维生素E对鲤鱼肠上皮细胞生长发育及抗氧化能力的影响
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摘要
本试验以原代培养鲤鱼肠上皮细胞为研究模型，研究了维生素E对鲤鱼肠上皮细胞生长发育及抗氧化能力的影响。研究共包括2个试验：试验一、维生素E对鲤鱼肠上皮细胞生长发育的影响；试验二、维生素E对鲤鱼肠上皮细胞抗氧化能力的影响。
试验一结果表明：2.5μg／ml维生素E显著增加了鲤鱼肠上皮细胞的MTT吸光值(P＜0.05)。维生素E对原代培养鲤鱼肠上皮细胞蛋白含量、细胞蛋白含量／MTT吸光值、Na~+-K~+-ATP酶活力、Na~+-K~+-ATP酶活力／MTT吸光值有极显著(P＜0.01)影响；且维生素E与细胞蛋白含量、Na~+-K~+-ATP酶活力、Na~+-K~+-ATP酶活力／MTT吸光值分别呈显著或极显著的正相关(r_1=+0.4810，P＜0.05；r_2=+0.6740，P＜0.01；r_3=+0.6440，P＜0.01)；维生素E与细胞Na~+-K~+-ATP酶活力呈极显著的二次回归关系(Y=0.092+0.014X-0.002X~2，R~2=0.4600，P＜0.01；其中Y为细胞Na~+-K~+-ATP酶活力，X为培养液中维生素E浓度)，当维生素E浓度为3.5000μg／ml时，鲤鱼肠上皮细胞Na~+-K~+-ATP酶活力最大。维生素E对原代培养鲤鱼肠上皮细胞AKP活力、蛋白消耗量、蛋白消耗量／MTT有极显著(P＜0.01)影响，当维生素E浓度达到最大值(7μg／ml)时，鲤鱼肠上皮细胞AKP活力、蛋白消耗量、蛋白消耗量／MTT吸光值均极显著(P＜0.01)降低。维生素E对原代培养鲤鱼肠上皮细胞培养液LDH活力、培养液氨浓度分别有显著(P＜0.05)或极显著(P＜0.01)影响，且维生素E与培养液氨浓度呈显著的负相关(r=-0.5100，P＜0.05)。蛋白消耗量／MTT吸光值与细胞AKP活力、培养液氨浓度分别呈显著或极显著的正相关(r_1=+0.4710，P＜0.05；r_2=+0.5540，P＜0.01)。
试验二结果表明：维生素E对原代培养鲤鱼肠上皮细胞抗超氧阴离子活力、抗羟自由基活力有极显著(P＜0.01)影响，维生素E与细胞抗超氧阴离子活力、细胞抗羟自由基活力呈极显著正相关关系(r_1=+0.5410，P＜0.01；r_2=+0.8100，P＜0.01)，且维生素E与细胞抗羟自由基活力呈极显著的二次回归关系(Y=98.718+17.237X-1.300X~2，R~2=0.7440，P＜0.01；其中Y为细胞抗羟自由基活力，X为培养液中维生素E浓度)，当维生素E浓度为6.6296μg／ml时，鲤鱼肠上皮细胞抗羟自由基活力最大。维生素E对细胞GSH浓度、GSH／GSSG比例有极显著影响(P＜0.01)，维生素E与细胞GSH浓度呈显著的正相关(r=+0.4070，P＜0.05)；且维生素E与细胞GSH浓度、GSH／GSSG比例分别呈极显著的二次回归关系(Y_1=85.925+18.547X-2.132X~2，R~2=0.4510，P＜0.01；Y_2=0.858+0.212X-0.024X~2，R~2=0.3690，P＜0.01；其中Y_1为细胞GSH浓度，Y_2为细胞GSH／GSSG比例，X为培养液中维生素E浓度)，当维生素E浓度为4.3497μg／ml、4.4167μg／ml时，鲤鱼肠上皮细胞GSH浓度、GSH／GSSG比例分别达到最大。维生素E对鲤鱼肠上皮细胞培养液丙二醛含量有显著(P＜0.05)影响，且维生素E与培养液丙二醛含量存在显著的负相关(r=-0.4450，P＜0.05；)。
结果说明：适量维生素E能够促进鲤鱼肠上皮细胞的增殖；维生素E能促进鲤鱼肠上皮细胞的生长发育，主要与维生素E促进单个肠上皮细胞发育和功能完善有关；维生素E维护了鲤鱼肠上皮细胞结构的完整性：同时维生素E能够通过清除自由基、增加细胞自身非酶性抗氧化物和减少脂质过氧化物质产生来提高鲤鱼肠上皮细胞的抗氧化能力。当维生素E浓度为6.6296μg／ml时，鲤鱼肠上皮细胞抗羟自由基能力最强；当维生素E浓度为4.3497μg／ml时，鲤鱼肠上皮细胞内GSH含量最高；当维生素E浓度为4.4167μg／ml时，鲤鱼肠上皮细胞GSH／GSSG比值最大。
The carp intestinal epithelial cells in primary culture were selected to evaluate effectsof vitamin E on the growth and development and anti-oxidation capacity. Firstly, the MTTabsorbance values, protein content, Na~+-K~+-ATPase activities, alkaline phosphataseactivities in the cells and lactate dehydrogenase activities and ammonia concentration inthe culture medium were investigated after vitamin E was added 96 h. 2.5μg/ml of vitaminE concentration significantly increased carp intestinal epithelial cells MTT absorbancevalues (P＜0.05). The results showed that the protein content, protein content/MTT OD,Na~+-K~+-ATPase activities, Na~+-K~+-ATPase activities /MTT OD in the cells were verysignificantly enhanced by vitamin E concentration (P＜0.01),andthere were significantcorrelation between vitamin E and protein content, Na~+-K~+-ATPase activities,Na~+-K~+-ATPase activities /MTT OD in the cells (r_1=+0.4810,P＜0.05; r_2=+0.6740,P＜0.01; r_3=+0.6440, P＜0.01).The regression results showed that