氧化应激在造影剂肾病中的作用及干预研究

发布时间：2019-05-22 00:08

【摘要】：背景和目的随着现代医学影像诊断和介入治疗的快速发展，造影剂肾病(CIN)的发病率呈上升趋势，其发病机制、干预措施也受到更多的重视。老年糖尿病人群中CIN的发病机制尚未完全明确，氧化应激以及信号传导是目前研究的热点。我们通过制作老年糖尿病大鼠模型，观察造影剂对肾功能和肾组织一氧化氮(NO)、一氧化氮合成酶(NOS)及谷胱甘肽(GSH)的影响，探讨了NO、NOS及GSH在CIN发病机制中的作用。继而构建表达了两种NOS的嵌合体酶，研究了嵌合体酶的类型和结构对NO合成活性的影响。临床工作中对于老年患者，，通过肌酐或者由肌酐计算得出的肾小球滤过率可能无法准确反映肾功能的变化，从而延误CIN的诊断。本论文通过对白细胞介素18和胱抑素C在老年糖尿病患者中的表达研究,寻找早期、敏感、特异预测CIN的生物学指标。考虑到氧化应激在CIN发病机理中的作用，抗氧化剂对于高危人群预防CIN的发生可能有一定的效果。GSH作为复杂的抗氧化网络的中心，其功能是通过缓冲活性氧、调节NO循环和改善控制着生长、发展和防御的细胞氧化还原信号传递系统来体现的。评价还原型谷胱甘肽联合生理盐水水化对老年糖尿病患者CIN干预效果。由于血液中的存在着大量GSH，本论文还研究了GSH与血红蛋白的结合过程的机理，进一步了解GSH发挥作用的机制。本论文的研究内容主要包括三个部分。第一部分：低渗造影剂对老年糖尿病大鼠肾毒性及一氧化氮合成酶对一氧化氮合成机理影响的研究一、材料与方法方法是通过建立老年糖尿病大鼠模型，观察尾静脉注射欧乃派克350(10ml/kg)后，大鼠血肌酐、尿素氮以及肾组织匀浆NO、NOS和GSH的含量。构建表达了两种NOS的一氧化氮合成酶，获取了一氧化氮合成酶的光谱，研究了一氧化氮合成酶的NO合成活性，测试了对铁氰化物、细胞色素C还原速率、黄素、NADPH氧化还原速率的影响。二、结果结果是老年糖尿病大鼠血肌酐、尿素氮与正常对照组比较均升高(P 0.05)，肾组织匀浆NO、NOS、GSH与正常对照组比较均下降。注射造影剂后，血肌酐与正常对照组比较明显升高(P 0.01)，肾组织匀浆NO和NOS与糖尿病对照组比较下降(P 0.05)。nNOSeFNR一氧化氮合成酶表现出较低的血红素还原速率。第二部分：白细胞介素18、胱抑素C对老年糖尿病患者造影剂肾病的预测作用研究一、材料与方法选择2011年1月到2011年10月在我院接受增强CT检查的年龄大于60岁、糖尿病史1年以上、无明显肾功能异常的患者共30例，患者均采用低渗非离子型造影剂。ELISA法测定造影前和造影后24小时、48小时IL-18以及血尿素氮(BUN)、血肌酐(SCr)、CysC。二、结果观察对象均无造影剂肾病的发生，造影后血IL-18与造影前比较无统计学意义(P0.05)，造影后24小时尿IL-18、血胱抑素C升高明显，与造影前比较差异有统计学意义(P 0.05)，48小时后SCr升高明显(P 0.05)。第三部分：还原型谷胱甘肽对老年糖尿病患者造影剂肾病影响及与血红蛋白结合过程的研究一、材料与方法将准备采用低渗造影剂进行增强CT扫描的96位糖尿病患者(年龄≥60岁)随机分为两组。一组静脉注射还原型谷胱甘肽(于扫描前静脉注射溶于100ml生理盐水中的1800mg还原型谷胱甘肽，扫描后同等剂量注射两天)，同时以1ml/kg BW/h的速度静脉注射生理盐水(GSH组，n=48)；另一组仅以相同速度静脉注射生理盐水(对照组，n=48)。SCr、BUN、CysC和尿IL-18分别于造影前和造影后48小时测量。肌酐高于基线0.5mg/dL或者比肌酐基线值增长25%即被确认为CIN。利用多种光谱法研究了还原型谷胱甘肽与Hb的结合过程，确证了GSH对血红蛋白的猝灭机制。根据所得到的结合常数等参数推断出GSH与血红蛋白之间的相互作用力的类型。由F ster能量转移理论可以得到GSH与血红蛋白的结合距离。利用多种分子光谱法研究了GSH的存在对血红蛋白构象的影响。二、结果 GSH组和对照组在基线特征和基线肾功能方面没有显著性差异。GSH组CIN的发病率稍低于对照组(2.08%vs.8.33%, P=0.364)。增强CT扫描后两组的SCr、BUN、CysC、尿IL-18均升高，估测肾小球滤过率(eGFR)均降低。造影后两组SCr(P=0.001)，eGFR(P=0.002), BUN(P 0.001)，CysC(P=0.001)和尿IL-18(P 0.001)有显著性差异。GSH浓度的增加导致Hb的荧光强度逐渐降低。依据热力学原理求得一系列热力学参数，从表中看出, ΔH O、ΔS 0、ΔG O,说明GSH与Hb作用过程是一个焓变减小、熵减、吉布斯自由能降低的自发过程，可推断出GSH与Hb之间的作用力是以氢键及范德华力结合。依据峰位置的红移或蓝移来说明氨基酸周围微环境的变化，最大发射波长发生了明显的红移现象，表明GSH改变了Hb中色氨酸残基所处的微环境，极性增强。结论 1.低渗造影剂可以引起老年糖尿病大鼠肾损伤，NOS、NO的代谢以及信号传递可能与其有关。 2.一氧化氮合成酶的种类和结构性质对一氧化氮的合成过程有显著影响。 3.尿IL-18和Cys C是可以早期反映肾损伤的敏感指标，对CIN的早期诊断及防治有重要作用。 4.还原型谷胱甘肽加生理盐水水化可能通过调节氧化和抗氧化系统预防造影剂肾病的发生。 5.确定了还原型谷胱甘肽与Hb的结合过程及相互作用类型。
[Abstract]:Background and Purpose With the rapid development of modern medical image diagnosis and interventional therapy, the incidence of contrast-contrast nephropathy (CIN) is on the rise. The pathogenesis of CIN in the elderly diabetic population is not well defined, oxidative stress and signal transduction are the heat of the present study. Objective: To study the effect of contrast agent on nitric oxide (NO), nitric oxide synthase (NOS) and glutathione (GSH) in renal and renal tissues by making a model of old diabetic rats, and to study the role of NO, NOS and GSH in the pathogenesis of CIN. The type and structure of the chimera enzyme and the effect of the structure on the activity of NO synthesis were studied. In response to a change in renal function that may not be accurately reflected in the clinical work for elderly patients, the rate of glomerular