【摘要】 目的:观察体外培养的小鼠神经母细胞瘤细胞A2无血清培养条件下分化成神经细胞过程中,细胞内脂质过氧化的主要产物-丙二醛和自由基清除剂超氧化物歧化酶的变化,探讨脂质过氧化反应程度和抗氧化能力与细胞老化的相关性。方法:实验于2004-04/2005-02在上海第二医科大学附属仁济医院神经内科神经生物学实验室进行。①建立神经细胞老化实验模型:传代培养的小鼠神经母细胞瘤细胞A2中加入无血清培养液后,在37℃、含体积分数为0.05的CO2的空气中培养,24h后换培养液,以后每隔3d更换培养液;按不同的培养天数分为培养1,7和14d组,各组在培养至相应的天数后终止培养。②评估神经细胞变化的指标检测:采用显微荧光分光光度计测定细胞老化指标脂褐素水平(相对荧光值)反映细胞老化程度;以比色法测定细胞内丙二醛浓度评估细胞脂质过氧化程度,亚硝酸盐比色法得出超氧化物歧化酶活性反映细胞的抗氧化能力。结果:①细胞内脂褐素相对荧光值:培养1,7和14d组分别是(11.55±0.9)%,(22.41±1.99)%和(29.51±4.57)%,组间比较差异显著(F=478.04,P<0.01)。②丙二醛浓度:培养1,7和14d组分别是(0.90±0.20),(1.91±0.19)和(2.98±0.20)μmol/L;组间比较差异显著(F=148.31,P<0.01)。③超氧化物歧化酶活性:培养1,7和14d组分别是(685.30±107.85),(372.07±74.35)和(175.04±29.67)μkat/L:组间比较差异显著(F=66.10,P<0.01)。结论:①随着无血清培养天数的增加,小鼠神经母细胞瘤细胞A2细胞内脂褐素逐渐增多,细胞在不断衰老。②细胞内丙二醛浓度随细胞老化而不断增加,而超氧化物歧化酶活性却随着细胞老化而下降。说明神经细胞的老化与细胞内脂质过氧化反应呈正相关,与细胞的抗氧化能力呈负相关。③体外神经细胞老化实验研究模型是科学和客观的,具有很强的应用价值。更多还原

【Abstract】 AIM: To observe the differentiation of neuroblastoma cell A2 cultured in vitro into neuroblast under the cultural condition without blood serum, the change of the main product of lipid peroxidation in cells-malondialdehyde (MDA) and superoxide dismutase (SOD), free radical cleaning dose, to explore the correlation of lipid peroxidation reaction degree and anti-oxidation ability with cell aging. METHODS: The experiment was done in Neurobiology Laboratory, Department of Neurology, Renji Affiliated Hospital, Shanghai Second Medical University from April 2004 to February 2005. ① The establishment of experimental model of neuronal cells aging: After the serum-free medium was added into the neuroblastoma cell A2 cultured for transftering of culture in mice, cultured at 37 ℃ and in the CO2 containing 0.05 volum number, and changed the culture medium after 24 hours, and changed the culture medium every 4 days; According to the different cultured days it was assigned into 1, 7 and 14 days groups, and the culture was terminated when the culture reached the corresponding number of days in every group. ② The evaluation of nerve cell aging index: The level of lipofuscin (relative fluorescent value) index of cells aging was detected by micro-spectrofluorometer to reflect the degree of cell aging; The concentration of malondialdehyde in cells was detected by chromatometry to evaluate the degree of cell lipid peroxidation, and the superoxide dismutase was detected by nitrite salt chromatometry to reflect the cell anti-oxidation ability. RESULTS: ① The corresponding luorescent value of lipofuscin in cells: It was (11.55±0.9)%, (22.41±1.99)% and (29.51±4.57)% in 1, 7 and 14 days groups respectively, and the difference was significant among the comparison of the groups (F=478.04, P < 0.01). ② The concentration of MDA: It was (0.90±0.20), (1.91±0.19) and (2.98±0.20) μmol/L in 1, 7 and 14 days groups respectively; The difference was significant among the comparison of the groups (F=148.31,P < 0.01). ③ The activity of SOD: It was (685.30±107.85), (372.07±74.35) and (175.04±29.67) μkat/L in 1, 7 and 14 days groups respectively; The difference was significant among the comparison of the groups (F=66.10, P < 0.01=. CONCLUSION: ① As the increase of number of days of serum-free culture, the lipofuscin in neuroblastoma A2 cells in rats increases gradually, and the cells were senility gradually. ② The concentration of MDA in cells increases continuously as the aging of cells, while the activity of SOD decreases as the aging of cells, which indicates that the aging of nerous cells shows positive correlation with lipid peroxidation reaction in cells, shows negative correlation with anti-oxidation ability of cells. ③ The established experimental study models of nerous cells aging in vitro are scientific and objective, and have powerful application value.更多还原