there were verysignificant quadratic relationship between Na~+-K~+-ATPase activities and vitamin E level(R~2= 0.4600, P＜0.01).The alkaline phosphatase activities, protein consumption, proteinconsumption/MTT OD in the culture medium was significantly depressed by vitamin Econcentration (P＜0.05). When the vitamin E concentration achieved the maximum (7μg/ml), alkaline phosphatase activities,protein consumption/MTT OD in the cells werevery significantly lower (P＜0.01). Vitamin E on the carp intestinal epithelial cell culturemedium LDH activity, ammonia concentrations were significantly (P＜0.05) orsignificantly (P＜0.01), and there were significantly negative correlation between vitamin Eand ammonia concentration in the culture medium (r=-0.5100, P＜0.05). The results ofcorrelation analysis showed that there were significant correlation between proteinconsumption/MTT OD in the cells and alkaline phosphatase activities in the cells,ammonia concentration in the culture medium (r_1=+0.4710, P＜0.05; r_2=+0.5540, P＜0.01).
Secondly, malondialdehyde concentration in the culture medium and anti-superoxideanion activitity,anti-hydroxy radical activitity, reduced glutathione concentration,the ratiobetween reduced glutathione and oxidized glutathione in the cells were investigated.Theresults showed that anti-superoxide anion activitity,anti-hydroxy radical activitity,reduced glutathione concentration,the ratio between reduced glutathione and oxidizedglutathione in the cells were very significantly enhanced and the malondialdehydeconcentration in the culture medium was significantly depressed with the vitamin Econcentration increasing(P＜0.01). The results of correlation analysis showed that therewere very significant correlation between vitamin E level and anti-superoxide anionactivities,anti-hydroxy radical activities in the cells(r_1=+0.5410, P＜0.01; r_2=+0.8100,P＜0.01;),and vitamin E and anti-hydroxyl radical activity was highly significant quadraticregression relationship(R~2=0.7440, P＜0.01). There were significant correlation betweenvitamin E level and reduced glutathione concentration in the cells (r=+0.4070, P＜0.05),but negative significant correlation between vitamin E level and malondialdehydeconcentration in the culture medium (r=-0.4450, P＜0.05;).
In summary, proper vitamin E can promote carp intestinal epithelial cellproliferation .But vitamin E can promote carp intestinal epithelial cell growth anddevelopment, with the major vitamin E for a single intestinal epithelial cell developmentand improve the function. Vitamin E safeguarded carp intestinal epithelial cells structuralintegrity. Vitamin E also can be passed to remove free radicals, own cells to increasenon-enzymatic antioxidants and reduction of lipid peroxidation to raise carp intestinalepithelial cells antioxidant capacity.
