【作者】 薛美兰；

【导师】 张秀珍； 马爱国；

【作者基本信息】 青岛大学 ， 营养与食品卫生学， 2004， 硕士

【摘要】 目的 通过对实验动物（大鼠）进行大剂量维生素C（VC）的干预，观察大剂量的VC对实验动物DNA氧化损伤、抗氧化和免疫系统的影响，并探讨其机制。 方法 选用健康初断乳Wistar大鼠，体重70～90克，雌雄各半，按体重、性别随机分为4组，每组12～14只，分别为对照组（基础饲料）、补充VC 2000mg／kg、5000mg／kg、10000mg／kg饲料四组，实验期为8周，自由进食及饮水。实验过程中，定期称大鼠体重，第6～7周留取尿液；实验结束后腹主动脉取血进行各项指标的检测分析。采用单细胞凝胶电泳法检测DNA自发及诱导氧化损伤的情况；采用荧光偏振法测定红细胞膜流动性；采用毛细管电泳法测定尿中O⁶-甲基鸟嘌呤的含量；采用2、4-二硝基苯肼法测定大鼠血浆和肾上腺中VC的含量；采用试剂盒测定血浆中SOD、MDA和GSH-Px以及红细胞膜中的MDA和GSH-Px。采用MTT法测定大鼠外周血淋巴细胞转化率，观察其增殖活性。 结果 经过8周VC补充后，对各组大鼠机体VC水平、抗氧化能力、细胞功能等进行了分析，结果显示：①血浆和肾上腺VC含量在VC补充各组明显增加，与对照组血浆VC含量（405.77μg／100mL）相比，2000、5000和10000mg／kg饲料组的血浆VC含量分别上升达425.69、460.00和515.97μg／100mL，其中10000mg／kg饲料组的VC含量比对照组升高27.2％（P＜0.01），比2000mg／kg饲料组升高21.2％（P＜0.01）。肾上腺VC含量的分析结果也显示VC补充各组大鼠肾上腺VC水平明显升高，10000mg／kg饲料组肾上腺VC含量比对照组升高40.1％（P＜0.01），比2000mg／kg饲料组升高28.7％（P＜0.05）。②DNA损伤分析结果显示：各组DNA自发损伤无差别（F=0.485，P＞0.05）。10μmol／L的H₂O₂诱发DNA损伤时，各组间有明显差别（F=3.310，P＜0.05），随剂量增加，DNA损伤逐渐加重，5000mg／kg饲料组DNA损伤比对照组升高28％（P＜0.05），10000mg／kg饲料组比对照组升高49％（P＜0.01）。各组O⁶-甲基鸟嘌呤的含量差别显著（F=4.276，P=0.01），10000mg／kg饲料组含量最高，比对照组升高36％（P＜0.05）。③抗氧化酶活性分析结果显示：各组血浆SOD含量有明显差别（F=6.213，P＜0.01），随着补充剂量的增加，各干 中文摘要预组有先上升后下降的趋势，Zooomg/kg饲料组SOD含量高于对照组（尸<。.05），I0000mg/kg饲料组SOD含量低于对照组（尸<。.05）。各组红细胞膜GSH一Px活性分别为1 82.22、213.58、138.13和102.11酶活力单位，Z000mg/kg饲料组GSH一Px活性最高，但与对照组比较无差别（尸>。.05），I000Omg/kg饲料组GSH一Px活性最低，比对照组下降44.0%（尸<。.05）。④血浆脂质过氧化产物MDA含量各组间有明显差别（F一9.466，P<0.01），2000 mg/kg饲料组MDA含量比对照组降低25.7%（尸<0.01），l000omg/kg饲料组MDA含量对照组增高20.3%（尸<0.05）。各组红细胞膜MDA含量有明显差别（F一4.54，尸<0.01），各VC干预组的MDA值均低于对照组，其中Z000mg/kg饲料组的MDA含量最低，比对照组下降44.4%（尸<0.01）。⑤细胞功能活性分析显示:红细胞膜流动性，无论P值和刀值，各组间比较均有差别（F值分别为4.272、3.155，P值均小于0.05）。其中Z000mg/kg饲料组红细胞膜流动性最高，明显低于对照组（尸<0.05），l0000mg/kg饲料组红细胞膜流动性最低，但与对照组比较无差别（尸>0.05）。外周血琳巴细胞增殖活性分析显示各组大鼠淋巴细胞转化率无明显差别（F一0.485，尸>0.05），但ZOO0mg/kg饲料组淋巴细胞转化率高于对照组和l000Omg/kg饲料组（尸均<0.05）。 结论①补充较高剂量（Z000mg/kg饲料）VC可使大鼠血浆SOD水平明显增高，使血桨和红细胞膜MDA含量明显降低，增加大鼠的红细胞膜流动性。②长期大剂量（10000mg/kg饲料）补充VC可明显增加大鼠血浆和肾上腺VC含量，使大鼠血浆SOD水平和红细胞膜GSH一Px的活性明显降低，增加大鼠体内脂质过氧化损伤，使血浆MDA水平升高，尿中06一甲基鸟嗦吟排出量增加。当VC补充剂量超过S000mg/kg饲料时，可使由10拜mol/L HZO:诱导的DNA氧化损伤增强。 总之，给大鼠补充Z000mg/kg饲料的vC对大鼠机体的抗氧化和细胞免疫功能有一定的促进作用，对DNA损伤没有影响;当补充剂量超过Z000mg/kg饲料时，在一定程度上，反而会促进大鼠机体的氧化损伤，尤其是H20:诱导的DNA损伤。更多还原

