摘要
目的 观察氯胺酮对谷氨酸引起神经元样嗜铬细胞瘤(PC12)细胞株凋亡的影响。方法PC12细胞株分别以2×103/孔和1×105/ml密度接种于96孔细胞培养板和60 mm细胞培养皿中,置于培养基,培养基中加10 nmol/L神经生长因子(7S-NGF),96孔细胞培养板和60 mm细胞培养皿的细胞均随机分为五组,A组暴露于20 mmol/L谷氨酸中,B组暴露于20 mmol/L谷氨酸+0.1 mmol/L氯胺酮(氯胺酮比谷氨酸提前1 min 加入)中,C组暴露于20 mmol/L 谷氨酸+1.0 mmol/L氯胺酮中,D组暴露于20 mmol/L 谷氨酸+100 μmol/L D-2-氨基-5-膦酸基戊酸(D-AP5)(细胞与D-AP5提前孵育2h)中,E组暴露于等容积的不含7S-NGF的新鲜培养液中,采用MTT法测定细胞培养板中PC细胞的细胞活力,采用死端比色TUNEL系统细胞检测培养皿中PC12细胞株的凋亡率。结果 A组、B组、C组和D组细胞活力分别为:37％±6％、65％±7％、99％±10％、90％±22％,与A组比较,B组、C组、D组细胞活力均升高(P<0.05或0.01)。A组、C组、D组、E组凋亡细胞率分别为66％±10％、20％±6％、22±7％、3.2％±1.8％,与A组比较,C组、D组、E组细胞凋亡率明显降低(P<0.01)。结论 氯胺酮通过抑制谷氨酸引起的神经元样PC12细胞株凋亡而发挥神经保护作用。
Objective To study the effects of ketamine on glutamate-induced apoptosis in neuronal cells using PC 12 pheochromocytoma cell line (provided by Chinese Academy of Phamacological Research) .Methods After being incubated in the culture medium containing 7S-NGF for 6 days. Over 95 % of the PC cells differentiated into neuron-type cells. The 7S-NGF induced differentiated neuronal PC 12 cells were seeded in 24-well plates pre-coated with poly-L-lysine(2×106 cells per well) .24 hours later the PC12 cells were exposed to glutamate 20 mrnol/ L(group A); glutamate 20 mmol/L + ketamine 0.1 mmo/L (group B); glutamate 20 mmol/L + ketamine 1.0 mmol/L (group C); glutamate 20 mmol/L + D-APS 100 )Ltmol/L(group D) and new culture medium containing no 7S-NGF(group E, control group), and incubated for 18 hours .The viability of the cells was evaluated by the ability of the cells to reduce the tetragotium derivative MTT into a blue formagan salt. DNA fragmentation indicative of apoptosis was detected using TUNEL technique. Results in group A following incubation with glutamate 20 mmol/L for 18 h , at 37℃, the viability was PC 12 cells was reduced to 37%± 6% However ketamine , when added to the culture medium to gather with glutamate , inhibited glutamate-induced cell death . The viability of PC 12 cell was 65 ± 7% in group B an 99±10% in group C. Ketamine appeared to attenuate the apoptotic process, because the number of the apoptotic cell bodies, determinated by YUNEL was also reduced by ketamine, with only 15-20% of neuronal cells staining positive after exposure to 20 mmol/L glutamate.The difference between group A and C was very significant (P< 0.01).In addition D-APS 100μmol /L ( a competitive antagonist) also exerted significant protective effects on neuronal PC 12 cells. Conclusion Ketamine protects neuronal PC 12 cells from glutamate neurotoxicity induced apoptosis.
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2004年第6期450-452,共3页
Chinese Journal of Anesthesiology