试验一结果表明：2.5μg／ml维生素E显著增加了鲤鱼肠上皮细胞的MTT吸光值(P＜0.05)。维生素E对原代培养鲤鱼肠上皮细胞蛋白含量、细胞蛋白含量／MTT吸光值、Na~+-K~+-ATP酶活力、Na~+-K~+-ATP酶活力／MTT吸光值有极显著(P＜0.01)影响；且维生素E与细胞蛋白含量、Na~+-K~+-ATP酶活力、Na~+-K~+-ATP酶活力／MTT吸光值分别呈显著或极显著的正相关(r_1=+0.4810，P＜0.05；r_2=+0.6740，P＜0.01；r_3=+0.6440，P＜0.01)；维生素E与细胞Na~+-K~+-ATP酶活力呈极显著的二次回归关系(Y=0.092+0.014X-0.002X~2，R~2=0.4600，P＜0.01；其中Y为细胞Na~+-K~+-ATP酶活力，X为培养液中维生素E浓度)，当维生素E浓度为3.5000μg／ml时，鲤鱼肠上皮细胞Na~+-K~+-ATP酶活力最大。维生素E对原代培养鲤鱼肠上皮细胞AKP活力、蛋白消耗量、蛋白消耗量／MTT有极显著(P＜0.01)影响，当维生素E浓度达到最大值(7μg／ml)时，鲤鱼肠上皮细胞AKP活力、蛋白消耗量、蛋白消耗量／MTT吸光值均极显著(P＜0.01)降低。维生素E对原代培养鲤鱼肠上皮细胞培养液LDH活力、培养液氨浓度分别有显著(P＜0.05)或极显著(P＜0.01)影响，且维生素E与培养液氨浓度呈显著的负相关(r=-0.5100，P＜0.05)。蛋白消耗量／MTT吸光值与细胞AKP活力、培养液氨浓度分别呈显著或极显著的正相关(r_1=+0.4710，P＜0.05；r_2=+0.5540，P＜0.01)。
试验二结果表明：维生素E对原代培养鲤鱼肠上皮细胞抗超氧阴离子活力、抗羟自由基活力有极显著(P＜0.01)影响，维生素E与细胞抗超氧阴离子活力、细胞抗羟自由基活力呈极显著正相关关系(r_1=+0.5410，P＜0.01；r_2=+0.8100，P＜0.01)，且维生素E与细胞抗羟自由基活力呈极显著的二次回归关系(Y=98.718+17.237X-1.300X~2，R~2=0.7440，P＜0.01；其中Y为细胞抗羟自由基活力，X为培养液中维生素E浓度)，当维生素E浓度为6.6296μg／ml时，鲤鱼肠上皮细胞抗羟自由基活力最大。维生素E对细胞GSH浓度、GSH／GSSG比例有极显著影响(P＜0.01)，维生素E与细胞GSH浓度呈显著的正相关(r=+0.4070，P＜0.05)；且维生素E与细胞GSH浓度、GSH／GSSG比例分别呈极显著的二次回归关系(Y_1=85.925+18.547X-2.132X~2，R~2=0.4510，P＜0.01；Y_2=0.858+0.212X-0.024X~2，R~2=0.3690，P＜0.01；其中Y_1为细胞GSH浓度，Y_2为细胞GSH／GSSG比例，X为培养液中维生素E浓度)，当维生素E浓度为4.3497μg／ml、4.4167μg／ml时，鲤鱼肠上皮细胞GSH浓度、GSH／GSSG比例分别达到最大。维生素E对鲤鱼肠上皮细胞培养液丙二醛含量有显著(P＜0.05)影响，且维生素E与培养液丙二醛含量存在显著的负相关(r=-0.4450，P＜0.05；)。
结果说明：适量维生素E能够促进鲤鱼肠上皮细胞的增殖；维生素E能促进鲤鱼肠上皮细胞的生长发育，主要与维生素E促进单个肠上皮细胞发育和功能完善有关；维生素E维护了鲤鱼肠上皮细胞结构的完整性：同时维生素E能够通过清除自由基、增加细胞自身非酶性抗氧化物和减少脂质过氧化物质产生来提高鲤鱼肠上皮细胞的抗氧化能力。当维生素E浓度为6.6296μg／ml时，鲤鱼肠上皮细胞抗羟自由基能力最强；当维生素E浓度为4.3497μg／ml时，鲤鱼肠上皮细胞内GSH含量最高；当维生素E浓度为4.4167μg／ml时，鲤鱼肠上皮细胞GSH／GSSG比值最大。
The carp intestinal epithelial cells in primary culture were selected to evaluate effectsof vitamin E on the growth and development and anti-oxidation capacity. Firstly, the MTTabsorbance values, protein content, Na~+-K~+-ATPase activities, alkaline phosphataseactivities in the cells and lactate dehydrogenase activities and ammonia concentration inthe culture medium were investigated after vitamin E was added 96 h. 2.5μg/ml of vitaminE concentration significantly increased carp intestinal epithelial cells MTT absorbancevalues (P＜0.05). The results showed that the protein content, protein content/MTT OD,Na~+-K~+-ATPase activities, Na~+-K~+-ATPase activities /MTT OD in the cells were verysignificantly enhanced by vitamin E concentration (P＜0.01),andthere were significantcorrelation between vitamin E and protein content, Na~+-K~+-ATPase activities,Na~+-K~+-ATPase activities /MTT OD in the cells (r_1=+0.4810,P＜0.05; r_2=+0.6740,P＜0.01; r_3=+0.6440, P＜0.01).The regression results showed that there were verysignificant quadratic relationship between Na~+-K~+-ATPase activities and vitamin E level(R~2= 0.4600, P＜0.01).The