【作者】 王静；

【导师】 罗萍；

【作者基本信息】 吉林大学 ， 内科学， 2004， 硕士

【摘要】 糖尿病肾病是糖尿病最常见的慢性微血管并发症之一，也是导致慢性肾功能不全重要病因之一。其病理特征为肾小球毛细血管硬化和肾小管间质纤维化，尿内微量蛋白出现异常变化。DN在早期存在可逆性，如能及时诊断并干预治疗，去除不利因素，肾脏病变有可能恢复。如有肾功能损害常呈进行性且不可逆转，而且目前尚未发现有效而特异的治疗药物。因此早期诊断并判断DN患者受损程度及损伤部位，预防DN发生，阻止其进展显得尤为重要。近年研究认为TGF-β1在糖尿病肾病发病机制中起重要作用，尿中TGF-β1排泄量增加可敏感反应糖尿病早期肾脏损害。因此我们检测尿中TGF-β1含量并探讨其作用机制，为早期糖尿病肾病检测寻找新方法奠定理论基础。目的：通过尿微量白蛋白、尿β2-微球蛋白及尿TGF-β1定量检测，并与常规肾功能检测指标比较，探讨TGF-β1对早期DN的诊断价值和病情评估。方法：研究对象选择我院2002-2003年72例2型糖尿病住院患者，均采用口服降糖药治疗。其中男37例，女35例，年龄38-87岁，平均60.8±9.2岁，病程平均12.8±9.7年，未服用过ACEI类及肾毒性药物，并排除肾脏高血压、泌尿系感染、心功能不全等影响尿蛋白因素。根据24小时尿白蛋白排泄率（UAER）分组：糖尿病组（Ⅰ组）：正常白蛋白尿UAER〈20ug/min；早期DN组（Ⅱ组）：微量白蛋白尿UAER 20ug/min-200ug/min；糖尿病肾病组（Ⅲ组）：大量白蛋白尿UAER〉200ug/min。选择正常对照组16例，其中男9例，女7例，平均年龄57.4±7.1岁，均为健康体检者无糖尿病、高血<WP=30>压及肾脏疾患。采用ELISA方法检测尿中TGF-β1，同时检测尿白蛋白(mALB) 、尿β2-微球蛋白(β2-M)、血糖、血肌酐、糖化血红蛋白、血脂等，并与16例正常对照组（NC）进行比较分析。收集所有受试者24小时尿液，加10ml甲苯防腐，准确记录尿量。混匀后留取6ml测量尿mALB及尿肌酐，其中mALB、β2-M测定选用ELISA方法测定，根据24小时尿量计算尿白蛋白排泄率。尿肌酐测定采用碱性苦味酸速率法。留取2ml尿液以2000转/分离心10min，取上清液置于-70oC冰箱保存，应用ELISA法方法测定 TGF-β1，应用Excel作图根据标准曲线得出其浓度，其原理是辣根过氧化酶（HRP）标记的TGF-β1和样品中未标记的TGF-β1竞争和酶标板中包被的TGF-β1抗体结合。因尿液样品中TGF-β1浓度低，将其浓缩后，再经酸化激活、NaOH预处理，严格按照试剂盒说明进行,测定尿液中总TGF-β1。尿TGF-β1与尿β2-M用尿肌酐浓度校正，以消除尿液稀释或尿液浓缩等对结果的影响。HbA1C使用比色法检测。血肌酐、血糖使用全自动生化分析仪测定。计量资料以均数±标准差（±s）表示，统计学分析应用SPSS10.0统计学软件。 P<0.05为有显著差异。P<0.01为有极显著差异。结果：正常对照组和糖尿病各组尿检测结果比较：可见与正常对照组比较糖尿病患者中正常白蛋白组尿TGF-β1明显升高（P〈0.05），而UAER、β2-M变化则无统计学差异。在本研究中，尿TGF-β1与UAER（r=0.736,p<0.01）、β2-M (r=0.732,p<0.01) 正相关。尿TGF-β1亦与SCR（r=0.687,p<0.01）、BUN（r=0.659,p<0.01）及LDL-C（r=0.361,p<0.05）正相关,与CCR呈负相关(r=-0.760,P<0.01)，而与 FBG、HbA1c、血压无相关性。讨论： 本研究结果显示：与正常对照组相比，2型糖尿病患者尿TGF-β1排泄在正常白蛋白尿组已升高(p<0.05)，其差异有统计学意义，提示尿TGF-β1排泄能更早期反映糖尿病肾病[5]。同时发现尿TGF-β1与尿白蛋白呈<WP=31>正相关。随尿白蛋白排泌量的增加而增加，随着糖尿病病变程度的增加明显增高。提示TGF-β1与糖尿病肾病的病变程度密切相关[6]。本研究结果与文献[7,8]报道相似，提示尿TGF-β1水平除可作为糖尿病肾病早期诊断的敏感指标，还可能具有反映不同程度肾脏病变的意义。本研究结果表明：糖尿病患者血脂代谢紊乱主要表达为总胆固醇（CHO）、低密度脂蛋白(LDL-C)、脂蛋白a（LPA）增高。尿TGF-β1和LDL-C、UAER、SCR及β2－M呈正相关，与CCR呈负相关，与HbAlc、FBG、血压均无相关性，与Sharma等的报道相符[7]。在高血糖状态下，多种生化异常及微循环障碍共同参与DN的发生，包括蛋白激酶C（PKC）的激活、蛋白非酶糖化作用、氧化应激、肾素－血管紧张素活性增加等。其可通过各种途径促进TGF-β1合成和活性增加[22]，导致细胞外基质积聚、肾小球硬化和小管间质纤维化。其中高血糖、血管紧张素Ⅱ、非酶糖化作用、NO及血栓调节素、脂代谢紊乱对TGF-β1的调节,均与PKC活性密切相关[17,21-24]。PKC诱导TGF-β1mRNA过度表达或者活性增加机制尚未完全明了，可能与其诱导TGF-β1合成与分泌增加，促进隐性TGF-β1的激活有关。尿TGF-β1出现是DM发展至ESRF的前提[17]，而与其伴行的特殊细胞外蛋白如纤维粘连蛋白、蛋白多糖及粘蛋白也相应增加，反过来也增加了TGF-β1在肾小球硬化的病理作用[16,18]。ECM增加作用于肾小球系膜及肾小管，导致肾小球进一步硬化，肾小管内皮纤维化，肾小球滤过率进行性下降，最终导致DN患者的肾功能衰竭。TGF-β1可能是发病机制的最后通道[8]。糖尿病肾病基本病理改变除肾小球硬化外，肾小管间质病变亦相当严重而且?更多还原

