摘要
目的建立具有激动样活性的抗α1肾上腺素能受体自身抗体(anti-αadrenergic receptor autoantibody,α1-AA)的糖尿病(diabetes mellitus,DM)大鼠模型,并观察该抗体对大鼠血清肌酐(serum creatinine,Scr)、尿蛋白(urine protein,Upro)、肾脏结构及Ⅳ型胶原、Smad2/3蛋白等表达及多沙唑嗪干预后的影响。方法用α1肾上腺素受体胞外第二肽段合成肽,于0、4、8、12、16周行鼠尾静脉注射法对DM大鼠进行α1-AA免疫介导(α1-AA注射量为100μg·100g-1),酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测α1-AA阳性率;电镜下观察肾脏的病理变化,免疫组织化学法检测大鼠肾脏组织中Ⅳ型胶原、Smad2/3蛋白分布及表达情况,并观察用受体阻断剂干预后受体阳性组大鼠的Scr、Upro、肾脏结构及Ⅳ型胶原、Smad2/3蛋白的表达情况。结果 1DM+α1-AA介导组阳性率91.7%(22/24),明显高于无介导组的27.30%及健康对照组的10.0%,差异具有统计学意义(P<0.01)。2DM+α1-AA介导组大鼠Scr和24 h Upro明显高于无介导组及健康对照组,差异具有统计学意义(P<0.01)。与无多沙唑嗪干预组相比,多沙唑嗪干预组中大鼠Scr和24 h Upro明显改善,差异具有统计学意义(P<0.01)。3电镜下观察肾脏病变发现DM+α1-AA介导组大鼠肾组织损伤最严重,DM无介导组损害不明显,DM+α1-AA介导组中干预组较无干预组病变明显减轻。4半定量分析大鼠肾组织Ⅳ型胶原、Smad2/3蛋表达结果显示,DM+α1-AA介导组MOD值显著高于无介导组(P<0.05)和健康对照组(P<0.01),DM+α1-AA介导组中干预组的上述表达明显减轻,与无干预组比较差异具有统计学意义(P<0.01)。结论通过免疫介导的方法可以建立具有激动样活性的α1-AA的大鼠模型;并证实抗α1-AA介导可致DM大鼠肾基质重构,免疫介导参与DM肾损害病理生理过程,受体拮抗剂可改善和逆转肾损害。
Objective To study renal matrix remodeling mediated by anti-α1-adrenergic receptor autoantibody（α1-AA）in rats with diabetes mellitus（DM）and investigate the effects of doxazosin intervention on renal matrix remodeling and renal functions.Methods Wistar rats were randomized into healthy blank control group,DM group,DM group withα1-AA,DM group withα1-AA and doxazosin intervention and healthy rats with doxazosin intervention.DM rat models were established by intraperitoneal injection of streptozocin and had been observed continually for 16 weeks.Rat models withα1-AA having agonist activity were established and Wistar rats in groups c and d were immunized using synthetic peptide obtained from the second extracellular peptide fragment ofα1-AA（α1-AA IV100μg·100 g-1 body weight,given as a single dose through the caudal vein at weeks 0,4,8,12,16.α1-AA was detected using enzyme-linked immunosorbent assay（ELISA）.24 h urine protein（Upro）was detected by radioimmunoassay and Scr by picric acid method.The collagen Ⅳand Smad2/3 expression in renal tissue was detected by immunohistochemical method.Pathological changes of the kidney were observed under optical and electronic microscopes.Results The positive rate ofα1-AA in DM rats withα1-AA mediation was 91.7%,significantly higher than DM rats withoutα1-AA mediation（27.30%）and healthy control group（10.0%,P〈0.01）.24 h Upro and Scr levels were both significantly higher in DM rats withα1-AA mediation than in DM rats without and healthy rats（P〈0.01）.Under electron microscope,microstructures of renal tissue of DM rats withα1-AA mediation were seriously damaged,while mild abnormality could be found in DM rats withoutα1-AA mediation.High expression ofcollagen IV and Smad2/3 was found in the renal cortex of DM rats withα1-AA mediation,which was significantly different compared to the other 2 groups（P〈0.01 or P〈0.05）.Following intervention of the receptor antagonist doxazosin,expression of collagen IV and Smad2/3significantly decreased,while renal functions and renalmatrix remodeling improved,the difference was statistically significant compared to groups without intervention（P〈0.01）.Conclusion Renal matrix remodeling of DM rats is possibly associated withα1-AA-mediated immune responses.The collagenⅣ and Smad2/3 is involved in renal matrix remodeling of DM rats and plays an important role in the pathological development and progression of the disease.Receptor antagonists can reverse and improve renal functions and renal matrix remodeling.
出处
《华南国防医学杂志》
CAS
2015年第10期730-734,738,共6页
Military Medical Journal of South China
基金
湖北省自然科学基金项目(2002AB116)
