treatment may be relate with the activation of P13K/Akt signal pathway.
