BMP2信号通路介导的肾动脉钙化对糖尿病肾病进展的影响

发布时间：2018-05-24 20:40

本文选题：骨形态蛋白2 + 糖尿病肾病　；参考：《西南医科大学》2016年硕士论文

【摘要】：目的:血管钙化是糖尿病患者并发心血管疾病的主要表现之一,也是糖尿病患者死亡的重要危险因素。糖尿病肾病(Diabetic nephropathy,DN)作为糖尿病的严重并发症之一,其发生发展和糖尿病血管损伤紧密相关。已有研究表明,糖尿病患者比非糖尿病患者更易发生动脉血管钙化,而DN患者的钙化程度更为严重及普遍。目前对DN血管钙化的研究大多集中在外周动脉及冠状动脉,而对肾动脉血管的改变知之甚少。本研究通过观察糖尿病肾病基础上给予增强血管钙化的因素后,肾动脉钙化程度与肾功能进展的关系,同时观察糖尿病肾病大鼠肾动脉上BMP2及其下游因子Smad1,Runx2和Osterix的基因及蛋白变化情况,探讨糖尿病肾病时肾动脉钙化严重程度与糖尿病肾病进展的关系,及BMP2/Smad1/Runx2/0sterix信号通路激活在糖尿病肾病大鼠肾动脉钙化的作用。方法:将64只SD大鼠随机分为对照组(CON组,18只))、糖尿病肾病组(DN组,22只)、糖尿病肾病合并血管钙化组(DN+VDN组,24只),DN组及DN+VDN组高脂饲料喂养4周后,予以单次腹腔注射1%链脲佐菌素(STZ)溶液35mg/kg诱导糖尿病,CON组注射同等体积的0.1mmol/L柠檬酸盐缓冲液。72小时后测血糖,连续三天血糖≥16.7 mmol/L为糖尿病成模。2周后,24 h尿蛋白量大于30 mg,尿量原尿量50%者为DN模型制备成功。DN成模第二天,DN+VDN组以维生素D3(300 000U/kg)肌肉注射,将尼古丁(25 mg/kg)完全溶解于花生油中给DN+VDN组大鼠灌胃,9 h后再灌胃一次,其余两组予等量花生油灌胃及生理盐水肌注。8、12和16周时收集各组大鼠尿液检测24小时尿白蛋白(24-h Upro);心脏采血检测血清尿素氮(BUN)、肌酐(Scr)、空腹血糖、胱抑素C(CysC)。大鼠肾脏组织病理检查:清洁取肾,石蜡包埋后行HE染色,观察肾小球和肾小管间质的基本病理改变情况。摘取双侧肾动脉用生理盐水冲洗后,Von Kossa染色观察大鼠肾动脉钙盐沉积,并用钙离子试剂盒检测肾动脉组织钙含量。免疫荧光双染检测各组大鼠肾动脉组织BMP2/α-SMA和Runx2/α-SMA蛋白表达变化。免疫组化检测大鼠肾动脉组织BMP2、Smad1、Runx2及Osterix蛋白的表达。实时荧光定量(RT-PCR)检测各组大鼠肾动脉组织BMP2和Runx2的基因表达。结果:1、大鼠一般情况①体重:与CON组比较,模型组体重明显减轻,但DN组及DN+VDN组各时间点体重无明显统计学差异。②血糖:CON组血糖在正常水平范围波动,DN组及DN+VDN组大鼠血糖值显著高于同时间点CON组,且两模型组间血糖无统计学差异。③血清尿素氮(BUN)、肌酐(Scr):与CON组相比,DN组及DN+VDN组大鼠BUN值于实验8周后明显升高,12周后DN组和DN+VDN组血Scr亦显著升高(P0.05),而DN组与DN+VDN组间BUN、Scr值均无明显统计学差异。④胱抑素C(CysC):实验期间,DN组大鼠血清CysC水平明显高于同时间点CON组,低于DN+VDN组(P0.05)。⑤24小时尿白蛋白(24-h Upro):8周后DN组及DN+VDN组大鼠24-h Upro较CON组开始上升,随时间的延长进行性加重,并于16周末DN+VDN组大鼠的24-h Upro较同期DN组明显升高(P0.05)。2、大鼠肾动脉钙化的特征:①Von Kossa染色:CON组大鼠肾动脉组织各时间点均未见明显异常,DN组及DN+VDN组大鼠8周末时即可见血管内膜及中膜弹性纤维间大量黑色颗粒,且随着时间延长,沉积的黑色颗粒逐渐增多聚集成团。②钙含量测定:随时间的进展,三组大鼠钙含量均逐渐增加,且DN+VDN组大鼠肾动脉组织各时间点钙水平较DN组显著升高,CON组大鼠较同期DN组明显下降(P0.05)。3、免疫荧光检测BMP2/α-SMA和Runx2/α-SMA的表达:CON组大鼠肾动脉组织中可见BMP2及Runx2均少量表达且各时间点无明显变化;与同期CON组大鼠相比,DN组和DN + VDN组大鼠肾动脉组织BMP2及Runx2表达的绿色荧光强度相对较强,而α-SMA表达的红色荧光相对较弱;与DN组相比,DN +VDN组大鼠肾动脉血管中可见BMP2及Runx2蛋白的表达较同期DN组增加,α-SMA的表达减少;此外,随时间的推移,DN和DN + VDN组大鼠α-SMA的荧光强度也随BMP2或Runx2蛋白表达的增强而减弱。4、BMP2/Smad1/Runx2/Osterix信号蛋白免疫组化表达:CON组肾动脉组织中BMP2、Smad1、Runx2及Osterix在血管中膜均有散在微量表达。DN组大鼠肾动脉组织中可见BMP2、Smad1、Runx2和Osterix蛋白在实验8周末时表达即开始明显增加,阳性着色深,胞质呈棕黄色,主要分布于动脉血管中膜平滑肌细胞中,随时间的进展,表达逐渐增强。与同期DN组大鼠相比,可见DN+VDN组大鼠肾动脉组织BMP2/Smad1/Runx2/Osterix信号通路的表达明显升高。5、肾动脉组织BMP2及Runx2 mRNA的表达:RT-PCR结果示,对照组大鼠肾动脉组织BMP2、Runx2 mRNA仅有微量表达,且各时间点无统计学差异;模型组大鼠肾动脉组织自实验第8周末起,BMP2和Runx2 mRNA水平开始增加,16周末时BMP2及Runx2 mRNA的表达量达最高值,此外,与DN组相比,DN+VDN组同期大鼠表现出更高的BMP2和Runx2 mRNA水平(P0.05)。6、肾脏HE染色显示:CON组大鼠各时间点未见明显异常;8周末时DN组大鼠可见肾小球体积增大,基底膜轻度增厚,系膜区增宽,肾小管上皮细胞偶见空泡样或颗粒状变性,随时间的延长,12周末时可见基底膜增厚更明显,偶见肾小动脉壁玻璃样变性,16周末时肾组织损伤进一步加重,基底膜重度增厚,并可见节段性硬化;与DN组相比,DN+VDN组同期大鼠上述病理改变明显加重。结论:1、DN在较早阶段时即已存在肾动脉等大血管的钙化,而在钙化血管中存在BMP2/Smad1/Runx2/Osterix信号通路的激活;2、肾动脉钙化的发生与严重程度与DN进展密切相关;3、BMP2/Smad1/Runx2/Osterix信号通路是DN肾动脉血管钙化重要的调节因素。
[Abstract]:Objective: vascular calcification is one of the major manifestations of cardiovascular disease in diabetic patients and an important risk factor for the death of diabetic patients. Diabetic nephropathy (DN) is one of the serious complications of diabetes. The development of diabetes mellitus is closely related to diabetic vascular injury. Non diabetic patients are more likely to have arterial calcification, and the degree of calcification in DN patients is more serious and widespread. Most of the studies on vascular calcification in DN are concentrated in the peripheral arteries and coronary arteries, but little is known about the changes in the renal artery. The relationship between the degree of renal artery calcification and the progression of renal function, and the changes in the gene and protein of BMP2 and its downstream factors Smad1, Runx2 and Osterix on the renal artery of diabetic nephropathy rats, and to explore the