【作者】 刘颖；

【导师】 蒋宝泉；

【作者基本信息】 第三军医大学 ， 营养与食品卫生学， 2007， 硕士

【摘要】 目的:糖尿病(DM)是一种全球范围内严重影响人类健康的常见内分泌代谢性疾病,其发病率和死亡率近年来呈逐步上升趋势。目前全世界已有糖尿病人近1.5亿,我国实际患病人数已达4000万人以上。DM主要是由于胰岛B细胞减少或B细胞功能减退引起的糖、脂肪、蛋白质、水及电解质代谢紊乱,并可由此产生各种并发症,如由大血管或微血管病变所引起的冠心病、脑血管疾病、视网膜病变、肾血管病变等,其中糖尿病患者有50%以上死于冠心病。因此,有效的改善糖尿病的代谢紊乱对预防糖尿病并发症、促进疾病康复和提高病人生活质量都具有十分重要的意义,而努力探索和积极寻找治疗和控制糖尿病的理想方法是医学界关注的重要课题。本研究以生物发酵作为切入点,通过观察我们所研制的糖尿病复合营养酸奶对保护糖尿病大鼠的胰腺功能、改善糖代谢、降低血糖血脂及对胰腺组织形态学、脂质过氧化状态和胰岛B细胞凋亡的影响,为糖尿病患者功效食品的研制提供实验依据。方法:清洁级成年Wister大鼠(雌雄各半)120只,体重190±10克,随机分为正常对照组、糖尿病对照组、治疗组、预防治疗组四组,每组30只。在常规饲料自由进食基础上,正常对照组给予生理盐水灌喂,糖尿病对照组和治疗组给予普通酸奶灌喂,预防治疗组给予糖尿病复合营养酸奶灌喂,隔日一次,每次2毫升,喂养至第15天按四氧嘧啶(ALX)50mg/k.w尾静脉注射制作糖尿病模型,正常对照组注射生理盐水。5天后测其空腹4小时后血糖,将血糖值>13.8mmol/L的大鼠做为糖尿病模型大鼠。糖尿病模型制作成功后除治疗组动物灌喂液由原来普通酸奶改为糖尿病复合营养酸奶外,其他组动物喂养方式不变,继续分组喂养,并于注射ALX后第30天、第90天再次进行空腹4小时血糖测定,注射ALX后第6天、第32天、第92天进行糖耐量测定,注射ALX后第33天、第93天分别从各组大鼠中随机取10只大鼠进行腹主动脉取血、处死大鼠取材进行以下指标测定:血清中的胰岛素(INS)、C-肽(C-P)、丙二醛(MDA)、超氧化物歧化酶(SOD)、肌酐(Cr)、尿素氮(BUN)、甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL)的含量,血浆中胰高血糖素(GLU)和糖化血红蛋白(GHb)含量,光镜下观察病理结构改变,TUNEL法测定胰岛B细胞凋亡,免疫组化法观察INS和GLU分泌的表达。结果:1.大鼠空腹血糖、糖耐量及糖化血红蛋白测定结果:1.1 FBS水平(mmol/L):1.1.1注射ALX后第5天,糖尿病对照组、治疗组、预防治疗组FBS非常显著高于正常对照组(P<0.01),预防治疗组非常显著低于糖尿病对照组和治疗组(P<0.01)。1.1.2注射ALX后第30天和90天,糖尿病对照组、治疗组、预防治疗组非常显著高于正常对照组(P<0.01),但治疗组、预防治疗组非常显著低于糖尿病对照组(P<0.01)。1.2糖耐量测定结果(mmol/L):注射ALX后第6天、第32天、第92天糖尿病对照组、治疗组、预防治疗组动物0.5h、1.0h、1.5h、2.0h血糖测定值显著或非常显著高于正常对照组(P<0.05或p<0.01),但治疗组、预防治疗组第32天、92天上述各时相点血糖测定值非常显著低于糖尿病对照组(P<0.01),并且预防治疗组第6天上述各时相点血糖测定值非常显著低于治疗组(P<0.01)。1.3糖化血红蛋白测定结果(%):注射ALX后第93天,糖尿病对照组、治疗组、预防治疗组糖化血红蛋白含量非常显著高于正常对照组(P<0.01),但治疗组和预防治疗组显著低于糖尿病对照组(P<0.01)。2.大鼠血清INS、C-P、GLU测定结果:2.1血清INS含量(μIU/ml):注射ALX后第33天、第93天,糖尿病对照组、治疗组、预防治疗组INS含量非常显著低于正常对照组(P<0.01),但治疗组、预防治疗组显著或非常显著高于糖尿病对照组(P<0.05或p<0.01)。2.2血清C-P含量(ng/ml):注射ALX后第33天和93天,正常组、治疗组、预防治疗组C-P含量显著或非常显著高于糖尿病对照组(P<0.01、P<0.05和P<0.01),而治疗组、预防治疗组与正常对照组没有显著差异。2.3血浆GLU含量(pg/ml):注射ALX后第93天,糖尿病对照组、治疗组血浆GLU显著或非常显著高于正常对照组(P<0.01、P<0.05),预防治疗组与正常对照组之间无显著差异(p>0.05),但预防治疗组显著低于糖尿病对照组(P<0.05)。3.大鼠血清SOD、MDA含量测定结果:注射ALX后第33天和93天,糖尿病对照组、治疗组、预防治疗组SOD含量非常显著低于正常对照组(P<0.01),MDA含量非常显著高于正常对照组(P<0.01),但治疗组、预防治疗组SOD含量非常显著高于糖尿病对照组(p<0.01),MDA含量显著或非常显著低于糖尿病对照组(P<0.05或p<0.01)。4.大鼠血清TG、TC、LDL测定结果:注射ALLOX后第93天,治疗组、预防治疗组血清TG、TC、LDL含量显著或非常显著低于糖尿病对照组(P<0.05或P<0.01)。5.大鼠血清Cr和BUN测定结果:注射ALLOX后第93天,糖尿病对照组、治疗组、预防治疗组Cr和BUN含量非常显著高于正常对照组(P<0.01),但治疗组、预防治疗组显著或非常显著低于糖尿病对照组(p<0.05或p<0.01)。6.大鼠胰岛病理结构变化:光镜下观察,正常对照组大鼠胰岛呈着色淡浅的细胞团,边界清楚,形态规则,胰岛内细胞排列整齐,胰岛数及岛内细胞数较多,大小一致分布均匀,细胞核呈紫蓝色,大而圆,核清晰,核仁明显,胞浆丰富呈淡粉色;糖尿病对照组大鼠胰岛细胞结构紊乱,胰岛数及岛内细胞数减少,细胞形态不规则,多数胞核变形,固缩,大小不等,出现空泡变性;治疗组胰岛数及岛内细胞数较糖尿病对照组为多,形态较为规则,少部分核固缩;预防治疗组大鼠胰岛细胞形态接近正常组,边界较清楚,细胞排列较整齐,胰岛数及岛内细胞数多于治疗组,细胞分布均匀,核大小基本相等,无核固缩。7.大鼠胰岛组织细胞胞浆胰岛素(INS)和胰高血糖素(GLU)免疫组化结果:胞浆镜下观察,正常对照组胰岛B细胞分泌INS最多,遍布胰岛中央,分泌颗粒丰富,胞浆着色深黄;糖尿病对照组胰岛B细胞分泌INS颗粒最少,胞浆着色较浅;治疗组胰岛B细胞分泌INS颗粒数量较糖尿病对照组微多,胞浆着色浅黄;预防治疗组B细胞分泌INS颗粒数量接近正常对照组,胞浆着色较深。正常对照组胰岛A细胞分泌GLU颗粒数量少均匀分布胰岛周围,着色较浅;糖尿病对照组胰岛A细胞分泌GLU颗粒丰富,大部分已经进入到胰岛中间区,胞浆着色深黄;治疗组A细胞分泌GLU颗粒数量较糖尿病对照组少,一部分进入到胰岛中间区,胞浆着色淡黄;预防治疗组A细胞分泌GLU颗粒大部分分布在胰岛周围,分泌颗粒较多,胞浆着色浅黄。8.大鼠胰腺组织胰岛B细胞凋亡:正常对照组可见少数凋亡细胞;糖尿病对照组有较多凋亡细胞;治疗组和预防治疗组也可见一些凋亡细胞,但明显少于糖尿病对照组;预防治疗组B细胞凋亡数也较治疗组少。