【作者】 彭海航；

【导师】 欧阳五庆；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2009， 硕士

【摘要】 目的:针对宠物毛发问题产生的原因,选取玉米幼芽提取物(Extracts of Maize Plunmule,EMP)作为本实验研究对象,通过建立在体和体外毛囊紫外(UVA)氧化损伤模型验证EMP对毛囊氧化损伤的保护作用,评价EMP对毛囊氧化损伤的保护效果,并在此基础上开发一种新型宠物美毛保健产品。方法:(1)在体实验:本实验以C57BL/6小鼠为实验动物,将30只小鼠随机分成五组,分别为:空白对照组、UVA模型组、EMP低剂量、EMP中剂量、EMP高剂量组,采用灌胃给药的方式,通过对小鼠毛囊表征观察、长度观察、组织化学观察、凋亡细胞检测等手段,研究EMP对UVA致毛囊氧化损伤后毛发生长周期的影响。(2)体外实验:对C57BL/6小鼠毛囊细胞进行体外培养,设六个实验组,分别为:空白对照组、UVA模型组、EMP低剂量组、EMP中剂量组、EMP高剂量组和阳性药对照组。以DMEM作为基础培养基,用酶消化法对小鼠毛囊球部细胞进行分离培养;用MTT法测定UVA照射体外培养小鼠毛囊细胞的存活率,并用相差显微镜观察各组细胞的形态学变化;用生物化学方法测定SOD、GSH-Px酶活性和MDA含量以及细胞内GSH/GSSG比值变化。(3)美毛产品制备:通过预试验及查阅大量相关文献,采用正交试验设计法,筛选最佳配方比例,并用直接压片的方法,制备出一种含有EMP的宠物美毛保健产品。结果:(1)在体实验结果EMP灌胃各剂量组较UVA模型组小鼠背部皮肤由粉红色变为黑色的时间提前(P<0.05),皮肤为黑色的总时间延长(P<0.05),从皮肤变为黑色到长毛的时间间隔缩短(P<0.05);组织学检查结果与肉眼观察的皮肤变化一致,但实验结束时EMP灌胃各剂量组小鼠背部新生毛发的长度与UVA模型组相比差异无显著性;关于组织中毛囊细胞凋亡水平,与UVA模型组相比EMP可显著抑制毛囊中细胞的凋亡(P<0.01)。(2)体外实验结果经UVA照射损伤后的细胞数量减少、形状皱缩,细胞贴壁能力下降,添加EMP保护的细胞有轻微的萎缩,细胞形态基本正常;EMP可显著提高UVA辐射成纤维细胞中的GSH/GSSG比值(P<0.01),以及SOD和GSH-Px的活性(P<0.01),降低MDA含量(P<0.01)。(3)美毛产品制备及质量评价结果正交试验设计筛选出最佳配比为EMP 1%、大豆粉与海藻粉的配比为1:0.5、蔗糖5%、羧甲基纤维素钠(CMC)1.5%、硬酯酸镁1.5%,烘干温度为35℃。制得的片剂对光和热都较稳定,但有吸湿现象,故应干燥保存。结论:通过EMP在动物水平和细胞水平对毛囊氧化损伤的保护实验可知,EMP可以有效维持氧化损伤后毛囊的毛发生长周期,使小鼠毛发进入生长期,并可自发经过退行期再进入休止期,诱导毛发生长和延长毛发的生长期,在生长期早期能加速小鼠毛发生长,但对毛发最终长度无影响;对毛囊细胞具有明显的抗氧化损伤作用,可以增强氧化损伤毛囊细胞中抗氧化酶的活力,维持细胞形态和功能的完整性,有效抑制了细胞凋亡的发生。通过相关指标、因素正交试验筛选,采用直接压片的方法制备了一种以EMP为功效组分的宠物美毛片,该片剂具有外观整洁、适口性好、方便宠物服用等优点,有望成为新一代可食用的宠物毛发保健产品。更多还原

