【作者】 陈璐；

【导师】 刘超；

【作者基本信息】 南京医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:研究宫内发育迟缓(intrauterine growth retardation,IUGR)大鼠出生后不同阶段胰岛功能以及胰岛素敏感性的变化特点,探讨IUGR与2型糖尿病之间的内在联系。方法:1、采用营养不良法(妊娠11天起予正常50%饲料)建立IUGR大鼠模型,以出生后不同阶段(1天、3周、7周、10周、15周及36周)雄性大鼠为研究对象,通过称量体重、胰重,并计算胰重/体重比值等手段,观察其生长发育状况。2、运用抗胰岛素抗体免疫组化染色法,对IUGR大鼠出生不同阶段胰腺组织进行形态学及量化分析,了解不同阶段胰岛形态和胰岛素表达的变化。3、采用腹腔糖耐量试验(intraperitoneal glucose tolerance test,IPGTT)及胰岛素释放试验(insulin releasing test,IRT),测定糖负荷后不同时点血糖和胰岛素的水平,检测IUGR大鼠出生不同阶段糖耐量及胰岛素释放的变化。4、以胰岛分离纯化技术获得IUGR大鼠出生不同阶段的胰岛,进行胰岛体外葡萄糖刺激下胰岛素释放(glucose-stimulated insulinsecretion,GSIS)试验,采用放射免疫法(Radioimmunoassay,RIA)检测上清中胰岛素浓度,以评价胰岛体外分泌功能。5、运用逆转录-聚合酶链反应(reverse transcription-polymerasechain reaction,RT-PCR)对第36周IUGR大鼠胰腺组织胰岛素合成相关基因进行检测,以进一步探讨胰岛细胞功能异常的机制。6、采用腹腔胰岛素耐量试验(insulin tolerance test,ITT),测定胰岛素负荷后不同时点的血糖值,以了解IUGR大鼠出生不同阶段胰岛素敏感性的变化。结果:1、IUGR组大鼠出生时体重、胰重和胰重/体重比值均显著低于正常组(P＜0.001);出生后体重逐渐追赶,但胰重及胰重/体重比值始终低于正常组(P＜0.05)。2、IUGR组新生鼠胰腺组织中胰岛素阳性表达细胞面积及胰岛素染色阳性率均明显小于正常组(P＜0.01);胰岛素阳性染色面积随年龄逐渐增大,至36周时IUGR组大鼠胰岛素阳性面积与正常组已无明显差异,但胰岛平均染色光密度显著降低(P＜0.05)。3、IUGR组新生鼠空腹血糖及胰岛素水平均低于正常新生鼠,但糖负荷后120min和180min血糖值明显高于正常组(P＜0.05)。随年龄增长,IUGR组大鼠逐渐出现糖耐量减退,表现为糖负荷后血糖下降减慢,胰岛素早期出现代偿分泌增多,以15周为著(P＜0.05),后期(36周)则分泌减少。4、胰岛体外GSIS功能检测结果提示,15周起IUGR组大鼠胰岛体外胰岛素刺激指数明显低于正常组(P＜0.01)。5、对胰岛素合成相关基因检测发现,第36周IUGR组大鼠胰腺组织胰岛素基因1(Insulin1)表达明显减少(P＜0.05),但其他基因[胰岛素基因2(Insulin2)、胰-十二指肠同源盒基因-1(pancreaticduodenal homeobox-1,PDX-1)]表达无明显差异。6、IUGR组大鼠第10周起即出现给予胰岛素负荷后血糖值下降迟滞于正常组(P＜0.05),且随年龄增长逐渐明显。结论:1、IUGR大鼠胰腺发育受损,胰岛β细胞功能减退,随年龄增长逐渐出现糖耐量减低及胰岛素敏感性下降。2、IUGR大鼠后期胰岛素分泌减少可能与胰腺组织Insulinl基因表达下调有关。更多还原

【Abstract】 Objectives:To investigate the association between intrauterine growth retardation(IUGR)and type 2 diabetes by studying pancreatic isletβcell function and insulin sensitivity at different stages of lifetime in rats born with IUGR.Methods:1.The IUGR rat model was established by maternal nutrition restriction(50%calorie restriction of the normal)during the 11th to 21st days of pregnancy.Male offsprings at different stages(newborn,3 weeks, 7 weeks,10 weeks,15 weeks and 36 weeks)were selected as research subjects.Body and pancreas weights were assessed and the ratio of pancreas weight to body weight was calculated in order to evaluate the growth and development status of rats.2.Immunohistochemistry analysis of pancreas was used to evaluate the changes of islet morphology and the expression of insulin in islets at different stages in IUGR rats.3.On the basis of intraperitoneal glucose tolerance test(IPGTT)and insulin releasing test(IRT),plasma glucose and serum insulin were detected after a glucose load to investigate the changes of isletβcell function at different stages in IUGR groups.4.Pancreatic islets of IUGR rats at different stages were isolated and purified.On the basis of glucose-stimulated insulin secretion(GSIS)test, supernatant insulin was tested by radioimmunoassay to investigate the changes of islet secretory function in vitro as age grows.5.RT-PCR was applied to detect the expression of pancreas genes relevant to insulin synthesis of IUGR rats at age 36 weeks in order to elucidate the mechanism of pancreatic islet dysfuncion.6.On the basis of insulin tolerance test(ITT),plasma glucose levels were detected after insulin load to study the changes of insulin sensitivity at different stages in IUGR rats.Results:1.Body weight,pancreas weight and pancreas/body weight of the newborns in IUGR group were much lower than those in normal group (P＜0.001).While body weight of IUGR rats caught up with normal rats after birth,pancreas weight and pancreas/body weight remained lower at all stages of lifetime(P＜0.05).2.The areas of insulin-expression positive cells and positive rate of cells staining with insulin in IUGR newborns were less than normal pups (P＜0.01).Insulin-staining positive areas were enlarged as age grows. There was no significant difference of insulin-staining positive areas in two groups,while the average photodensity of islets was remarkably decreased in IUGR rats at 36 weeks of age(P＜0.05).3.The fasting glucose and insulin levels of IUGR newborns were both lower than those of normal controls,whereas glucose levels at 120min and 180min after glucose load were significantly higher in IUGR group(P＜0.05).Glucose tolerance was impaired with time in IUGR rats, which was manifested as elevated glucose levels and compensatory increment of insulin secretion after glucose administration at early stage, especially at age 15 weeks(P＜0.05),while insulin secretion decreased at later stage(36 weeks).4.Compared with normal group,the stimulating index of pancreatic islets in vitro of IUGR rats was significantly decreased form 15 weeks of age(P＜0.01),indicating the impaired pancreatic islet function.5.Although no significant differences were observed in gene expression of insulin2 and PDX-1,the expressions of insulin1 were reduced markedly in IUGR rats(P＜0.05)at 36 weeks of age.6.There was a significantly blunted glycemic response of IUGR group to insulin load compared with normal group(P＜0.05)from 10 weeks of age,which was impaired gradually as age grows.Conclusions:1.IUGR rats have both impaired pancreatic development and isletβcell dysfunction.Glucose tolerance and insulin sensitivity deteriorate with age.2.Decreased gene expression of insulinl in pancreas may be responsible for the reduced secretion of insulin in IUGR rats at later stages of life.更多还原