【作者】 马丽云；

【导师】 张宏； 林来祥； 孙蓓；

【作者基本信息】 天津医科大学 ， 内科学， 2009， 硕士

【摘要】 目的:糖尿病(Diabetes Mellitus,DM)是冠心病的等危症,除左心室外,DM患者亦存在右心室收缩和舒张功能异常。肺动脉高压是引起右心室功能异常的主要原因之一。DM患者在排除冠状动脉疾病、高血压、心力衰竭或吸烟因素之后,肺动脉高压发生率仍明显增加。内皮素-1(endothelin-1,ET-1)、降钙素基因相关肽(calcitonin gene-related peptide,CGRP)及P物质(substance P,SP)是肺动脉高压病理过程中的关键因子。本研究通过短期高脂饲料喂养联合链脲佐菌素制备2型糖尿病(Type 2 Diabetes Mellitus,T2DM)大鼠模型,观察糖尿病大鼠肺组织超微电镜和光镜下结构,测量肺血管参数和ET-1、CGRP及SP的表达变化,探讨DM肺动脉高压和右心病变的可能发病机制。方法:3月鼠龄的成年雄性Wistar大鼠12只,随机分为两组:正常对照组(normal,N)(n=6)和DM组(n=6)。N组给予普通饲料,DM组给予高脂饲料。喂养8周后给予链脲佐菌素(streptozotocin,STZ)30mg/kg体重尾静脉一次注射制备DM模型,N组尾静脉注射柠檬酸-柠檬酸钠缓冲液。72小时后测DM组大鼠血糖均大于16.65mmol/L,造模成功。成模后20周处死大鼠,留取肺组织:(1)2.5%戊二醛和1%四氧化锇溶液进行双重固定后,以JEOL-100CX透射电镜摄片并观察肺组织超微结构变化;(2)10%中性福尔马林固定:①HE染色光镜下观察肺组织结构变化;②进行Weigert来复红弹力纤维染色后运用MIAS2000图像分析系统测量与呼吸性细支气管、肺泡管伴行的肺小动脉管壁厚度占血管外径百分比(ratio of vascularwall thickness/vascular external diameter,WT%)和管壁面积占血管总面积百分比(ratio of vascular wall area/total vaseular area,WA%),来评价DM大鼠肺动脉结构变化;③用免疫组织化学方法定位肺组织ET-1、CGRP及SP的表达,然后运用MIAS2000图像分析系统测定平均光密度和阳性表达面积/视场总面积以评价阳性表达强度。结果:①DM组大鼠于造模后3天开始出现多饮多尿,体重不增加或明显减轻,活动减弱,一般状态差。成模20周后杀检时,DM组大鼠体重明显低于N组(P＜0.01);血糖明显高于N组(P＜0.01)。②超微病理结构:DM大鼠肺泡Ⅱ型上皮细胞微绒毛减少,嗜锇板层小体少见,肺间质不清,气血屏障基膜较明显,内皮细胞不明显。③光镜下观察发现,N组大鼠肺泡结构正常,DM组与N组比较,肺泡腔缩小,肺泡隔增厚,肺泡数量增多。肺泡隔面积与总视场面积比增加(P＜0.01)。④Weigert来复红弹力纤维染色显示DM组大鼠肺动脉壁增厚,血管周围炎性细胞浸润。DM组与N组比较,肺动脉WT%和WA%均增加(均P＞0.05),差异无统计学意义。⑤免疫组织化学显示N组和DM组肺血管内皮细胞、肺泡巨噬细胞、支气管上皮细胞、肺泡上皮细胞、气道和血管平滑肌细胞、肺神经内分泌细胞可见ET-1阳性表达;肺泡上皮细胞、各级支气管粘膜上皮的神经内分泌细胞和血管平滑肌层可见CGRP阳性表达;肺神经内分泌细胞、支气管上皮细胞和血管平滑肌细胞可见SP阳性表达。DM组与N组比较,ET-1平均光密度增加(P＜0.05),ET-1表达阳性面积/视场总面积增加(P＜0.05),差异有统计学意义;SP平均光密度增加(P＜0.05),SP表达阳性面积/视场总面积增加(P＜0.05),差异有统计学意义;CGRP平均光密度增加(P＞0.05),差异无统计学意义,CGRP表达阳性面积/视场总面积增加(P＞0.05),差异无统计学意义。结论:①高脂饲料喂养联合小剂量链脲佐菌素尾静脉一次性注射可成功制备T2DM大鼠模型。②DM肺组织超微病理示肺泡上皮细胞受损;光镜示肺泡隔增厚,肺泡腔缩小,肺泡数量增多,提示肺组织可能存在缺氧。③DM组大鼠与呼吸性细支气管及肺泡管伴行的肺动脉WT%和WA%有增加趋势,提示肺动脉结构改变可能是糖尿病肺动脉高压发生的病理基础。④ET-1、CGRP和SP表达有增加趋势,提示可能与糖尿病肺动脉高压和右心功能改变有关。更多还原

