【作者】 吴守彩；

【导师】 高海青；

【作者基本信息】 山东大学 ， 老年医学， 2010， 硕士

【摘要】 糖尿病肾病(diabetic nephropathy, DN)是糖尿病慢性微血管并发症之一,是一类以进行性纤维化为病理特征的肾脏疾病,其发病机制复杂。近期研究发现,结缔组织生长因子(connective tissue growth factor, CTGF)在糖尿病肾病中可能是骨形态发生-7(bone morphor genetic p roteins-7, BMP-7)信号通路的抑制剂,两者在肾脏纤维化过程中均发挥关键作用。传统中药通心络已广泛用于心脑血管病的防治,随着对通心络新用途的不断开发,其临床应用也在不断扩大。本实验研究通心络对DM大鼠肾脏中BMP-7及CTGF的影响,探讨其对DN大鼠肾脏的保护作用及其可能机制。目的研究通心络对糖尿病大鼠肾脏组织保护作用及结缔组织生长因子(CTGF)及骨形态蛋白7(BMP-7)表达的影响,并探讨其对DN大鼠肾脏的保护机制。方法雄性Wistar大鼠70只,随机选出15只作为正常对照组(C组,n=15)。剩余55只采用链脲佐菌素(STZ)诱导建立糖尿病大鼠模型；禁食12小时,一次性尾静脉注射STZ 55mg／kg,72h后测定血糖,以空腹血糖水平≥16.7mmol／l(300mg／l)为糖尿病模型成功的标准。造模成功后大鼠随机分为糖尿病组(DM组),通心络治疗组(TXL组),于24周末处死大鼠检测各组大鼠24h尿微量蛋白、采血测空腹血糖(FPG)、糖化血红蛋白(HbAlc)、糖基化终末产物(AGEs)和肝肾功能等血清学指标,肾重／体质量(KW／BW),光镜PAS染色,观察肾脏病理改变及细胞外基质(ECM)含量的变化,采用逆转录-聚合酶链式反应(reversetranscriptase coupled to polymerase chain reaction, RT-PCR)和Western blotting分别用于大鼠肾脏组织CTGF和BMP-7mRNA和蛋白质表达水平。结果1.一般指标及血清AGEs等的变化：C组大鼠表现活泼,体质量增长迅速；DM组大鼠出现多饮多食多尿及消瘦表现,较C组体质量明显下降(P<0.01)；TXL组DM组上述症状减轻,体质量明显升高(P<0.05)。DM组与C组比较,FPG、HbA1C、血清AGEs均明显升高(P<0.05)；TXL组较DM组有下降趋势,但差异无统计学意义(P>0.05)。2.各组大鼠肾功能的变化：DM组较C组大鼠BUN、Scr、Kw／Bw升高、24h尿微量蛋白升高,表明DM组大鼠存在肾脏肥大及早期肾病表现。TXL组与DM组比较,24 h尿微量蛋白含量减少,Kw／Bw亦降低,差别有统计学意义(P<0.05),其他肾功能指标的改变无统计学意义(P>0.05)。3.各组大鼠肾脏组织病理学观察镜下可见,C组大鼠肾小球及肾小管、间质未见明显病理改变。DM组大鼠大部分肾小球基底膜增厚,毛细血管腔受压变窄,肾小管上皮细胞空泡样变明显。TXL组大鼠肾小球球囊与正常组比较无明显增大,毛细血管丛内可见清晰的血管腔,部分毛细血管丛基底膜稍增厚,系膜区稍增宽,部分肾小管上皮细胞胞浆疏松,轻度水肿,未见空泡样改变。4.各组大鼠肾脏组织中ECM含量的变化DM组较C组大鼠肾脏组织ECM明显升高(P<0.01)。TXL组与DM组比较,ECM含量减少(P<0.05)。5.肾组织CTGF、BMP-7mRNA的变化RT-PCR结果示,DM组较C组CTGF mRNA上调、BMP-7mRNA下调明显(P<0.01)；TXL组较DM组CTGFmRNA下降,BMP-7mRNA上调,差异有统计学意义(P<0.05)。6.Western blot肾组织CTGF、BMP-7蛋白和内参β-actin蛋白检测条带光密度分析显示：DM组肾组织CTGF表达升高、BMP-7表达降低(P<0.01)；给药24周后可见,TXL组较DM组CTGF表达下降,BMP-7表达升高(P<0.05)。结论1、通心络可以减轻糖尿病大鼠微量蛋白尿、改善肾脏组织病理及减少肾组织ECM含量,对DN大鼠早期具肾脏保护作用。2、通心络抑制糖尿病大鼠肾脏组织CTGF表达,增加BMP-7表达,可能为通心络对糖尿病肾病大鼠肾脏的保护机制。更多还原