filtration through the muscle or by the muscle tone may not accurately reflect changes in renal function, thereby delaying the diagnosis of the CIN In this paper, the expression of interleukin-18 and cystatin C in elderly patients with diabetes is studied, and the biological means of early, sensitive and specific prediction of CIN are found. The antioxidant has a certain effect on the prevention of CIN in high-risk people, taking into account the role of oxidative stress in the pathogenesis of CIN. The function of GSH as the center of the complex antioxidant network is to reflect the active oxygen, regulate the NO circulation and improve the control of the growth, development and defense of the cell oxidation-reduction signal delivery system Evaluation of the effect of reduced glutathione combined with normal saline on the intervention of CIN in elderly patients with diabetes The present paper also studies the mechanism of the binding process of GSH and hemoglobin in the blood, and further studies the mechanism of GSH. The research content of this paper mainly includes three parts: The first part: hypotonic contrast agent on the renal toxicity and nitric oxide synthase in the aged diabetic rats One, the material and the method of the study is to establish the model of the old diabetic rats, and observe the tail vein injection of Opike 350 (10 ml/ kg). The serum levels of NO, NOS and NO, NOS in the rat's blood muscle, the urine and the kidney tissue were detected. And the activity of NO synthesis of the nitric oxide synthase was studied, and the rate of reduction of iron cyanide and cytochrome C, and the oxidation of the emodin and NADPH were tested. reduction rate The results were as follows: The results showed that the blood myostatin and urea nitrogen in the old diabetic rats were higher than those in the normal control group (P 0.05), and the renal tissue homogenate NO, NOS, and GSH. Compared with the normal control group, the control group of the normal control group decreased significantly (P 0.01), the renal tissue homogenate NO and the NOS and the diabetes control group. Comparative decrease (P 0.05). nNOSeFNR nitric oxide synthase expression lower heme reduction rate, second part: interleukin-18, cystatin C, A study on the predictive effect of contrast-contrast nephropathy in patients was conducted in our hospital from January 2011 to October 2011 in our hospital for enhanced CT examination. A total of 30 patients with a age of more than 60 years, a history of diabetes more than 1 year, and no obvious renal function Low-permeability non-ionic contrast agents were used in the patients. The results of the ELISA method were 24 hours,48 hours IL-18 and blood urea nitrogen (BUN) before and after contrast. ), the blood muscle The results of SCr and CysC.2 showed no contrast-related nephropathy, and there was no significant difference in blood IL-18 after contrast (P0.05). IL-18 and cystatin C in 4-hour urine were significantly higher than those before contrast (P 0.05). SCr increased significantly after 48 hours (P 0.05). Part 3: Reduced glutathione for old-age diabetes Patient contrast nephropathy The study I, materials and methods of the