【作者】 赵海军；

【导师】 刘娅；

【作者基本信息】 吉林大学 ， 放射医学， 2006， 博士

【摘要】 糖尿病是以高血糖为主要特征的慢性代谢性疾病,各国的发病率呈明显上升的趋势,但是现有的治疗药物多是通过单一途径发挥作用,并且存在一定的副作用。因此,寻找一种通过多种途径发挥作用、高效低毒的治疗药物显得尤为重要。胰高血糖素样肽-1(GLP-1)作为一种机体内具有多种生理功能的生物活性多肽,成为糖尿病治疗药物研究的热点。本研究拟通过固相多肽合成技术生产出新型的GLP-1类似物,并探讨其治疗糖尿病的作用,力图开发一种全新的糖尿病治疗药物。研究结果表明:合成的GLP-1类似物粗品纯度达26.03%,纯品纯度达92.38%,质谱检测显示化合物的分子量为3297.4。GLP-1类似物的最大耐受剂量达到2500μg·kg-1,因此可以安全的用于糖尿病治疗。给予Wistar大鼠40mg·kg-1的STZ,同时饲以高脂饲料,可以获得稳定的具有高血脂为特征的糖尿病动物模型。GLP-1类似物治疗2周可以明显地减少糖尿病大鼠的进食量和饮水量;降低糖尿病大鼠的血糖和血脂水平;提高糖尿病大鼠的血清胰岛素和C肽含量;GLP-1类似物对于STZ引起的胰岛损害有明显的保护作用,可以增加糖尿病大鼠表达胰岛素阳性细胞率;GLP-1类似物对于糖尿病引起的皮肤改变有明显的改善作用。因此,本研究中的GLP-1类似物是通过多种途径在糖尿病治疗中发挥作用的。更多还原

【Abstract】 ObjectiveDiabetes mellitus(DM) is chronic metabolic disease with glucosemetabolism abnormal. Morbidity of DM in many countries is increasing.Nowadays ,the effects of many therapeutic drugs are limited. So discovery ofnew therapeutic drug with many effects is very important. Glucagon-likepeptide-1(GLP-1) become a focus of DM therapeutic drug research as a kind ofbioactive peptide with many Physiologic functions. This study will synthesis anew type GLP-1 analog by Solid Phase Peptide Synthesis(SPPS),and explore thetherapeutic effects of it on diabetes mellitus.Material and Methods1 synthesis of Glucagon-like peptide-1(GLP-1) analogGlucagon-like peptide-1(GLP-1) analog is synthesized by Solid Phase PeptideSynthesis(SPPS). The purity of GLP-1` was detected by High PerformanceLiquid Chromatography (HPLC) and molecular weight by MassSpectrometer(MS) .2 study on therapeutic effects of GLP-1 analog on DMThe tolerant dose-range of GLP-1 analog is investigated in mice beforetherapeutic experiment for confirmation of therapeutic dose . 84 Wistar rats areprepared which 12 rats are raised by common forage as negative control groupand 72 rats raised by high fat forage as experiment group. The level of serumtotal cholesterols (TC)、triglyceride (TG)、low-density lipoprotein (LDL) andhigh-density lipoprotein (HDL) ,of all rats ,are detected on the 28th day . Afterhyperlipemia was confirmed ,12 rats as high fat forage (HFF) group are raisedhigh fat forage and 60 rats of experiment group rats are given 40mg/kgstreptozotocin(STZ)through intraperitoneal injection(ip). The fasting plasmaglucose(FPG) level of experiment group rats was measured on the 7th day ofgiven STZ. The rats are determined DM which value of fasting plasma glucosereach 16.7mmol·L-1.Then, the DM rats are randomly clustered 5 experimentgroups:GLP-1 high dose(100ug·kg-1) group、GLP-1 medium dose(10ug·kg-1)group、GLP-1 low dose (1ug·kg-1) group、DM group、Exendin-4(10ug·kg-1)group. All rats are given therapeutic material through Subcutaneous injection(sc) .negative control (NC) group、high fat forage(HFF) group and DM group areonly given equal volume phosphate buffer solution (PBS) . All rats are killed onthe 2nd week of therapy, blood is taken out for measure of the level of FPG、TC 、TG 、LDL、HDL、serum insulin and C-peptide;liver、kidney、pancreatic 、Thymus and Spleen of all rats are take out for calculating organ index. TLymphocyte of Spleen is cultured for Lymphocyte Transformation testing,andstimulation index(SI)is calculated. The pathological and histological change ofliver、kidney、pancreatic and skin are observed in light microscope;thepercentage of insulin expressing positive cell in pancreas islet and TransformingGrowth Factor beta1(TGFβ1)expressing positive cell in kidney is investigatedby immunohistochemical methods.Through the researching process ,water intake and food intake of all groups