【作者】 全正莉；

【导师】 刘建社；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 硕士

【摘要】 目的观察糖尿病肾病(diabetic nephropathy DN)大鼠肾组织中TOLL受体4(Toll-like receptor4 TLR4)的表达变化情况,并对TLR4在DN发生发展中所起的可能作用进行初步的探讨。方法试验分两组-模型组和对造组。模型组大鼠应用腹腔内一次性大剂量注射链脲佐菌素(streptozoticin STZ)溶液制作糖尿病模型,而对造组仅给予腹腔注射不含STZ的溶液。于造模成功后2w、4w、6w、8w、12w分别测定模型组和对造组24h尿蛋白排泄量、血肌酐(Cr)、尿素氮(BUN)、肾重/体重(KW/BW)、尿白蛋白/肌酐比值(A/C)、血清CRP及TNF-α滴度;观察肾脏损伤后病理形态学变化并测定细胞外基质增生程度;RT-PCR法测定肾组织TLR4的mRNA表达;免疫组化法检测肾组织TLR4、核因子кB(NF-кB)、生长转化因子-β1(transforming growth factor-β1 TGF-β1)、纤维连接蛋白(fibronectin FN)的蛋白表达。结果糖尿病大鼠2w后即发现24h尿蛋白排泄量增加等早期肾脏病征象,此时肾组织TLR4以及NF-кB表达即开始增加,与对照组相比,差异具有显著性,并随病变程度的加重呈持续性增加。12w时试验大鼠相关炎症介质的血清滴度明显增高,并出现肾组织纤维化及肾功能损伤。相关性分析提示TLR4的表达与炎症反应程度、肾组织纤维化程度及肾功能损伤程度均呈显著正相关。结论TLR4及其信号传导通路可能通过激活免疫炎症反应参与了糖尿病肾病的发生和发展。目的观察氯沙坦对糖尿病肾病大鼠的炎症抑制作用。方法采用链脲佐菌素(streptozotocin STZ,65mg·kg-1)腹腔注射建立糖尿病大鼠模型。试验分3组:对照组、模型组、治疗组,后一组于成模后一周给予氯沙坦20mg·kg-1·d-1灌胃。分别测定对照组和模型组4、8、12周24h尿蛋白、尿白蛋白/肌酐比值(A/C)、血CRP及TNF-α水平;观察肾脏病理形态学变化;应用免疫组化法检测肾组织TOLL样受体4(TLR4)、核因子-кB(NF-кB)的蛋白表达;应用RT-PCR法测定肾组织TLR4的核酸表达;12周后测定治疗组的上述指标。结果模型组与对照组相比:模型组大鼠24h尿蛋白、A/C、血CRP及TNF-α含量显著增高;肾组织TLR4、NF-кB的表达明显增加。相关性分析提示TLR4的表达与NF-кB的表达、24h尿蛋白、A/C、血CRP及TNF-α含量均呈正相关关系。氯沙坦治疗组大鼠较模型组大鼠24h尿蛋白、A/C、血CRP、TNF-α含量下降,肾组织TLR4、NF-кB的表达亦减弱,治疗前后比较存在差异(P<0.05)。结论氯沙坦对糖尿病肾病具有保护作用,部分作用机制可能是通过下调NF-кB、TLR4的表达,降低血CRP、TNF-α含量,抑制炎症反应而实现的。更多还原

【Abstract】 Objective To investigate the changes of Toll-like receptor4(TLR4) expression in renal tissue and regulating possible mechanism in rats with diabetic nephropathy. Methods Male SD rats were randomly divided into two groups as follows: control group and model group . Model group were induced to be diabetic nephropathy by intraperitoneal injection with streptozoticin(STZ). 24h urine protein, creatinine, urea nitrogen, profile of kidney hypertrophy(KW/BW), urine albumen/ creatinine value(A/C), the plasma concentration of CRP and TNF-αwere determined at week 2, 4, 6, 8 and 12 respectively. The transmutation of renal pathomorphology was observed and the hyperplastic degree of extracellular matrix was evaluated. mRNA expression of TLR4 were detected by reverse transcript(RT) -PCR. Renal protein expression of TLR4, nuclear factor-кB( NF-кB), transforming growth factor-β1 (TGF-β1) and fibronectin (FN) were detected by immunohistochemical method.Results 24 hours urine protein excretion was increased, mng symptom of renal disease, at 2 week in model group. At same time, the TLR4 expression in renal tissue was markedly increased compared with control group. To follow the aggravation of the disease, the expression of TLR4 was durative increased. At 12 week, the plasma concentration of CRP and TNF-αof the rats which with diabetic nephropathy were raised up obviously and it emerged nephridial tissue fibrosis and injury of renal function .Positive correlation is found between TLR4 expression and nephridial tissue fibrosis, the same correlation also existed between TLR4 expression and injury degree of renal function.Conclusion TLR4 possibly via adjustment inflammatory reaction to play a pivotal role in the pathogenesis of renal damage in diabetic nephropathy. Objective To observe the inflammation depressant effect of losartan on experimental diabetic nephropathy in rat.Methods SD rats were randomly divided into three groups: control group, model group, losartan-treatment group. The latter two groups were induced to be diabetic nephropathy by intraperitoneal injection with 65 mg/kg streptozoticin(STZ). Then, 20 mg·kg-1·d-1 losartan was continuously given 1 week after STZ injection in losartan-treatment group. The 24h urine protein, urine albumen/ creatinine value (A/C), plasma concentration of CRP and TNF-αwere detected at week 4, 8 and 12 respectively. Renal protein expression of TLR4 and NF-кB were detected by immunohistochemical method. TLR4 mRNA expression in the renal was detected by reverse transcript (RT)–PCR. All of these were detected in losartan-treatment group at week 12.Results The 24h urine protein,A/C,plasma concentration of CRP and TNF-α,the renal expression of TLR4 and NF-кB increased in diabetic rats, compared with control group. Furthermore, TLR4 expression has positive correlation with NF-кB expression, 24h urine protein, A/C, plasma concentration of CRP and TNF-α. The plasma concentration of creatinine , CRP and TNF-αdecreased in losartan-treatment group , and the renal expression of TLR4 and NF-кB were also down-regulated (P<0.05).Conclusions Losartan can lessen the diabetic nephropathy impairment through down-regulation of the TLR4 and NF-кB expression in renal, reduction of CRP and TNF-αplasma concentration.更多还原