结论:1.大鼠空腹血糖、糖耐量、糖化血红蛋白测定结果表明,该糖尿病复合营养酸奶具有明显改善糖代谢和降血糖效应;2.胰岛病理结构观察、胰岛组织细胞胞浆免疫组化和胰岛B细胞凋亡实验结果表明,糖尿病复合营养酸奶对ALX所致糖尿病大鼠胰岛结构和功能的损伤具有保护作用;3.血清MDA和SOD含量的测定表明,糖尿病复合营养酸奶能明显减轻糖尿病大鼠机体过氧化损伤,此作用可能是糖尿病复合营养酸奶对胰岛结构和功能具有保护作用的机制之一;4.血清TG、TC、LDL测定结果表明,糖尿病复合营养酸奶具有改善糖尿病大鼠脂代谢紊乱,此作用对防治糖尿病血清疾病并发症是有益的作用;5.研究结果还表明,该糖尿病复合营养酸奶具有减轻ALX所致糖尿病大鼠肾脏损伤、预防感染和降低死亡率的作用;6.由于预防治疗组动物在注射ALX建立糖尿病模型前即以摄入糖尿病复合营养酸奶,动物实验的各项研究指标显示预防治疗组较治疗组具有更明显的效果,表明该糖尿病复合营养酸奶对预防糖尿病具有潜在的应用前景;7.该糖尿病复合营养酸奶对糖尿病大鼠具有的上述作用可能是配方组成中含有的多种营养功效成分综合作用的结果;更多还原

【Abstract】 OBJECTIVE:Diabetes mellitus(DM) was a endocrine metabolic disease and severe harm the health of body. The attack rate and death rate of DM increased more and more quickly. At present there were 1.5 hundred million diabetic in the world .It was more than 40000 thousand diabetic in china. Because of the decrease of islet cell and action of islet cell ,it caused the disorder of fat ,protein, water and electrolyte and produced severe complication ,for example, coronary heart disease、cerebrovascular disease、peripheral vascular disease、retinopathy and renal vascular disease ect. More than fifty percent of diabetic who succumbed to coronary heart disease. Therefore, it was very important significance for us to improve the health status of diabetic and enhence the quality of life of diabetic and prevent the complication of diabetic and promote the rehabilitation of diabetic. So the ideal method of searching for treating and controlling diabetic was very important topic of medical science boundary. So in this study we observed and researched the protective effects of Compound acidophilous milk for diabetic on Alloxan induced pancreatic gland damage and glycometabolism in rats and cut down the influence of pancreatic tissue histomorphology and lipid peroxidation and pancreatic island B apoptotic .We also Attempted to provid some significant experimental based for developing and exploiting efficiency food.METHODS:120 Adult Wistar rats(190±10) were randomly divided into Control group ,DM group ,therapy group and protective therapy group ,30 in each group. Each group were fed a basic diet and Control group were intragastric administration with Sodium Chloride, DM group and therapy group were intragastric administration with ordinary acidophilous milk, protective therapy group were intragastric administration with compound acidophilous milk of diabetic .Each guoup was intragastric administration every other day. After 15 days, the rats of DM group, therapy group and protective therapy group have been given a single shot of alloxan (50mg per 1 kg), while Control group have been given a single shot of Sodium Chloride (50mg per 1 kg) . After 5 days , determining fasting 4 hours blood sugar,then the rats of blood sugar more than 13.8 mmol/L were diabetic model rats. After the diabetic model have been induced by alloxan , the rats of therapy group were intragastric administration with compound acidophilous milk of diabetic ,and other groups have not been changed . In 30 th days