aremeasured once a day and body weight is measured once a week.Results1 Synthesis of Glucagon-like peptide-1(GLP-1) analogGLP-1 analog is synthesis by SPPS. The purity of crude GLP-1 analog is26.03%.the purity of purified GLP-1 analog is 92.38%.molecular weight is3297.4 .2 Study on therapeutic effects of GLP-1 analog on DM2.1 tolerant dose-range of GLP-1 analog in micemaximal tolerant dose of GLP-1 analog in mice is higher than 2500ug·kg-1.2.2 Building of rat diabetes mellitus model.After 28 days of raised high fat forage, TC、LDL、HDL level of Experimentgroup rats are higher than the negative control group(P<0.05) . On the 7th day ofSTZ injection,level of FPG of experiment group rats increase significantlycompared with NC group and HFF group(P<0.01).During 7 days of STZinjection, water intake of experiment group rats increase significantly and bodyweight decrease compared with NC group and HFF group (P<0.01).2.3 Effects of GLP-1 analog on food intake and water intake of diabetesmellitus rats.During 2 weeks of GLP-1 analog injection, food intake of GLP-1 high dosegroup and Exendin-4 group is less than DM group(P<0.01);Food intake of DMgroup is more than NC group and HFF group(P<0.05).During 1 week of GLP-1 analog injection, water intake of GLP-1 high dosegroup is less than DM group(P<0.05). During 2 weeks of GLP-1 analoginjection, water intake of GLP-1 medium dose group、high dose group andExendin-4 group is less than DM group(P<0.01).2.4 Effects of GLP-1 analog on body weight of diabetes mellitus rats.During 7 days of STZ injection, body weight of DM group and all therapeuticgroups deduce significantly compared with NC group and HFF group(P<0.01);During 2 weeks of GLP-1 analog injection, body weight of DM group and alltherapeutic groups deduce significantly compared with NC group and HFF group(P<0.01),but there are no statistical differences compared between alltherapeutic groups and DM group(P<0.01).2.5 Effects of GLP-1 analog on organ index of diabetes mellitus rats.After 2-week GLP-1 analog therapy, liver index and kidney index of DM andall therapeutic groups are higher than NC group and HFF group(P<0.01),andThymus index is lower than NC group and HFF group(P<0.01);Pancreas indexof DM group is lower than NC group and HFF group(P<0.01),but Pancreasindex of GLP-1 high dose group and Exendin-4 group is higher than DM group(P<0.05);there are no significant differences compared among spleen index ofall groups(P>0.05).2.6 Effects of GLP-1 analog on immune function of diabetes mellitus rats.After 2-week GLP-1 analog therapy,Thymus index of DM and all therapeuticgroup is lower than NC group and HFF group(P<0.01),but there are nosignificantly difference spleen index compared among all groups(P>0.05);thereare no significant difference compared among stimulation index(SI)of all groups( P>0.05) .2.7 Effects of GLP-1 analog on fasting plasma glucose (FPG) of diabetesmellitus rats.On the 1st week of GLP-1 analog injection, level of FPG of high dose groupis lower than DM group(P<0.01);On the 2nd week of GLP-1 analog injection,level of FPG of high dose group 、medium dose group and Exendin-4 group islower than DM group(P<0.01),but level of FPG of both DM group and alltherapeutic groups is lower than both NC group and HFF group (P<0.01).2.8 Effects of GLP-1 analog on blood lipids of diabetes mellitus rats.On the 2nd week of GLP-1 analog injection, level of TC of both DM group andall therapeutic group is higher than both NC group and HFF group(P<0.01);level of TG of high dose