【作者】 杨光敏；

【导师】 欧阳五庆；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2007， 硕士

【摘要】 目的为了探讨玉米幼芽提取物（Extract from Maize Plumule）对果蝇寿命、抗氧化能力的影响;对小鼠睾丸组织结构、功能和抗氧化酶活性的影响;对原代培养大鼠睾丸Leydig细胞睾酮合成和分泌功能的影响。方法1.果蝇衰老模型的建立。收集羽化8 h内的黑腹果蝇,随机分组,以添加D-半乳糖（6.5%）及不同浓度玉米幼芽提取物（0㎎/kg,20㎎/kg,40㎎/kg,80㎎/kg）的培养基饲喂,分别测定10、20、30、40日龄时果蝇体内超氧化物歧化酶（superoxide dismutase, SOD）活性及丙二醛（malondialdehyde, MDA）含量。果蝇寿命实验:每天定时记录果蝇存活数,直至最后一只果蝇死亡,计算果蝇平均寿命和最高寿命。果蝇体内水分含量的测定:饲养至4 d后,将果蝇称初重,放入干燥箱中（55℃）,干燥1 h后再次称重,测定果蝇体内水分含量;果蝇耐饥饿能力实验:饲养至4 d后,放入恒温培养箱中（25℃）,每天定时记录果蝇存活数,计算果蝇耐饥饿能力。果蝇抗热应激实验:饲养至10 d后,将果蝇放入洁净的玻璃培养瓶,润湿棉塞,置于恒温培养箱中（40℃）,每隔1 h观察并记录果蝇死亡数量,计算各组存活率。2.小鼠衰老模型的建立。小鼠颈背部皮下注射D-半乳糖（5 g/L,125㎎/kg·BT）,连续6周,制备D-半乳糖亚急性衰老小鼠模型。同时灌胃不同浓度玉米幼芽提取物,剂量依次为10㎎/kg、20㎎/kg、40㎎/kg,按20 ml/kg·BW灌胃。灌胃结束后第二天,检测小鼠血清睾酮（testosterone,T）含量和睾丸组织SOD、谷胱甘肽过氧化物酶（glutathione perocidase, GSH-PX）活性及一氧化氮（nitric oxide, NO）、MDA含量。取各组小鼠附睾,检测附睾精子功能,包括精子计数、精子活率测定。睾丸组织切片HE染色,显微镜观察组织结构变化。透射电镜观察超微结构变化。3.睾丸Leydig细胞衰老模型的建立。取SD大鼠（250～300 g）睾丸,用消化法制备细胞悬液,10 %血清DMEM/F12培养基培养,3β-羟类固醇脱氢酶（3β-HSD）染色鉴定睾丸Leydig细胞。原代培养细胞加入不同浓度的玉米幼芽提取物（5㎎/L,10㎎/L,20㎎/L）培养18 h后,再加入H₂O₂（30 %H₂O₂,50μM培养基）制备细胞衰老模型,6 h后取各组细胞培养上清液,检测T、SOD、MDA,细胞HE染色。四噻唑蓝（MTT）实验测定细胞的存活率。结果1.玉米幼芽提取物80㎎/kg能极显著延长果蝇平均寿命和最高寿命（P<0.01）,提高30、40日龄时果蝇SOD活性、降低MDA含量（0.01< P<0.05或P<0.01）,提高果蝇抗热应激的能力（0.01< P<0.05或P<0.01）。2.玉米幼芽提取物10㎎/kg·BW能极显著升高衰老小鼠睾丸组织SOD活性、GSH-PX活性及NO含量、血清T水平、附睾精子数和精子活率（P<0.01）,极显著降低MDA含量（P<0.01）。给药组小鼠睾丸生精细胞数目增多、排列有序,管腔内精子数目增多。生精细胞细胞器数目增多,线粒体形态完整,细胞核清晰。Leydig细胞数目增多,细胞核清晰、颗粒状分布。3. 3β-羟类固醇脱氢酶（3β-HSD）染色鉴定原代培养细胞为睾丸Leydig细胞。玉米幼芽提取物20㎎/L能极显著提高原代培养大鼠睾丸Leydig细胞睾酮合成和分泌的能力（P<0.01）,保持细胞结构完整性。结论1.玉米幼芽提取物具有延长果蝇寿命、提高果蝇抗氧化能力的作用。2.玉米幼芽提取物可提高D-半乳糖致衰老小鼠睾丸抗氧化能力,提高血清T水平,对睾丸组织超微结构有保护和促进作用,具有延缓睾丸衰老的作用。3.玉米幼芽提取物可提高原代培养大鼠睾丸间质细胞合成和分泌睾酮的能力,维持细胞结构完整性,具有延缓雄性性腺衰老的作用。综上所述,玉米幼芽提取物具有延长果蝇寿命、延缓小鼠睾丸衰老、维持并提高原代培养大鼠睾丸Leydig细胞睾酮合成和分泌能力的作用。更多还原

