益气养阴法对IgA肾病大鼠AT1和Ⅳ型胶原的影响

发布时间：2018-02-28 03:08

本文关键词： IgA肾病 RAS系统血管紧张素Ⅱ（AngⅡ）血管紧张素ⅡⅠ型受体（AT1） Ⅳ型胶原肾小球硬化　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：IgA肾病（IgAN）是世界范围内常见的原发性肾小球疾病，近年来研究发现15%～20%的患者在确诊后10年内、30%～35%的患者在确诊后20年内进入终末期肾衰竭,严重威胁着患者的身心健康。IgAN在我国发病率较高，其临床表现多样，以反复发作性血尿为主要特点，可伴有蛋白尿、高血压等。IgAN的诊断目前主要是依靠病理检查并结合血尿、蛋白尿等临床表现，其病理特征主要包括肾小球系膜区多聚IgA1分子伴少量C3、IgM、IgG沉积及系膜细胞增殖、系膜基质的增多。关于其发病机制仍然知之甚少，许多学者认为其是多种因素综合作用的结果。近年来，细胞因子的作用引起人们的关注，研究发现肾脏局部的RAS系统激活，可以刺激系膜细胞增殖、系膜基质增宽及细胞外基质的合成，在IgAN的进展过程中发挥重要作用。目前IgAN的治疗仅限于对症支持及免疫抑制等治疗，因此发现有效的特异性治疗方案成为研究热点。因此我们提出“益气养阴法”治疗IgAN，应用于临床上后取得了不错的治疗效果，但其具体的治疗作用机制仍然不是很清楚。本文首先建立IgAN动物模型，应用益气养阴中药按照相应的药物剂量进行干预治疗。通过观察大鼠24小时尿蛋白定量、尿红细胞计数、肾功能等，同时检测血清中AngⅡ的水平及肾组织AT1、Ⅳ型胶原的表达情况，旨在探讨益气养阴中药治疗IgA肾病的作用机制及药物作用靶点。方法：40只雄性清洁级SD大鼠被选入实验。我们将这40只大鼠随机分成正常对照组10只、模型组10只、贝那普利组10只、中药组10只。除正常对照组外的后三组均运用BSA、脂多糖、蓖麻油、四氯化碳复制IgAN大鼠动物模型。具体方法如下：将免疫原BSA用蒸馏水配制成100g/L浓度，隔天灌胃，剂量为400mg/kg，持续8周；每周一次皮下注射四氯化碳0.1ml、蓖麻油0.3ml，连续9周；并于第六周尾静脉注射脂多糖0.05mg。正常对照组给予相同剂量的蒸馏水隔日灌胃8周；每周一次皮下注射生理盐水0.4ml；第六周尾静脉注射生理盐水0.2ml。然后从第10周起，贝那普利组、中药组分别予以盐酸贝那普利、中药灌胃，，正常对照组、模型组予以等量生理盐水灌胃，持续8周。实验过程中，我们将大鼠置于代谢笼内分别于实验的各个时间点留取尿液标本，测定尿红细胞计数及24小时尿蛋白定量。实验第17周末，麻醉动物，留取血液标本，迅速采集大鼠肾脏标本。血液标本离心后用半自动生化仪测定大鼠肾功能及放射免疫法测定血清中AngⅡ的水平。最后，于光镜、电镜下观察大鼠肾脏组织病理形态学改变，免疫荧光法检测大鼠肾脏IgA免疫复合物沉积，免疫组织化学技术检测Ⅳ型胶原的表达情况，RT-PCR技术检测AT1的基因表达水平。结果: 模型组、贝那普利组、中药组大鼠ATI基因表达较正常组明显升高（P0.01），贝那普利组、中药组大鼠这个指标的表达较模型组有所降低（P0.01），贝那普利组、中药组间无明显区别。正常组大鼠Ⅳ型胶原主要在肾小球及肾小管基底膜表达，系膜区、肾间质几乎不表达；模型组、贝那普利组、中药组大鼠较正常组Ⅳ型胶原的表达明显增多，可见其在系膜区表达明显升高；贝那普利组、中药组较模型组表达较模型组降低，两者之间无明显差异。结论: 1益气养阴中药可以降低IgA肾病大鼠尿蛋白及尿红细胞计数，从而保护肾功能； 2益气养阴中药可以显著改善IgA肾病大鼠肾小球上皮细胞足突的融合、系膜细胞的增生及系膜基质的增宽等病理损害； 3模型组大鼠血清中AngⅡ水平、肾脏局部AT1mRNA的表达明显增加，提示肾脏局部RAS系统的激活在IgAN的进展中起重要作用。益气养阴中药可能是通过阻断这一途径而达到对IgA肾病大鼠治疗作用的； 4IgA肾病大鼠肾组织AT1mRNA的表达与Ⅳ型胶原的合成呈正相关，提示RAS系统的激活可以促进细胞外基质的合成。益气养阴中药可以显著抑制这一作用，延缓疾病的进展。
[Abstract]:Objective: IgA nephropathy (IgAN) is a worldwide common primary glomerular disease, recent studies have found that 15% to 20% of patients in the 10 years after diagnosis, 30% ~ 35% of the patients in the 20 years after diagnosis in end-stage renal failure, a serious threat to physical and mental health of patients with.IgAN incidence in our country high rate of clinical manifestations with recurrent hematuria as the main character, may be associated with proteinuria, hypertension and other.IgAN diagnosis mainly depends on pathological examination combined with hematuria, proteinuria, the major pathological features including mesangial poly IgA1 molecules with a small amount of C3, IgM, IgG, proliferation deposition and mesangial cells, increased mesangial matrix. The pathogenesis is still poorly understood, many scholars believe that it is the result of many factors. In recent years, the role of cytokines to cause the attention of people, the study found that kidney Bureau The activation of RAS system, can stimulate the proliferation of mesangial cells and mesangial matrix synthesis and broadening of the extracellular matrix, play an important role in the progression of IgAN. The treatment of IgAN is limited to symptomatic and immunosuppressive therapy, so that specific therapy effective scheme so we become a research hotspot. Put forward the "supplementing Qi and nourishing Yin" in the treatment of IgAN, the application has achieved good curative effect in clinic, but the treatment mechanism is still not very clear.
In this paper, we establish the IgAN animal model, application of Tonifying Qi and nourishing Yin were treated according to the drug dosage accordingly. Through the observation of the rat 24 hour urinary protein, urine red blood cell count, renal function, and serum levels of Ang II and renal tissue to detect AT1 expression of type IV collagen, mechanism and medicine the target to explore Yiqiyangyin medicine for the treatment of IgA nephropathy.