and 90th days after every group were given a single shot of alloxa determines fasting 4 hours blood sugar and In 6th days、32 th days and 92th days after every group were given a single shot of alloxa carbohydrate tolerance test .In 33th days and 93th days after every group were given a single shot of alloxa, randomly drawing out 10 rats in every groups that were killed and the pancreatic gland samples and blood serum were collected .Tentative index include INS ,C-P ,MDA ,SOD ,Cr ,BUN ,TG ,TC , LDL in blood serum and GLU, glycosylated hemoglobin in blood plasm and so on.The pancreatic gland morphological structure was also obserced under lingt microscope .The degree of apoptosis was assessed with the terminal deoxynucleotidy1 transferase (TdT)-mediated d UTP nick end labeling (TUNEL) by using an in situ Apoptosis Detection Kit.The expression of INS in the cytoplasm of B cell and GLU in the cytoplasm of A cell was detected by immunohistochemistry.RESULTS:1.Effect of compound acidophilous milk for diabetic on blood-fasting sugar and sugar tolerance and glycosylated hemoglobin1.1 evel of FBS(mmol/L):1.1.1 th days after every group were given alloxa, the level of FBS in the DM group,the therapy group and the protective therapy group increased very significantly more than control group(P<0.01), but the level of FBS in the protective therapy group is lower than the DM group,the therapy group(P<0.01);1.1.2 0th days and 90th days after every group were given alloxa, the level of FBS in the DM group,the therapy group and the protective therapy group increased very significantly more than control group(P<0.01), but the level of FBS in the therapy group and the protective therapy group is lower than the DM group (P<0.01);1.2 el of sugar tolerance(mmol/L): on 6th day、32th day、92th day , compare with Control group, the difference of sugar tolerancein 0.5h、1.0h、1.5h、2.0h of DM group , therapy group, protective therapy group is very significantly (P<0.05) while compare with DM group ,the difference of sugar tolerance in 0.5h、1.0h、1.5h、2.0h of therapy group, protective therapy group is very significantly (P<0.01). sugar tolerance of 0.5h、1.0h、1.5h、2.0h of 6th and sugar tolerance of 1.0h、1.5h of 32th of protective therapy group were different from DM group’and therapy group’,and the difference were very significantly(P<0.05)(p<0.01).1.3 evel of glycosylated hemoglobin(%):93th days after every group were given alloxa, the level of glycosylated hemoglobin in the DM group,the therapy group and the protective therapy group increased very significantly more than control group(P<0.01), but the level of glycosylated hemoglobin in the therapy group and the protective therapy group is lower than the DM group (P<0.01);2.Effect of compound acidophilous milk for diabetic on pancreatic function2.1 evel of serum INS (μIU/ml):33 th days and 93th days after every group were given alloxa, the level of INS in the DM group ,the therapy group and the protective therapy group is more lower than control