relationship between the severity of renal artery calcification and the progression of diabetic nephropathy and the activation of BMP2/Smad1/Runx2/0sterix signaling pathway in diabetic nephropathy. Methods: the role of renal artery calcification in diabetic nephropathy rats. Methods: 64 SD rats were randomly divided into control group (group CON, 18), diabetic nephropathy group (group DN, 22), diabetic nephropathy combined with vascular calcification group (group DN+VDN, 24), DN group and DN+VDN group high fat feed for 4 weeks, and single intraperitoneal injection of 1% streptozotocin (STZ) solution 35mg/kg Induced diabetes, group CON was injected with the same volume of 0.1mmol/L citrate buffer for.72 hours after.72 hours. After three days of blood glucose more than 16.7 mmol/L for.2 weeks, the protein amount of 24 h was greater than 30 mg, and the urine volume of primary urine was 50% of the DN model for second days. The DN+VDN group was injected with vitamin D3 (300 000U/kg). Nicotine (25 mg/kg) was completely dissolved in peanut oil to gavage to rats in group DN+VDN and reperfusion after 9 h. The rest two groups were given equal amount of peanut oil for gastric perfusion and.8,12 and 16 weeks of physiological saline injection. Urine albumin (24-h Upro) was collected in each group of rats, and serum urea nitrogen (BUN), creatinine (Scr), fasting blood glucose, cystine, and cystine were suppressed by heart blood sampling. C (CysC). Pathological examination of kidney tissue in rats: after cleaning the kidneys and after paraffin embedding, HE staining was performed to observe the basic pathological changes of the interstitium in the glomeruli and renal tubules. After the extraction of bilateral renal arteries, the calcium salt deposition in the renal arteries was observed by Von Kossa staining and the calcium content of the renal artery was detected by the calcium ion kit. The expression of BMP2/ alpha -SMA and Runx2/ alpha -SMA protein in renal artery tissue of rats was detected by double staining. The expression of BMP2, Smad1, Runx2 and Osterix protein in renal artery tissue of rats was detected by immunohistochemistry. The expression of BMP2 and Runx2 in renal artery tissues of rats in each group was detected by real-time fluorescence quantitative (RT-PCR). Results: 1, the general condition of rats: and C In group ON, the body weight of the model group was significantly reduced, but there was no significant difference in weight at all time points between the DN group and the DN+VDN group. 2. The blood sugar in the group CON was fluctuated in the normal level, and the blood sugar of the DN group and the DN+VDN group was significantly higher than that in the CON group at the same time, and there was no statistical difference between the two model groups. (3) the serum urea nitrogen (BUN) and creatinine (Scr): and C. Compared with group ON, the BUN value of rats in group DN and DN+VDN group increased significantly after 8 weeks, and the blood Scr in DN and DN+VDN groups increased significantly after 12 weeks (P0.05), but there was no significant difference between the DN group and DN+VDN group. (5) 24 hour urinary albumin (24-h Upro): after 8 weeks, 24-h Upro in group DN and group DN+VDN began to rise and increased with time, and the 24-h Upro of DN+VDN group rats increased significantly at the end of the 16 week than that in DN group (P0.05). There was no obvious abnormality. In group DN and group DN+VDN, a large number of black particles were seen between the intima and the middle membrane elastic fibers at the end of 8 weeks. And with the time prolonging, the black particles were gradually increased and aggregated. The calcium content was measured