【Abstract】 Diabetic nephropathy (DN) is one of the chronic microvascular complications of diabetes and a kind for fibrosis to pathological features of renal disease, its pathogenesis is complexity, many mechanisms are still under explored. Recent studies found that connective tissue growth factor (CTGF) in diabetic nephropathy may be the bone morphogenetic-7 (BMP-7) signaling pathway inhibitor, The both play a key role in the kidney fibrosis. Tongxinluo,the components of traditional herb has been widely used in cardiovascular and Cerebrovascular disease prevention and treatment. With Tongxinluo continuous development of new uses, its clinical application are also expanding. This study on Tongxinluo is to explore the the protective effect of Diabetic mellitus (DM) rats and its possible mechanism through the changes of the expression of CTGF and BMP-7 in DN rats’kidney.Objective To study the effects of Tongxinluo on the expression of connective tissue growth factor CTGF) and bone morphogenetic protein-7 (BMP-7) in Diabetic rats,and to explore the protective effects and mechanism on the kidney of Diabetic nephropathy rats.Methods Randomly selected 15 as the normal control group (C group, n= 15) from 70 healthy male Wistar rats.The other 55 rats was established the diabetic (DM) rats model by one-dose introperitoneal injection of streptozotocin(STZ).After 72h,the determination of blood glucose to fasting glucose levels≥16.7mmol/1 (300mg/1) that were diabetes model of success. After animal models were successfully established,they were randomly divided into diabetic groups (DM group), Tongxinluo (500mg-kg-1-d-1) treated groups(TXL group). The ratio of kidney weight to body weight,24h urine microprotein, plasma fasting blood glucose (FPG), glycosylated hemoglobin (HbAlc), advanced glycation end products (AGEs) and kidney function were determined. The renal pathological changes were examined with light microscope PAS staining. The mRNA and protein expression of CTGF and BMP-7 in kidney were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western bloting.Results 1. Weight and blood indices:Compared with C group,DM group weighed significantly less(P<0.01), After treated with Tongxinluo capsules for 24 weeks, rats in TXL group also weighed significantly less than those in C group (P <0.01),but weighed more than those in DM group with statistic significance(P<0.01). As to FPG、HbA1c and AGEs, DM group and C group, FBG, HbA1C, serum AGEs were significantly higher (P<0.05); TXL group was a downward trend than DM group, but the difference was not statistically significant.2. The changes of renal function in rats:DM group than in C group rats with decreased of plasma BUN、Scr and Kw/Bw higher,24h urine protein increased; TXL group than DM group,24h urine protein content decreased, Kw/Bw is also reduced (P<0.05).3. Light microscopic findings of renal morphological changes in rats (PAS×400):C group rats glomerular and tubular interstitial were no significant pathological changes.. In DM group, Most of the glomerular basement membrane were thickened, capillary was narrowed, tubular epithelial cells were vacuolated changes significantly. TXL group compared with DM group, some of glomerular basement membrane is thickened and widened mesangial areas slightly, some of renal tubular epithelial cells were no vacuolar changes.4.The changes of extracellular matrix (ECM) in the rars’kidney: DM group than C group, ECM were significantly higher (P<0.01); TXL group was reduced than DM group, (P<0.05).5.The expression of CTGF and BMP-7mRNA in kidney:RT-PCR results showed, The expression of CTGF mRNA in DM group than C group were significantly increased (P<0.01), TXL group than DM group decreased (P<0.05);The expression of BMP-7mRNA in DM group than C group were significantly decreased (P<0.01), TXL group than DM group increased (P <0.05).6.The expression of CTGF and BMP-7 protein in kidney:Western blotting results were that the expression of CTGF in DM group than C group, were significantly higher (P<0.01), TXL group was lower than the DM group (P<0.05); The expression of BMP-7 protein, DM group than C group, were significantly decreased (P<0.01), TXL group was increased than DM group (P<0.05).Conclusion 1.Tongxinluo can reduced microalbuminuria in diabetic rats and improved the renal pathology changes, and decreased kidney deposition of ECM in diabetic nephropathy rats.2.Its protective effects and mechanism may be related to downregulating expression of CTGF and upregulating expression of BMP-7.更多还原