group(P<0.01), but the level of INS in the therapy group and the protective therapy group is very significantly higher than the DM group(P<0.05 and P<0.01);2.2 evel of serum C-P(ng/ml): 33th days and 93th days after every group were given alloxa, the level of C-P in the control group, the therapy group and the protective therapy group is very significantly higher than the DM group(P<0.01、P<0.05 and P<0.01);furthermore, the level of C-P in the therapy group and the protective therapy group doses not have significant difference.2.3 evel of plasm GLU(pg/ml): 93th days after every group were given alloxa , the Level of plasm GLU in the control group and the protective therapy group is very significantly lower than the DM group(P<0.01 and P<0.05);the Level of plasm GLU in the therapy group is higher control group ,but also significantly lower than the DM group(P<0.05).3.Effect of compound acidophilous milk for diabetic on serum SOD、MDA 33th days and 93 days after every group were given alloxa,the level of SOD in DM group, the therapy group and the protective therapy group is very significantly lower than control group(P<0.01),the level of MDA in the DM group,the therapy group and the protective therapy group is more higher than control group(P<0.01),but the level of SOD in the therapy group and the protective therap is higher than DM group(P<0.01), the level of MDA in the therapy group and the protective therapy group is very significantly lower than the DM group (p<0.05 or P<0.01);4.Effect of compound acidophilous milk for diabetic on sersum TG、TC and LDL 93 days after every group were given alloxa,the level of TG、TC and LDL in the therapy group and the protective therapy group is very significantly lower than DM group(P<0.05 and P<0.01).5.Effect of compound acidophilous milk for diabetic on sersum Cr and BUN(μmol/L) 93 days after every group were given alloxa, the level of serum Cr and BUN in the DM group ,the therapy group , the protective therapy group is very significantly higher than control group(P<0.01),but the level of serum Cr and BUN in the therapy group , the protective therapy group is very significantly lower than DM group(P<0.05,P<0.01)6.Effent of compound acidophilous milk of diabetic on the pancreatic gland morpholological structureExamination of histological structure under a light microscope indicated that pancreatic islets of control group were colored to pale colour and bouncary distinctness, morphous regulation, lineing up in order.The number of pancreatic islet and isle cells in control group is more than DM group .Cell nucleus of control group were blue and limpid. Chromatospherite is conspicuous. Pancreatic islet cellular structure of DM group disordered and morphous is inordinate. Lots of cell nucleus of DM group were deformed, pycnotic and emerged to vacuolar degeneration. The number of pancreatic islet and isle cells in the therapy group was more than DM group but lower than control group. A little of cell nucleus were deformed and pycnotic. Islet