【Abstract】 Diabetic nephropathy(DN) is one of the most common chronic microvascular complications of Non-insulin dependent diabetes mellitus (NIDDM) and it also can lead to chronic renal failure .It’s pathological traits are glomerular sclerosis , tubulointerstitial fibrosis and trace protein in urine. The DN in early stage is reversible with timely diagnosis and treatment .If renal function injury exsists,it proceeds quickly and is unreversible.However there are no effective drugs for it, so it is important to diagnose earlier and prevent progressing.Recently ,more and more people regard that transforming growth factor-β1(TGF-β1) plays an important role in DN pathogenesis.In that , we detect the urinary excretion rate of TGF-β1 to find a new way to diagnose early DN. Objective: To explore the clinical significance of microalbumin(mALB) 、β2-microglobulin(β2-M) and TGF-β1 in patients with diabetic nephropathy by investigation of their urinary excretion rates and compared with conventional renal function markers .Methods: Seventy-two cases of NIDDM patients were divided into three groups according to urinary albumin excretion rate (UAER): normoalbuminuric group(Ⅰ)(<20ug /min ,n=22), microalbuminuric group (Ⅱ) (20-200ug/min, n=29), macroalbuminuric group (Ⅲ) (>200ug/min ,n=21).The excretion rates of mALB、TGF-β1and β2-M were detected by ELISA in 72 cases of NIDDM and 16 controls respectively. Fasting blood glucose (FBG),HbA1c,BUN,SCr,and Ccr were simultaneously detected in all cases. <WP=34>Results: The urinary excretion rates of TGF-β1 in various DM groups were markedly higher than those of controls(p<0.05).Moreover ,the excretion rates of TGF-β1 increased with the increase of UAER and there were significant differences among the three groups(p<0.01).Urinary TGF-β1 was also positively correlated with UAER and β2-M (p<0.01).Discussion: The study demonstrated that the urinary excretion rates of TGF-β1 in normoalbuminuric group (Ⅰ) were markedly higher than those of controls (p<0.05).It hinted that urinary excretion rates of TGF-β1 reflected diabetic nephropathy earlier. Moreover ,the excretion rates of TGF-β1 increased with the increase of UAER and there are significant differences among the three groups(p<0.01).Urinary TGF-β1 was also positively correlated with UAER and β2-M、SCr、BUN、LDL-C(p<0.01),which matched reference[7].In that the excretion rates of TGF-β1 was one of the sensitive markers of early DN,and it can reflect various renal injuries.This study also showed that the disturbance of lipoprotein metabolism in DM were the total cholesterol(TC) ,low density lipoprotein (LDL) and lipoprotein A(LPA) increased, and there is positive relation betweeen LDL and TGF-β1 .Under the situation of hyperglycemia, various abnormalities in biological chemistry and microcirculatory disorders take part in DN procession,including the activation of protein kinase C(PKC), protein nonezyme saccharification，oxidative stress,the increase activity of renin-angiotensin.They all accelerated the synthesis and activation of TGF-β1 by all means ,which lead to extracellular matrix accumulation,glomerulosclerosis, tubulointerstitial fibrosis. Among them , hyperglycemia、angiotensinⅡ、nonenzyme saccharification、NO、thrombomodulin and lipoprotein metabolism <WP=35>disturbance regulated the TGF-β1 involving the activity of PKC.Maybe it could increase the synthesis and excretion of TGF-β1,and activated the recessive TGF-β1.The occurrence of urinary TGF-β1 is the premise of DM developing into diabetic renal failure.And the special extracellular protein such as fibronectin proteoglycan and mucoprotein multiplied,which enhanced pathological action of the TGF-β1 on glomerulosclerosis.The increase of ECM acted on glomerular mesangium and renal tubule,resulting in glomerular scleroses proceeding , tubule endothelium fibrosis formation,glomerular filtration rate descending,and finally developing to renal failure. Perhaps TGF-β1 is the last passage of DN pathogenesis.The tubulointerstitial lesion in t更多还原