【作者】 杨建；

【导师】 班博； 闫波； 孙琳；

【作者基本信息】 天津医科大学 ， 内科学， 2009， 硕士

【摘要】 目的:自噬体-溶酶体系统是细胞利用溶酶体降解自身受损的细胞器和大分子物质的过程,是真核细胞特有的生命现象,在细胞的生长、发育和疾病发生中起着重要的作用。自噬消化过程既广泛存在于正常的生理过程中,又是细胞对不良环境的一种防御机制,同时还参与多种疾病的病理过程,无论是自噬过度还是自噬不足都可能导致疾病发生。目前研究表明,自噬体-溶酶体系统异常与肿瘤、神经退行性疾病以及病原微生物的入侵等相关。但自噬体-溶酶体系统与2型糖尿病(T2DM)发病相关性的研究尚少。有研究认为自噬体-溶酶体系统与T2DM相关,该系统数量与功能改变时,都会影响T2DM的发生与发展,但是目前研究报道结果尚不一致。本研究旨在研究自噬体-溶酶体系统在T2DM患者体内的变化,探讨其在T2DM发生、发展中的作用,从而为2型糖尿病的病因、干预与治疗提供理论基础。方法:本研究收集自2007年12月至2008年4月期间在济宁医学院附属医院内分泌科病房住院确诊的T2DM患者作为病例组,从医院健康查体中心选取相同年龄组健康者作为对照组。实验采用病例-对照观察,分为健康对照组、T2DM组和微血管并发症组,各40例,共计120例,其中对照组男22例,女18例,平均年龄55.72岁,糖尿病组男24例,女16例,平均年龄51.2岁;并发症组男21例,女19例,平均年龄55.08岁。收集外周血液标本并分离白细胞,分别提取蛋白及RNA,分别用免疫蛋白印迹(western blot)及逆转录聚合酶链式反应(RT-PCR)方法,对自噬体特异性标记LC3及溶酶体特异性标记LAMP2进行检测,从而观察自噬体-溶酶体数量的变化。所有数据输入采用SPSS13.0统计软件进行统计分析,设P＜0.05为有统计学意义。结果:western blot及RT-PCR结果均显示,对照组、T2DM组及并发症组之间自噬体的数量呈递减趋势,对照组与T2DM组及对照组与并发症组之间均有明显差异,有统计学意义(P＜0.05),而T2DM组与并发症组之间无明显差异,无统计学意义。三组间溶酶体有下降趋势,但无明显差异,无统计学意义。多因素非条件Logistic回归分析结果:与2型糖尿病发生、发展成正相关的指标有年龄、FBG、TG、TC及LDL,成负相关的有自噬体数量及尿酸,均有统计学意义(P＜0.05)结论:自噬体数量的改变对T2DM及其并发症的发生、发展之间存在显著相关性,自噬体对糖尿病患者有保护作用。在2型糖尿病患者及其合并并发症患者体内自噬体数量是减少的,自噬体的数量减少使糖尿病患者胰腺β细胞内受损的细胞器得不到及时清除,从而不能维持胰岛β细胞功能,异常的、畸形的及功能缺陷的β细胞线粒体以及膨胀的内质网的存在可能会降低细胞产生胰岛素的能力,导致糖尿病的加重及其并发症的发生。虽然三组间溶酶体数量有下降趋势,但无显著性差异。这可能与溶酶体变异较大有关,另外溶酶体不仅接受来自自噬体的物质,也同样肩负内源性和外源性大分子物质的消化。因此,虽然我们未观察到本组病人中溶酶体数量的显著变化,尚不能排除溶酶体与T2DM之间的相关关系。年龄、肥胖、高血糖及高血脂为T2DM的发生及发展的危险因素,而细胞自噬体可作为其保护因素之一。更多还原

【Abstract】 Objective:Autophagic-lysosmal system involves degradation of cellular components and macromolecular substances through lysosomal digestion,which is a specific vital phenomenon in eukaryotic cells.Autophagy plays an important role in cell growth,development and disease.Autophagy functions widely in normal physiological processes,cellular defense mechanism under certain circumstances and pathological processes.Excessive or reduced autophagy cause a great number of diseases.Accumulating evidence shows that autophagy has been associated to cancer, neurodegenerative diseases,invasion of pathogenic microorganisms and other diseases.To date,very few studies have been reported on the association between autophagic-lysosomal system and type 2 diabetes.Some studies have revealed the quantitaty and functional changes of autophagic-lysosomal system in the patients with T2DM,suggesting its role in the pathogenesis and development of T2DM.However,negatvie efftects have also been reported,indicting the comlex effects of the system.This study was designed to investigate the quantity changes of the autophagic-lysosomal system in the T2DM patients and explore its role in the onset and development of T2DM.The results will provide a theoretical basis for understaning the etiology and developing novel intervention and treatment measures for the disease.Methods:From Dec.2007 to Apr.2008,we collected blood samples of T2DM patients,without or with diabetic microangiopathy,in the Endocrinology Department of Affiliated Hospital of Jining Medical College.Healthy volunteers as normal control were from the hospital physical-examination center.We employed case-control method with 3 groups:normal control(40 cases),T2MD patients(40 cases),T2MD patients with microangiopathy(40 cases).Blood samples were collected and the leukocytes were isolated.Cellular protein and total RNA were parepared for western blotting and RT-PCR,respectively.All data were analysed with software SPSS13.0 and P value＜0.05 set as statistically significant.Results:The results of western blotting and RT-PCR showed that the LC3 protein levels and LC3 gene expression levels were significantly decreased in T2DM patient group and T2DM patients with with microangiopathy group,compared with normal controls,indicating that autophagosome formation were reduced.There was not significant difference between T2DM pateint group and T2DM patient with microangiopathy group.However,we did not observe the differences of LAMP-2 protein level and LAMP-2 gene expresion levels among these three groups.Logistic analyses showed that age,FBG,TG,TC and LDL were significantly positively correlated with the onset of T2DM,but uric acid were significantly negative correlated with them.Conclusions:Our study showed that number and formation of autophagosome were associated with the pathogeneis,development of T2DM and its complications, suggesting that autophagosomes has protective effects in T2DM patients.As the formation and number of autophagosome were reduced and the damaged cell organelles in the pancreaticβ-cell could not be cleared promptly,theβ-cell functions are not well maintained in T2DM patients.As a result,uncleared and damaged mitochondria and swelled endoplasmic reticulum ofβ-cell may reduce its ability to produce insulin.Although LAMP-2 protein,a mark of lysosomes,were decreased in T2DM patients,but there was no significant difference among the three groups, which may be related to the great variation of lysosomes.Lysosomes not only fuse with autophagosome,but also directly digest both endogenous and exogenous macromolecules.Therefore,the assocition of lysosomes and T2DM can not be ruled out and needs to be further investigated.In addition,age,obesity,high blood sugar and high blood lipids were the risk factors for the onset of T2DM.更多还原