in the three groups of rats with the progress of time, and the renal artery tissues of the group DN+VDN rats were all increased. Compared with the DN group, the level of calcium was significantly higher in the CON group than that in the DN group (P0.05).3. The expression of BMP2/ alpha -SMA and Runx2/ alpha -SMA were detected by immunofluorescence. The expression of BMP2 and Runx2 in the renal artery tissues of the rats of the CON group was small and there was no obvious change at each time point. The green fluorescence intensity expressed in BMP2 and Runx2 was relatively strong, while the red fluorescence expressed by alpha -SMA was relatively weak. Compared with the DN group, the expression of BMP2 and Runx2 protein in the renal artery of DN +VDN group was increased and the expression of alpha -SMA decreased. Besides, the fluorescence intensity of alpha -SMA in DN and DN + groups also followed the time. The enhancement of the expression of MP2 or Runx2 protein weakened.4 and the expression of BMP2/Smad1/Runx2/Osterix signal protein: BMP2, Smad1, Runx2 and Osterix in the renal artery tissues of group CON were scattered in the microexpression of the vascular membrane, and the BMP2 was seen in the renal artery tissue of the rats in the.DN group, and the expression began to increase obviously at the end of the 8 week of the experiment. In addition, the positive staining was deep and the cytoplasm was brown and yellow, mainly distributed in the membrane smooth muscle cells in the arterial blood vessels. The expression gradually increased with the development of time. Compared with the DN group, the expression of BMP2/Smad1/Runx2/Osterix signaling pathway in the renal artery tissue of the DN+VDN group was obviously elevated, and the expression of BMP2 and Runx2 mRNA in the renal artery tissue: RT-PC R results showed that the renal artery tissue of the rats in the control group was BMP2 and Runx2 mRNA only micro expression, and there was no statistical difference at all time points. The level of BMP2 and Runx2 mRNA in the renal artery tissue of the model rats began to increase at the end of the eighth week, and the expression of BMP2 and Runx2 mRNA at the 16 weekend reached the highest value. In addition, the DN+VDN group was compared with the same period of the DN+VDN group. The higher BMP2 and Runx2 mRNA level (P0.05).6 and renal HE staining showed that there was no obvious abnormality in the time points of the CON group. At the 8 weekend, the glomerular volume increased, the basement membrane was slightly thickened, the mesangial area widened, and the renal tubular epithelial cells had vacuolated or granular degeneration. The basement membrane was seen at the 12 weekend and the basement membrane was visible with time. The thickening is more obvious. I see the hyaline degeneration of the renal arteriole wall, the renal tissue damage is further aggravated at the end of the 16 week, the basal membrane is thickened and the segmental sclerosis is seen. Compared with the group DN, the pathological changes in the DN+VDN group are obviously aggravated. Conclusion: 1, the calcification of the large vessels, such as the renal arteries, is present in the early stage, and in the calcified blood vessels, in the earlier stage, the DN is in the calcified vessel. The activation of BMP2/Smad1/Runx2/Osterix signaling pathway is found, 2, the occurrence and severity of renal artery calcification are closely related to the progress of DN, and 3, BMP2/Smad1/Runx2/Osterix signaling pathway is an important regulatory factor for the calcification of the DN renal artery.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2

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