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目的:探讨乳清蛋白酶解物通过抑制过氧化氢,减轻过氧化氢对神经母细胞瘤细胞损害的机制。方法:将培育的人神经母细胞瘤细胞随机分为四份,对照组:未添加H_2O_2以及乳清蛋白酶解物的正常培养基中的细胞;过氧化氢组:神经母细胞瘤细胞+50μLH_2O_2,乳清蛋白观察组:神经母细胞瘤细胞+50μLH_2O_2+200μg/mL乳清蛋白酶解物;乳清蛋白对照组:神经母细胞瘤细胞+200μg/mL乳清蛋白酶解物,观察四组细胞存活和凋亡情况,以及细胞内钙离子和线粒体膜电位变化情况。结果:①细胞存活率对比上,过氧化氢组细胞存活率和凋亡率与其他三组相比,差异有统计学意义(F=24.62和32.82,P<0.01);乳清蛋白观察组细胞存活率和死亡率与过氧化氢组相比差异有统计学意义(t=7.65和9.82,P<0.05)。②过氧化氢组钙离子荧光强度最强,差异有统计学意义(Z=5.12,P<0.05);而对照组和乳清蛋白对照组荧光强度明显低于过氧化氢组和乳清蛋白组,差异有统计学意义(Z=5.57和4.82,P<0.05),乳清蛋白组荧光强度强于两对照组(Z=6.68,和4.15,P<0.05),弱于过氧化氢组(Z=6.12,P<0.05);③膜电位变化比较,正常对照组和乳清蛋白对照组荧光强度最强,提示膜电位变化幅度最小,过氧化氢组荧光强度最弱,提示膜电位变化幅度最大;乳清蛋白组荧光强度低于对照组而高于过氧化氢组,提示乳清蛋白酶解物能够阻止过氧化氢引起的线粒体膜电位改变。结论:乳清蛋白酶解物能够减少因过氧化氢诱导的神经母细胞瘤细胞内钙离子升高,减少线粒体内外膜电位差,从而抑制细胞凋亡。
OBJECTIVE: To investigate the mechanism of whey proteolysis by inhibiting hydrogen peroxide and reducing hydrogen peroxide damage to neuroblastoma cells. Methods: The cultured human neuroblastoma cells were randomly divided into four groups: control group: cells in normal culture medium without H 2 O 2 and whey protease; hydrogen peroxide group: neuroblastoma cells + 50 μL H 2 O 2, Whey protein observation group: neuroblastoma cells + 50μL of H_2O_2 + 200μg / mL whey protein hydrolyzate; whey protein control group: neuroblastoma cells + 200μg / mL whey protein hydrolysates, to observe the survival of four groups of cells and Apoptosis, as well as intracellular calcium and mitochondrial membrane potential changes. Compared with the other three groups, the difference was statistically significant (F = 24.62 and 32.82, P <0.01); whey protein observation group cells The difference of survival rate and mortality rate was statistically significant compared with hydrogen peroxide group (t = 7.65 and 9.82, P <0.05). (2) Hydrogen peroxide had the strongest Ca (superscript 2 +) fluorescence intensity (Z = 5.12, P <0.05), while the fluorescence intensity of the control group and the whey protein control group was significantly lower than that of the hydrogen peroxide group and the whey protein group (Z = 5.57 and 4.82, P <0.05). The fluorescence intensity of whey protein group was stronger than that of the two control groups (Z = 6.68 and 4.15, P <0.05) = 6.12, P <0.05). ③Compared with the changes of membrane potential, the fluorescence intensity of the normal control group and the whey protein control group was the strongest, suggesting the smallest change of membrane potential and the weakest fluorescence intensity of the hydrogen peroxide group, suggesting the largest change of membrane potential ; Whey protein group fluorescence intensity lower than the control group and hydrogen peroxide group, suggesting that whey proteolysate can prevent hydrogen peroxide caused mitochondrial membrane potential changes. CONCLUSION: Whey protein hydrolysates can reduce the apoptosis of neuroblastoma cells induced by hydrogen peroxide and increase the intracellular Ca2 + concentration, decrease the potential difference between mitochondrial inner membrane and outer membrane.