group and Exendin-4 group are lower thanDM group(P<0.01);level of LDL of medium dose group and high dose group ofGLP-1 analog and Exendin-4 group is lower than DM group(P<0.01);there isno significant difference compared among level of HDL of all groups(P>0.05).2.9 Effects of GLP-1 analog on level of serum insulin and C-peptide ofdiabetes mellitus rats.On the 2nd week of GLP-1 analog injection,level of serum insulin of DMgroup and all therapeutic group is lower than NC group and HFF group(P<0.01).while level of serum insulin of medium dose group and high dosegroup of GLP-1 analog and Exendin-4 group is higher than DM group(P<0.05).On the 2nd week of GLP-1 analog injection,level of serum C-peptide of DMgroup and all therapeutic groups is lower than NC group and HFF group(P<0.01). level of serum C-peptide of medium dose group and high dose groupof GLP-1 analog and Exendin-4 group is higher than DM group(P<0.05).2.10 Effects of GLP-1 analog on pathological and histological change of organand tissue of diabetes mellitus rats.GLP-1 analog can not improve significantly fatty liver and kidney injury ofdiabetes mellitus rats,but can increase significantly number of pancreas islet ofdiabetes mellitus. GLP-1 analog can improve the pathological and histologicalchange of skin of diabetes mellitus rats.2.11 Effects of GLP-1 analog on expression of insulin in pancreatic islet andTGFβ1 in kidney of diabetes mellitus rats.There are significant difference compared Number of β cell in pancreaticislet and distribution and staining grade of brown-staining particle in cytoplasmbetween all therapeutic groups and DM group(P<0.05).Immunohistochemical investigation show that expression of TGF-β1 inglomerulus and renal tubule epithelial cell in rat is significantly differentbetween DM group and NC group(P<0.05), but there are no significantdifference compared between all therapeutic groups and DM group(P>0.05).ConclusionsThis study has synthesized GLP-1 analog by SPPS and explored therapeuticeffects of it on diabetes mellitus in rats. we has gotten conclusions as following:1、GLP-1 analog has synthesized by SPPS. The purity of crude GLP-1analog is 26.03%.the purity of purified GLP-1 analog is 92.38 %.molecularweight is 3297.4 .2、tolerant dose-rang test shows maximal tolerant dose of GLP-1 analog inmice >2500ug· kg-1 and it can been Safely used in study on therapeutic effectsin diabetes mellitus rats.3、Stable diabetes mellitus model with hyperlipemia can been acquired afterWistar rats are given 40mg·kg-1 STZ and high fat forage.4、GLP-1 analog can decrease food and water intake of diabetes mellitusrats, but can not effect significantly body weight of diabetes mellitus rats.5、GLP-1 analog can decrease fasting plasma glucose(FPG) and serumTG 、LDL of diabetes mellitus rats, but can not effect significantly serum TC、HDL of diabetes mellitus rats.6、GLP-1 analog can not effect significantly Thymus index 、 Spleen indexand Lymphocyte Transformation of diabetes mellitus rats.7、GLP-1 analog can upgrade level of serum insulin and C-peptide。8、GLP-1 analog can not improve significantly fatty liver and kidney injuryof diabetes mellitus rats,but can increase significantly number of pancreas isletof diabetes mellitus. GLP-1 analog can improve the pathologic-al andhistological change of skin of diabetes mellitus rats.9、GLP-1 analog can increase Percentage of insulin positive cell inpancreatic island of diabetes mellitus, but can not effect Percentage of TGFβ1positive cell in kidney of diabetes mellitus.更多还原