Methods: 40 male SD rats were selected in the experiment. The 40 rats were randomly divided into normal control group 10 rats, 10 rats in model group, Benner Pury group 10, Chinese medicine group 10. Except the normal control group after three groups using BSA, lipopolysaccharide, castor oil, preparation IgAN rat animal model of complex carbon tetrachloride. The method is as follows: the immunogenicity of BSA diluted in distilled water 100g/L concentration, the intragastric dose of 400mg/kg for 8 weeks; weekly subcutaneous injection of carbon tetrachloride 0.1ml, castor oil, 0.3ml, for 9 weeks; and the control group was given the same dose of distilled water by gavage every day 8 weeks of normal at sixth weeks of intravenous injection of lipopolysaccharide 0.05mg.; weekly subcutaneous injection of saline 0.4ml; sixth weeks of intravenous injection of saline 0.2ml. and then from the tenth week, Benner Pury group, Chinese medicine group were given intragastric administration of Benner Pury hydrochloride, Chinese medicine, normal control Group and model group were given normal saline, lasted 8 weeks. During the experiment, we the rats were placed in metabolism cages at the each time point of urine samples, determination of urine red blood cell count and urine protein in 24 hours. At the end of the seventeenth week experiment, anesthesia animal blood samples rapidly collecting samples of rat kidney. The level of blood serum Ang determination of renal function and radioimmunoassay for rat samples after centrifugation by semi-automatic biochemical analyzer. Finally, in addition, the pathological changes of kidney tissue were observed under electron microscope, detection of renal IgA in rats immune complex deposition immunofluorescence, expression of IV type collagen immunohistochemistry, detect the expression of AT1 RT-PCR gene.
Result:
The expression of ATI gene in model group, Benner Pury group and Chinese medicine group was significantly higher than that in the normal group (P0.01). The expression of this index in the Benner Pury group and the traditional Chinese medicine group was lower than that in the model group (P0.01), and there was no significant difference between the Benner Pury group and the Chinese medicine group.
The rats in the normal group of type IV collagen is mainly expressed in renal tubular and glomerular basement membrane and mesangial region, renal interstitial almost no expression; model group, Benner Pury group, the expression of type IV collagen than normal group rats increased significantly, the expression was significantly increased in the mesangium; benazepril group. The Chinese medicine group than in the model group expressed lower than model group, no significant difference between the two.
Conclusion:
1 supplementing qi and nourishing Yin can reduce the urine protein and urine red blood cell count of IgA nephropathy rats, and protect the renal function.
2, Chinese herbs for supplementing qi and nourishing Yin can significantly improve the fusion of glomerular epithelial cells, foot processes, proliferation of mesangial cells and widening of mesangial matrix in IgA nephropathy rats.
3, the level of Ang II in serum and the expression of AT1mRNA in kidney of rats in model group increased significantly, suggesting that activation of RAS system in kidney plays an important role in the progress of IgAN. Yiqi Yangyin herbs may play a role in the treatment of IgA nephropathy by blocking this pathway.
The expression of AT1mRNA in renal tissue of 4IgA nephropathy rats is positively correlated with the synthesis of collagen type IV, suggesting that activation of RAS system can promote extracellular matrix synthesis. Yiqi Yangyin herbs can significantly inhibit this effect and delay the progression of disease.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.3

【引证文献】