【Abstract】 Objectives:Diabetes Mellitus(DM) is a Coronary Heart Disease Equivalent.Besides left ventricular dysfunction,right ventricular dysfunction also exists in the diabetic. Pulmonary hypertension is the main reason of right ventricular dysfunction.Patients with DM have significantly higher prevalence of pulmonary hypertension, independent of coronary artery disease,hypertension,heart failure or smoking. Endothelin-1(ET-1),Calcitonin gene-related peptide(CGRP) and Substance P(SP) are the key factors in the pathogenesis of Pulmonary hypertension.To make T2DM model by short-term high fat diet feeding and small amouts streptozotocin(STZ) via the tail vein.By observing ultrastructure of lung tissue and pathological changes under light microscope,measuring lung vascular parameters and the expression of ET-1,CGRP and SP,we studied the possible mechanism of pulmonary hypertension and right ventricular dysfunction in DM.Methods:12 male Wistar rats were randomly divided into normal(N) group(n=6) and DM group(n=6).N group rats were fed with normal diet.After fed with high fat diet for 8 weeks,DM group rats were injected with 30mg STZ/Kg WB via the tail vein.N group rats were injected with buffer solution.After 72 hours,blood glucose was more than 16.65mmol/L.20 weeks later,the rats were sacrificed and the lung samples were obtained:(1)Put samples in the solution of 2.5%glutaric dialdehyde and 1%osmium tetroxide to fixate and observed with transmission electron micro-scope; (2)Fixated with 10%formalin:①Observed the morphological changes of the lung by light microscope.②After lastic fiber staining,measuring the ratio of the vascular wall thickness/vascular external diameter(WT%) and ratio of vascular wall area/total vaseular area(WA%) of the lung arteries,companied by respiratory bronchioles and alveolar ducts.③Studied the expression of ET-1,CGRP and SP by immunohistochemistry,then mearsured average optical density and the ratio of the positive area and the total area of field of view with Image Analysis System to evaluate the intensity of positive expression.Results:①STZ injection for 3 days later,the rats became polydipsia,polyphagia, polyuria,weight loss or no obvious weight gain.After 20 weeks,compared with N group,the body weight decreased in DM group(P＜0.01),while the fasting blood glucose increased(P＜0.01).②Ultrastructure:The typeⅡalveolar epithelial cells microvilli of DM rats decreaed and osmiophilic lamellar bodies were rare.The interstitium of lung was unclear.The basement membrane of air-blood barrier was clear but the endothelial cells was not.③light microscope:The alveolar structure of N group rats were normal.Compared with N group,DM group rats had smaller alveolar space,thicker alveolar septa and more alveolars.Compared with N group, the ratio of the total area of alveolar septa and the total area of field of view in DM group rats increased(P＜0.05).④Weigert lastic fiber staining:In DM group,the lung arteries walls were thickened and inflammatory cells infiltrated.Compared with N group,WT%and WA%of DM group increased(P＞0.05),but it had no statistical significance.⑤Immunohistochemistry:ET-1 was expressed in lung vascular endothelial cells,alveolar macrophages,bronchial and alveolar epithelial cells,airway and vascular smooth muscle cells,lung neuroendocrine cells of both N and DM group rats.CGRP was expressed in alveolar epithelial cells,bronchial neuroendocrirte cells and vascular smooth muscle cells of both N and DM group rats.SP was expressed in lung neuroendocrine cells,bronchial epithelial cells and vascular smooth muscle cells of both N and DM group rats.Compared with N group,the DM group average optical density of ET-1 increased(P＜0.05),the ratio of the positive area and total area of field also increased(P＜0.05),the DM group average optical density of SP increased (P＜0.05),the ratio of the positive area and total area of field also increased(P＜0.05), the DM group average optical density of CGRP increased(P＞0.05) and the ratio of the positive area and total area of field also increased(P＞0.05).Conclusion:①The model of diabetic rats could be made by short-term high fat diet then small amounts STZ via the tail vein.②The DM rats lung ultrastructure showed the alveolar epithelial cells injured.Light microscopy showed the alveolar septa thickened,the alveolars volume reduced and the number of alveolar increased, suggesting that lung tissue might have hypoxia.③WT%and WA%of the lung artery had an upward trend.So,the changes of lung vascular structure might be the pathological basis of pulmonary hypertension in DM.④The expression of ET-1, CGRP and SP had an upward trend,that might be associated with the diabetic pulmonary hypertension and the right ventricular dysfunction.更多还原