【Abstract】 Objective To investigate the effects of high-dose vitamin C(VC) on DNA oxidative damage and cellular function.Methods 54 Wistar rats, weighed from 80g to 100g, were randomized into 4 groups, each of which was supplemented with amount of 0, 2000, 5000, 10000mg/kg of VC in diet respectively. The first group was control. The period of the trail was 8 weeks. The samples of the blood were collected by the end of the trial, and the urine in the middle of the trial. The levels of SOD and MDA, and GSH-Px activity in serum or erythrocyte membrane were examined by Kits. The erythrocyte membrane fluidity was determined by fluorescence polarization method. DNA damage was analyzed by SCGE, and O6-MeG was analyzed by high performance capillary zone electrophoresis. The transformation of Lymphocytes was detected by the method of MTT.Results After 8 weeks’ trial, the results were showed that the contents of vitamin C in serum and adrenal gland were increased in VC supplement groups. The VC contents of the 5000 and 10000mg/kg diet groups were 460. 00 and 515. 97 g/100mL respectively, which was higher than one in the control (P<0. 05, P<0. 01). The serum vitamin C level in 10000mg/kg diet group was 27. 2% higher than one in the control group (P<0. 01) and 21.2% higher than that of 2000 mg/kg diet group (P<0. 01). The differences between levels of vitamin C in adrenal gland were found in the four groups (F=3. 683,P<0. 05). The VC content in adrenal gland of 10000mg/kg diet group was increased by 40. 1% compared to one in the control group (P<0. 01) and increased 28. 7% compared to that of 2000mg/kg diet group (P<0. 05).Spontaneous DNA damage had no differences in the four groups (P>0. 05), while DNA damage induced by 10 mol/L H2O2 had significant differences(F=3. 310, P<0. 05). The DNA damage was gradually aggravated when the dose of VC supplementation was increased. The level of DNA damage in 5000mg/kg diet VC group was 28% higher than one in the control group (P<0. 05)and that of the 10000mg/kgdiet VC group was 49% higher than one in the control (P<0. 01). There were great differences in the level of O6-megth-ylguanine in urine among the four groups(F=4. 276, P = 0. 01). The level of O6-MeG in the 10000mg/kgdiet VC group was the highest and 36% higher than one in the control (P<0. 05).The differences between levels of serum SOD in those groups were found (F=6. 213, P<0. 01). The serum level of SOD in 2000 mg/kg diet group was higher than one in the control group (P<0. 05), while the serum level of SOD in 10000mg/kg diet group waslower than one in the control group (P<0. 05). The GSH-Px activity in erythrocyte membrane of those groups was 182. 22, 213. 58, 138. 13 and 102. 11U respectively. The GSH-Px activity of erythrocyte membrane in 2000 mg/kg diet group was the highest, but there was no difference compared to control group(P>0. 05). The GSH-Px activity of erythrocyte membrane in l0000mg/kgdiet group was the lowest and reduced 44. 0% compared to that of control group (P<0. 05).The level of serum MDA in the 2000mg/kgdiet group was the lowest and decreased 25. 7% than control group (P<0. 01). The level of serum MDA in 10000mg/kg diet group was the highest and 20. 3% higher than one in the control (P<0. 05). The level of MDA in erythrocyte membrane of 2000mg/kg diet group was the lowest and decreased 44. 4% compared to one in the control (P<0. 01).No matter P or , there were significant differences in the four groups (P<0. 05). The fluorescence polarization of erythrocyte membrane in the 2000mg/kgdiet groups was the lowest and lower than one in the control (P<0. 05). The fluorescence polarization of erythrocyte membrane in 10000mg/kg diet was the highest, but there was no difference compared to that of the control(P>0. 05). There were no differences in the transformation of Lymphocytes among those groups (F=0. 485,P>0. 05), while the transformation of Lymphocytes in the 2000mg/kgdiet group was significantly increased compared to that of the control and the 10000mg/kgdiet group (P<0. 05).Conclusion (1)Supplementing sl更多还原