【作者】 楚能武；

【导师】 章秋； 王佑民； 王长江；

【作者基本信息】 安徽医科大学 ， 内科学， 2009， 硕士

【摘要】 目的:本研究旨在观察胰岛素增敏剂吡格列酮对伴胰岛素抵抗的糖尿病(diabetes mellitus,DM)大鼠的肾脏结构及c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)蛋白的表达的影响,并探讨其保护DM大鼠肾脏的机制,为糖尿病肾脏病变的防治提供新思路。方法:①将长期高脂喂养、伴胰岛素抵抗(insulin resistance,IR)的DM大鼠随机分为模型组、吡格列酮(pioglitazone,PIO)组,PIO组予吡格列酮10mg/kg.d灌胃给药;②治疗6周后颈椎脱臼处死大鼠,取多块肾脏新鲜组织放入冻存管在液氮罐中保存备用。制备肾脏组织石蜡切片。取肾脏组织匀浆,?20℃储存备用;③HE、PAS、Masson’s、PASM染色后观察各组大鼠的肾脏病理情况。④免疫组化对JNK,p-JNK进行半定量分析;⑤逆转录聚合酶链反应(reverse transcription-polymerase chain raction ,RT-PCR)测定JNKmRNA在肾脏中的表达;⑥Westem免疫印迹方法定量分析JNK,p-JNK蛋白的表达。结果:肾脏病理:HE、PAS、Masson’s、PASM染色后观察,与NC组对比,DM组的肾小囊腔狭小,肾小球体积增大,胶原增多,基底膜不规则增厚,基质增多;系膜区扩大,系膜细胞增多,有局部溶解现象;肾小管变形,管腔狭窄;肾间质不均匀伴有散在纤维化。PIO组与DM组比较:肾小球大小均匀,基底膜规则伴轻度增生,系膜区染色欠均匀,肾小管形态少数异常,肾间质未见纤维化。免疫组化:①NC组肾小球,肾小管及肾间质的JNK及P-JNK表达甚微。②DM组肾小球,肾小管及肾间质均有不同程度的JNK及P-JNK表达,与NC组相比,差异有显著性(P<0.05)③PIO组肾小球,肾小管及肾间质均有微量的JNK和P-JNK表达,与NC组相比差异无显著性(P>0.05),但与DM组相比差异有显著性(P<0.05)。RT-PCR:①在NC组中JNKmRNA几乎不表达②与NC组相比,DM组JNKmRNA的表达量明显增高③与DM组相比,PIO组JNKmRNA的表达量明显下降④PIO组与NC组相比,JNKmRNA的表达量相差不明显。WESTERN BLOT:①与DM组相比,PIO组P-JNK与总JNK蛋白的表达量均降低差异有显著性(P<0.05)②DM组与正常对照组相比P-JNK与总JNK蛋白的表达量明显升高,差异有显著性(P<0.05)③与DM组相比,PIO组P-JNK与总JNK蛋白量的比值明显降低,差异有显著性(P<0.05)④PIO组P-JNK与总JNK蛋白的表达量以及P-JNK与总JNK蛋白量的比值与正常对照相近,差异无显著性(P>0.05)。结论:①吡格列酮能减轻糖尿病大鼠的肾脏病理状况。②正常情况下S-D大鼠肾脏JNK和p-JNK表达量很低,伴IR的T2DM肾脏组织的JNK和p-JNK表达上升明显,pJNK/JNK也有所上升。③经吡格列酮治疗后可降低JNK和p-JNK表达,同时pJNK/JNK也有所下降。④吡格列酮对伴IR的T2DM大鼠的肾脏保护作用可能与降低JNK磷酸化表达有关。更多还原

【Abstract】 Objectives:to observe the effect of insulin sensitizer pioglitazone (PIO) on kidney of Diabetic rats with insulin resisitance,and its effect on c-Jun N-terminal kinase(JNK)expression.To probe the protective mechanism of PIO on renal function of diabetic rats and to provide a new thought for the prevention and cure of Diabetic Nephropathy.Methods:①After animal model of diabetic rats with insulin resistance (IR)was established,rats were randomly divided into three groups: normal control group(NC),diabetes mellitus group(DM),and pioglitazone group(PIO)in which rats were treated with PIO at dose of 10mg/kg.d by intragastric administration.②Rats were executed through the method of cervical spine dislocation after 6-week’s intervention. Kidney specimens were harvested were stored in liquid nitrogen for use.③Pathological changes was observed by means of HE, PAS, Masson’s, and PASM.④JNK, p-JNK was analyzed semiquantitatively by using the method of immunol histochemistry.⑤JNK mRNA expression of kidney tissue was examined by reverse transcription-polymerase chain raction(RT-PCR).⑥Protein expression of JNK and p-JNK was examined by using Western Blot.Results: 1.Pathological changes of kidney structure:We observed the pathological changes of rat kidney tissue in the diabetes mellitus group (DM), and found that,compared with normal control group(NC), Bowman’s space of the former group became small,the size of glomeruli increased, the amount of collagen and extracellular matrix increased, and basement membrane was irregularly thickened.We also found that extra glomerularmesangial region expanded, and the amount of mesangial cells increased and part of those cells dissolved. In addition, renal tubules deformed with narrowing,and nterstitium distributed unevenly with scattered fibration.Compared with DM group,glomeruli of the PIO group were uniform,and basement membrane distributed evenly with slight hyperplasia. Besides, dyeing of extra glomerularmesangial region was uneven,and slight paramorphia was found in renal tubules, moreover there was no fibration in nterstitium. 2.Immunol histochemistry(IHC):①There was little JNK and P-JNK expression in the glomeruli , renal tubules, and nterstitium of NC group.②JNK and P-JNK expression in these three parts of DM group were variant , and compared with that of NC group the expression increased.The difference of expression levels between these two groups was statistically significant(P<0.05).③There was no statistical significance between the JNK and P-JNK expression levels of the PIO group and that of NC group(P>0.05),but compared with DM group the change level of JNK and P-JNK expression in PIO group was statistically significant(P<0.05).3.RT-PCR:①There was almost no JNK mRNA expression in NC group.②The JNK mRNA expression in DM group increased dramatically when compared with that in NC group.③JNK mRNA expression level of PIO group decreased remarkably in contrast to that of DM group.④There was no evidently difference between the JNK mRNA expression of the PIO group and that of NC group.4.Western Blot:①Compared with that of DM group ,P-JNK and JNK protein expression of PIO group decreased with statistical significance (P<0.05).②P-JNK and JNK protein expression of DM group increased dramatically when compared to that of NC group,and the difference was statistically significant(P<0.05).③The ratio of JNK protein expression level to P-JNK protein expression level in PIO group increased with statistical significance(P<0.05),compared with that in DM group.④In PIO group,JNK and P-JNK protein expression level and their ratio was similar to what we observed in NC group and there was no statistical significance(P>0.05).Conclusions:①PIO can improve the Pathological changes of kidney structure of diabetic rats.②JNK and P-JNK are less expressed in kidney tissue of SD rat under normal condition, but their expression level are increased dramatically in kidney tissue of DM group with Insulin resistance(IR).Furthermore, the ratio of P-JNK to JNK are also increased.③JNK and P-JNK expression can be down regulated by the treatment of pioglitazone (PIO),along with the reduced ratio of P-JNK to JNK.④Suppressed expression level of phosphorylated JNK may play roles in the mechanism of the protective effect of pioglitazone(PIO)on rat kidney in DM group with insulin resistance.更多还原