impact and integration with the hemoglobin will be prepared to use hypotonic contrast agents for enhanced CT The scanned 96-bit diabetic patients (aged 60 years) were randomly divided into two groups. A group of intravenous reduced glutathione (1800 mg of reduced glutathione in 100 ml of saline prior to the scan, two days after the same dose of injection), and at the same time at 1 ml/ kg B The velocity of W/ h was injected intravenously (GSH group, n = 48); the other The group was given normal saline at the same speed (control group, n = 48). SCr, BUN, Cy SC and urine IL-18 were measured at 48 hours prior to and after contrast, respectively, and the muscle strength was higher than the baseline of 0.5 m. A 25% increase in g/ dL or greater than the baseline value of the muscle was confirmed to be CIN. Reduced glutathione was studied using a variety of spectrometry In the process of binding to Hb, the quenching mechanism of GSH on the hemoglobin was confirmed. The type of interaction force between the GSH and the hemoglobin is deduced by the parameters such as F st. The combined distance of GSH and hemoglobin can be obtained by the theory of energy transfer. molecular light The effect of the presence of GSH on the conformation of hemoglobin was studied in this paper. There was no significant difference between the baseline characteristics and the baseline renal function in the GSH group and the control group. Compared with the control group (2.08% vs. 8.33%, P = 0.364), SCr, BUN, and Cys in the two groups after enhanced CT scan C and urinary IL-18 both increased, and the estimated glomerular filtration rate (eGFR) was decreased. Two groups of SCr (P = 0.001), eGFR (P = 0.002), BUN (P 0.001), CysC (P = 0.001), CysC (P = 0.001), CysC (P = 0.001), CysC (P = 0.001), CysC (P = 0.001), CysC (P = 0.001), P = 0.001) and urine IL-18 (P 0.001 There is a significant difference. The increase of the concentration of GSH results in a gradual decrease of the fluorescence intensity of Hb. A series of thermodynamic parameters are obtained from the thermodynamic principle. b The process of action is a spontaneous over-reduction, entropy reduction, Gibbs free energy reduction, It can be concluded that the force between the GSH and the Hb is combined with the Van der Waals force. The change of the micro-environment around the amino acid is explained according to the red shift or the blue shift of the peak position, and the maximum emission wavelength has obvious red shift. like, Conclusion 1. The low-permeability contrast agent can be cited. The metabolism of NOS and NO and the transmission of signal may be related to the renal injury, NOS and NO in the old diabetic rats. 2. The type and structure of nitric oxide synthase have a significant effect on the synthesis of nitric oxide. Cys C is a sensitive index that can reflect the renal injury in an early stage, and plays an important role in the early diagnosis and control of CIN. . The hydration of reduced glutathione and physiological saline may be regulated by the regulation of oxidation and anti-oxidation.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

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