类黄酮和维生素E协同对抗细胞膜损伤的作用

Effect of flavone and vitamin E against injury of cell membrane

目的:观察类黄酮和维生素E在拮抗壬基酚的致损伤作用方面的表现。方法:实验于2005-03在湖南环境生物学院实验中心完成。实验动物为5月龄SD大鼠30只。①抽取大鼠的外周血0.5mL种入含植物血凝素的RPMI1640培养基培养后,按照加入试剂的不同分为4组,空白对照组犤注入蒸馏水(5,10,15,20mL)犦,壬基酚组犤注入壬基酚(5,10,15,20mL)犦,类黄酮组犤注入类黄酮(1×10-6,1×10-5,1×10-4mol/L)0.5mL犦,维生素组犤注入维生素E(0.25,0.50,1.00,2.00mg)犦。不同处理后用姐妹染色体色差法制成色差法染色体玻片,镜检淋巴细胞姐妹染色单体交换率。②抽取SD大鼠的外周血种入含植物血凝素RPMI1640培养基培养。按照加入试剂的不同分5个组,壬基酚组(注入壬基酚10μL),壬基酚+类黄酮1组(注入壬基酚10μL+类黄酮10-6mol/L,经温育),壬基酚+类黄酮2组(注入壬基酚10μL+类黄酮10-6mol/L,不经温育),壬基酚+维生素组(注入壬基酚10μL+维生素E0.5mg),壬基酚+类黄酮+维生素E组(注入壬基酚10μL+类黄酮10-6mol/L+维生素E0.5mg)。按照不同处理后用姐妹染色体色差法制成色差法染色体玻片,在荧光显微镜下观测50个细胞,镜检姐妹染色单体交换率。③取相对固定部位的肝组织块,经处理后制成超薄切片经醋酸铀-枸椽酸铅双重染色,在透射电镜下观察膜系统。结果:经不同试剂处理培养外周血用姐妹染色体色差法制成的染色体玻片全部进入结果分析。①当每瓶加入的壬基酚达到15μL时,细胞数明显减少,即外周血淋巴细胞分裂增殖的趋势明显减缓或停滞,累积的分裂相不多。各种壬基酚剂量处理下的姐妹染色单体交换率/细胞的数值与对照组相比差异有显著性。②未经37℃、4h温育的10-6mol/L的类黄酮没有显示出对壬基酚的拮抗作用,与相应经过温育的处理组相比差异有显著性。经温育处理的10-6mol/L的类黄酮溶液可以明显降低姐妹染色单体交换率,与对照组比较差异呈显著性(P<0.05)。而0.5mg的维生素E对壬基酚并未呈现显著的拮抗致损伤作用。但10-6mol/L类黄酮溶液0.5mL和维生素E0.5mg同时处理,与对照组比较细胞姐妹染色单体交换率出现大幅下降,差异呈现显著性。③透射电镜观察可见,壬基酚染毒组与拮抗物处理组比较肝细胞失去正常形态,细胞膜消失,核膜破碎,细胞胞质未见结构完整清晰的细胞器,膜结构损伤严重。结论:类黄酮可显著降低壬基酚处理过的淋巴细胞姐妹染色体交换率,类黄酮单因素存在一定的拮抗作用。终浓度0.9×10-7mol/L的类黄酮和90.9mg/L的维生素E在降低壬基酚处理过的淋巴细胞姐妹染色体交换率方面存在显著的协同效应。即一定剂量的类黄酮和维生素E在拮抗壬基酚的致损伤作用中存在着显著的协同效应。

AIM： To observe the manifestations of flavonoid and vitamin E in the effect against nonyl phenel＇s damage. METHODS： The experiment was carried out in the experimental center of Hunan Environment Biological polytecnic College in March 2005. Thirty SD rats of 5 months old were used. （1） 0.5 mL peripheral blood was drawn from rats and put it RPMI 1640 culture medium containing plant hamag- glutinin, which were divided into 4 groups according to the different reagent： blank control group [to inject distilled water （5, 10, 15 and 20 mL）], nonyl phenol group [ to inject nonyl phenot （5, 10, 15 and 20 mL）], flavonoid group [ to inject flavonoid （1×10^-6, 1×10^-5, 1×10^-4 mol/L） 0.5 mL], vitamin E group [ to inject vitamin E （0.25, 0.50, 1.00, 2.00 mg）]. After different disposal achromatic chromosome sheet glass was made by sister chromosome achromatics, the exchange rate of sister chromatids of lymph corpusdes was checked under microscope. （2） The peripheral blood of SD rats was drawn and put in RPMI 1640 culture medium containing plant hamagglutinin, which were divided into five groups according to different reagent： nonyl phenol group （to inject 10 μL nonyl phenot）, nonyl phenol and flavonoid group one （to inject 10 μL nonyl phenol and 10^-6 mol/L flavonoid, with Warm culture）, nonyl phenol and flavonoid group two （to inject 10 μL nonyl phenol and 10^-6 mol/L flavonoid, without warm culture）, nonyl phenol and vitamin E group （to inject 10 μL nonyl phenol and 0.5 mg vitamin E） and nonyl phenol and flavonoid and vitamin E group （to inject 10 μL nonyl phenol and 10^-6 moL/L flavonoid and 0.5 mg vitamin E）. After different disposal achromatic chromosome sheet glass was made by sister chromosome achromatics to observe 50 cells under fluoroscope and check the exchange rate of sister chromatids under microscope. （3） The relatively regular liver tissue was removed to make ultrathin section after disposal under double staining of uranium acetate and lead citrate and observe epicyte system under transmissive electron microscope. RESULTS： Analysize the whole result of chromosome sheet glass made by sister chromosome achromatics to culture peripheral blood by different reagent. （1）When nonyl phenol reached 15 μL in each bottle, the number of ceils apperenthy increased, that is the tendency of peripheral blood lymph corpusdes division and proliferation reduced or stopped and there was not more accumulated division. There was significance between the exchange rate of sister chromatids under the disposal of various nonyl phenol reagent and the difference of cell. numerial value compared with the contrast group. （2） 10^-6s mol/L flavonoid without warm culture of 37 ℃ for 4 hours did not show any resistant effect against nonyl phenol, which there was significance as compared with the disposal group with warm culture. 10^6 mol/L flavonoid solution with the disposal of warm cultune could apperently reduce the exchange rate of sister chromatids which had significance （P 〈 0.05） as compared with the control group. However 0.5 mg vitamin E did not show significant damage effect against nonyl phenol. But 0.5 mL of 10^-6 mol/L flavonoid solution and 0.5 mg vitamin E were disposed at the same time, and the exchange rate of.cell sister chromotids reduced greatly as compared with the control group, which there was significance. （3） It could be observed under transmissive electron microscope that the nonyl phenol group with virtue as compared with the resistance disposal growp, liver cell lost its normal shape and cell membrane disappeared. karyolmma broke, there was no whole and clear organella seen in cytoplasm and membrane＇s structure damaged seriously. CONCLUSION： Flavonoid can greatly reduce the exchange rate of lymph corpusdes sister chromosone under the disposal of nonyl phenol and it has a certain resistant effect. There is significant synergism between concentration 0.9×10^-7 mol/L flavonoid and 90.9 mg/L vitamin E reducing the exchange rate of lymph corpuscles sister chromosome under the disposal of nonyl phenol, that is a certain reagent of flavonoid and vitamin E have significant synergism against nonyl phenol＇s damage effect.