alkaline phosphatase activities, protein consumption, proteinconsumption/MTT OD in the culture medium was significantly depressed by vitamin Econcentration (P＜0.05). When the vitamin E concentration achieved the maximum (7μg/ml), alkaline phosphatase activities,protein consumption/MTT OD in the cells werevery significantly lower (P＜0.01). Vitamin E on the carp intestinal epithelial cell culturemedium LDH activity, ammonia concentrations were significantly (P＜0.05) orsignificantly (P＜0.01), and there were significantly negative correlation between vitamin Eand ammonia concentration in the culture medium (r=-0.5100, P＜0.05). The results ofcorrelation analysis showed that there were significant correlation between proteinconsumption/MTT OD in the cells and alkaline phosphatase activities in the cells,ammonia concentration in the culture medium (r_1=+0.4710, P＜0.05; r_2=+0.5540, P＜0.01).
Secondly, malondialdehyde concentration in the culture medium and anti-superoxideanion activitity,anti-hydroxy radical activitity, reduced glutathione concentration,the ratiobetween reduced glutathione and oxidized glutathione in the cells were investigated.Theresults showed that anti-superoxide anion activitity,anti-hydroxy radical activitity,reduced glutathione concentration,the ratio between reduced glutathione and oxidizedglutathione in the cells were very significantly enhanced and the malondialdehydeconcentration in the culture medium was significantly depressed with the vitamin Econcentration increasing(P＜0.01). The results of correlation analysis showed that therewere very significant correlation between vitamin E level and anti-superoxide anionactivities,anti-hydroxy radical activities in the cells(r_1=+0.5410, P＜0.01; r_2=+0.8100,P＜0.01;),and vitamin E and anti-hydroxyl radical activity was highly significant quadraticregression relationship(R~2=0.7440, P＜0.01). There were significant correlation betweenvitamin E level and reduced glutathione concentration in the cells (r=+0.4070, P＜0.05),but negative significant correlation between vitamin E level and malondialdehydeconcentration in the culture medium (r=-0.4450, P＜0.05;).
In summary, proper vitamin E can promote carp intestinal epithelial cellproliferation .But vitamin E can promote carp intestinal epithelial cell growth anddevelopment, with the major vitamin E for a single intestinal epithelial cell developmentand improve the function. Vitamin E safeguarded carp intestinal epithelial cells structuralintegrity. Vitamin E also can be passed to remove free radicals, own cells to increasenon-enzymatic antioxidants and reduction of lipid peroxidation to raise carp intestinalepithelial cells antioxidant capacity.
引文
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