【Abstract】 ObjectiveFor the cause of pet hair problem, select Extracts of Maize Plunmule(EMP) as the experimental subjects, through the establishment of hair follicles in vivo and in vitro ultraviolet (UVA) model validation EMP oxidative damage to the hair follicle protective effect on hair follicles EMP evaluation of the protective effect of oxidative damage, and on this basis ,to develop a new type of pet hair protection products.Method(1) In vivo experiment: Taking C57BL / 6 mice as experimental animals, 30 mice were randomly divided into five groups, namely: control group, UVA model, EMP low-dose, EMP medi-dose, EMP high-dose group, the use of oral administration, through hair follicles in mice Characterization of observation, the length of observation, histochemical observation, by means of detection of apoptotic cells to study the hair growth cycle after EMP used on the UVA-induced oxidative damage hair follicles.(2) In vitro experiment: The C57BL / 6 mouse hair follicle cells cultured in vitro, for the six experimental groups, respectively: control group, UVA model, EMP low-dose group, EMP medi-dose group, EMP high-dose group and positive drug control group. Taking DMEM as basal medium, to make mouse hair follicle bulb cells isolated and cultured by enzymatic digestion, to determine the survival rate of hair follicel cell with UVA irradiation by MTT, and to observe the morphological changes of cells by phase contrast microscope; to determine the activities of SOD, GSH-Px and the cotent of MDA by biochemical methods, as well as the change of GSH / GSSG ratio.(3) Preparation of hair protection products:Through the pre-test and hunting a large number of the relevant literature, using orthogonal design, selecting ratio of the best formula, using direct compression methods, to prepare an EMP pet hair protection products.Result(1) The experimental results in vivo: EMP oral administration of various doses of UVA group than in model group mice back skin from pink to black, the mere mention earlier (P <0.05), the skin is black to extend the total time (P <0.05), from the skin into the long-haired black shorten the time interval (P <0.05); the results of histological examination with the naked eye to observe changes in the skin, but the end of the experiment EMP mice administered various doses of the length of the back of new hair model group compared with the UVA there was no significant difference; on the tissue levels of follicle cell apoptosis, compared with the UVA model EMP can significantly inhibit cell apoptosis in hair follicles (P <0.01).(2) The experimental results in vivo: By UVA irradiation of cells after injury to reduce the number, shape, shrinkage, decreased cell adhesion, EMP protection, add a slight shrinkage of cells, normal cells; EMP can significantly increase the radiation UVA in fibroblast GSH / GSSG ratio (P <0.01), as well as SOD and GSH-Px activity (P <0.01), lower MDA content (P <0.01).(3) Hair protection product preparation and quality evaluation results: Orthogonal experimental design selected for the best ratio of EMP 1%, soybean meal and seaweed powder ratio of 1:0.5, 5% sucrose, sodium carboxymethyl cellulose (CMC) 1.5%, the dolomol 1.5% , drying temperature of 35℃. The tablets obtained are more stable to light and heat, but the phenomenon of moisture and should be preserved dry.ConclusionThrough the EMP levels in animal and cellular levels of oxidative damage to the hair follicle, we can see the protection of experimental, EMP can be effectively maintained after oxidative damage of the hair follicle growth cycle of hair into the growth of mice, and spontaneous catagen after re-entering the rest period, induced hair, hair growth and prolong the growing season, early in the growing season can accelerate hair growth in mice, but had no effect on the length of the final hair; of hair follicle cells have a clear role in anti-oxidative damage, oxidative stress can enhance the follicle cells of antioxidant enzymes vitality, maintenance of cell morphology and function of the integrity and effective inhibition of cell apoptosis.Through the relevant indicators, the selection of orthogonal factors, the use of direct compression tablets were prepared by an EMP for the effective component of the products, the tablet has a clean appearance, palatability, and convenience of taking the advantages of pets is expected to become the new generation of edible pet hair protection products.更多还原