【Abstract】 ObjectiveThis study was designed to investigate the effect of Extract from Maize Plumule on life-span, antioxydative ability of Drosophila melanogaster , the effect of testis ultrastructrue and function and antioxydative enzyme activity of aging mice by D-gal, the effect of testosterone synthesis and secretion of Leydig cells in primary culture from rats testis.Methods1.Establishment of aging model of Drosophila melanogaster. Collect eclosion imago in 8 h and divided into different groups. Life-span team: feed with different concentration medium（0 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg）,then count the number of the survival Drosophila Melanogaster in every group everyday until the last one died, then work out the mean lifetime and maximum lifetime. SOD and MDA team: feed with D-gal and different concentration medium（0 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg）, then test SOD activity in every group and MDA content on 10 d、20 d、30 d、40 d, every group. D-gal substitutes sucrose in normal medium, D-gal concentration is 6.5%, with sucrose compensate. Water content research: the fruitflies in 4 days were put into baking oven（55℃） for 1 hour, then got the content. Starvation: the fruitflies in 4 days were put into baking oven（25℃）, then count the number of living fruitflies, three times everyday, until the last fruitflies died. Heat stress: the fruitflies in 10 days were put into baking oven（40℃）, then count the number of living fruitflies, every 1 hour, until the last fruitflies died.2.Establishment of aging mice model caused by D-gal. Sub-acutely aging mice model was made by subcutaneous injection（sc.） with D-gal for six weeks continually, while the mice were treated trough injecting Extract from Maize Plumule into their gaster with three different concentration: 10 mg/kg, 20 mg/kg, 40 mg/kg. After six weeks, measured SOD, GSH-PX, NO, MDA and serum T, then observed the testis tissue changes under the light microscope ,and the ultrastructural changes of testis under transmission electron microscope.Then test sperm function in epididymis, including sperm number and motility rate.3.Establishment of aging Leydig cells model caused by H₂O₂. We got cells suspension from SD rats （250～300 g） testis with enzymatic digestion. Leydig cells were being cultured in DMEM/F12 with different concentration Extract from Maize Plumule for 18 hours. Then H₂O₂（30 %H₂O₂,50μM）was added to the medium to make cell aging model. After 6 hours, collected the medium to detect T and SOD and MDA. Leydig cells stained with haematoxylin- eosin. Survival rate of Leydig cells was tested by MTT test.Results1.Anti-aging effect of Extract from Maize Plumule on Drosophila Melanogaster.Extract from Maize Plumule（80 mg/kg） had the ability of longer anverage lifetime and longest lifetime of Drosophila Melanogaster, and increasing SOD activity（P<0.01）in 30 and 40 days, and reducing MDA content in 30 and 40 days（0.01< P<0.05）, and increasing ability of anti-heatstress.2.Extract from Maize Plumule could increase SOD activity, GSH-PX activity, NO content, serum T content, and decrease MDA content remarkably, and increase sperm number and motility rate, specially group of 10 mg/kg. The ultrastructrue of testis of the mice with Extract from Maize Plumule changed obvirously.3.3β-HSD identifed primary cultured cells as Leydig cells. The group in 20 mg/L could help protect primary cultured Leydig cells from H₂O₂（30 % H₂O₂, 50μM）, and maintained Leydig cells integrality in morphology, and increased the testosterone synthesis and secretion ability of Leydig cells from rat testis.Conclusions1.Extract from Maize Plumule have the effect of increasing lifetime and anti-oxidation ability on Drosophila melanogaster.2.Extract from Maize Plumule could increase anti-oxidation ability of testis, increase serum T level, and protect the ultrastructrue of aging mice testis caused by D-gal. Therefore Extract from Maize Plumule have the effect of anti-aging on testis.3.Extract from Maize Plumule could increase increase the testosterone synthesis and secretion ability for testis Leydig cells in primary culture.更多还原