cells of the protective therapy group were closed to control group and bouncary distinctness, morphous regulation, lineing up in order.7.Express of the INS and GLU in the endochylema of pancreatic IsletExamination of immunohistochemistry under a light microscope indicated that Islet B cells of control group secretory INS were more than other groups and secreted lots of grains .Endochylema of cells were colored to deep color. Islet B cells of DM group secreted a little of grains. Endochylema of cells were colored to tint. The number and color of grains in the therapy group was more than DM group , furthermore ,the number and color of grains in the protective therapy group was closed to control group.Islet A cells of control group secretory GLU were less than other groups and the GLU grains situated In the fringe of pancreatic islet .Endochylema of cells were colored to tint color. Islet A cells of DM group secreted lots of grains that a part of have intruded into the middle of Islet. The number and color of grains in the therapy group was less than DM group , furthermore ,the number and color of grains in the protective therapy group was closed to control group.8.The alloxan-induced TUNEL-positive B cells in pancreatic islet in the DM group were particularly more than in the therapy group and protective therapy group. Further,there was only a few TUNEL-positive B cells in the control group.CONCLUSIONS:1.The result of FBS, OTGC ,HGb of the rats indicated compound acidophilous milk of diabetic could obviously improve glycometabolism and decease effect of blood sugar ;2.The observation of pancreatic island patho-structure, immunity class of pancreatic island histiocyte endochylema and the B apoptosis of pancreatic island indicated compound acidophilous milk of diabetic have protection for the structure and function of pancreatic island;3.The result of sersum MDA and SOD indicated that compound acidophilous milk of diabetic could bviously lessen oxidative damage of organism in DM rats. It may be that compound acidophilous milk of diabetic had protection for the structure and function of pancreatic island;4.The result of serumtTG, TC and LDL indicated that compound acidophilous milk of diabetic could have action of improving disorder of lipid metabolism in DM rats;5.The finding indicated compound acidophilous milk of diabetic could have the protection of lessening kidney damage of the DM, infection prevention and cutting down death rate rats;6.Beause of protective therapy group were intragastric administration with compound acidophilous milk of diabetic before founding DM model ,every index of animal experiment indicated protective therapy group had best effect .This indicated that compound acidophilous milk of diabetic had potential application prospect for preventing diabetes; 7.Compound acidophilous milk of diabetic had above-mentioned effect that because of there were lots of nutrition efftective ingredient in prescription of compound acidophilous milk of diabetic;更多还原