大鼠海马神经细胞体外缺糖缺氧模型制备方法的改进

Improvement in the preparation of hypoxia/reperfusion models of rat hippocampal neurons in vitro

目的:改进培养大鼠海马神经细胞体外缺糖缺氧模型制备方法,并通过兴奋性氨基酸N-甲基-D-天冬氨酸受体拮抗剂进行验证。方法:实验于2004-09/2005-06在南方医科大学基础医学院神经生物学教研室进行。实验材料:出生1d内清洁级SD大鼠,由南方医科大学实验动物中心提供(合格证号为粤证监字2004B023号)。N-甲基-D-天冬氨酸受体拮抗剂5-甲基二氢丙环庚烯亚胺马来酸(MK-801)和D-2-氨基-5-磷酰基戊酸(d-APV)购自Sigma公司。实验方法:取新生1dSD大鼠海马组织作神经细胞分散细胞原代培养,培养到13d时进行氧/葡萄糖剥夺模型的制备:将neurobasal培养基更换为不含葡萄糖的BSSo培养基,连续充以50mL/LCO2+950mL/LN2(体积比)混合气体。缺氧30,45,60,90min后取出细胞,更换为正常neurobasal培养基,恢复正常条件继续培养。将神经细胞随机分为正常对照组、单纯缺氧组、无糖缺氧组、MK-801组和d-APV组。将10μmol/LMK-801和500μmol/Ld-APV在通50mL/LCO2+950mL/LN2混合气前加入到BSSo培养基,缺氧结束后随BSSo培养基一起去掉。复氧24h后采用MTT比色法测神经细胞成活率及Hoechst荧光染料法测神经细胞凋亡率。结果:①随着氧/葡萄糖剥夺时间延长,神经细胞存活率下降。氧/葡萄糖剥夺30,45,60,90min再复氧24h,神经细胞存活率分别为(81.48±3.84)%、(63.14±3.14)%、(41.73±2.97)%和(16.78±2.12)%。②N-甲基-D-天冬氨酸受体拮抗剂MK-801(10μmol/L)和d-APV(500μmol/L)均能明显增加神经细胞存活率(P<0.05),并且两组细胞存活率与正常对照组比较,差异无统计学意义(P>0.05)。结论:实验制备的海马神经细胞短时间氧/葡萄糖剥夺模型可对神经细胞造成迟发性死亡,兴奋性氨基酸N-甲基-D-天冬氨酸受体拮抗剂可保护氧/葡萄糖剥夺诱导的神经细胞损伤,说明本实验建立的缺氧模型有效并简便可靠,并且缩短了缺氧时间。

AIM： To improve the preparation method of hypoxia/reperfusion model of cultured rat hippocampal neurons, and identify the model with N-methyI-D-aspartate receptors （NMDAR） antagonist. METHODS： The experiment was performed in the laboratory of Neurobiology Department, School of Basic Medical Science, Southern Medical University from September 2004 to June 2005. NMDAR antagonist MK-801 and d-APV were purchased from Sigma Company. Primary hippocampal neurons were harvested from the neonatal SD rats within one day, which were provided by the experimental animal center of Southern Medical University, No. 2004B023. Neurons cultured for 13 days were subjected to oxygen-glucose deprivation （OGD） by exchanging the culture medium with deoxygenated glucose-free Earle＇s balanced salt solution （BSSo）, and then the neurons were placed into an anaerobic chamber containing a gas mixture of 50 mL/L CO2 and 950 mL/L N2. Cells were taken out after deprived of oxygen and glucose for 30, 45, 60, and 90 minutes, then washed back into normal neurobasal media for culture under normoxic incubator. Neurons were assigned randomly into control group, hypoxia group, OGD group, MK-801 group and d-APV group. 10 μmol/L MK-801 and 500 μmol/L d-APV were added in exposure media, then washed back into normal neurobasal media after termination of OGD. The apoptotic rate of neurons after 24-hour normal oxygen was detected by MTT colorimetry and Hoechst staining. RESULTS： ①Cell survival rate decreased with OGD duration. Cell survival rates were （81.48±3.84）%, （63.14±3.14）%, （41.73±2.97）% and （16.78±9.12）% under 24-hour normal oxygen after OGD for 30, 45, 60 and 90 minutes, respectively. ②NMDAR antagonist MK-801 （10 μmol/L） and d-APV （500 μmol/L） could both enhanced cell survival （P 〈 0.05）, and there was no significance in cell survival between control group and two NMDAR antagonist groups （P 〉 0.05）. CONCLUSION： This OGD model could induce neuronal delayed death. NMDAR antagonist could protect neurons against OGD-induced neuronal damage, indicating that this OGD model is effective, credible and convenient, and more importantly, is very transitory.