清热止血方、GTW通过干预肾小球系膜细胞、足细胞轴中TNF-α、nephrin治疗HSPN蛋白尿的体外研究

发布时间：2018-05-18 07:53

本文选题：紫癜性肾炎 + 清热止血颗粒　；参考：《河南中医学院》2013年硕士论文

【摘要】：【目的】本实验通过体外纯化HSPN（血尿蛋白尿型）患儿致病性IgA1作用于实验动物肾小球系膜细胞、足细胞损伤模型，比较清热止血颗粒、GTW、清热止血颗粒+GTW、强的松+肝素四组药物对aIgA1刺激后实验动物肾小球系膜细胞、足细胞损伤模型TNF-α、nephrin的分泌和表达的影响；了解清热止血方、GTW是否通过阻断系膜细胞－足细胞轴和（或）直接保护足细胞而在HSPN治疗中起到快速降蛋白尿疗效的作用；验证在清热止血方联合GTW应用过程中，清热止血方、GTW的作用孰轻孰重或是两药的协同作用。【方法】本实验通过制备清热止血颗粒、GTW、清热止血颗粒+GTW、强的松+肝素四组实验动物含药血清；将体外纯化的HSPN(血尿蛋白尿型）患儿致病IgA1作用于SD大鼠系膜细胞、SPF级小鼠永生化足细胞，制备HSPN系膜细胞、足细胞损伤模型；将各组药物含药血清干预系膜细胞、足细胞损伤模型。观察HSPN患者和健康对照者血清aIgA1对系膜细胞、足细胞的增殖影响；检测系膜细胞、足细胞损伤模型及药物干预后两种细胞损伤模型TNF-a分泌情况；运用RT-PCR检测足细胞损伤模型和含药血清干预后足细胞损伤模型的Nephrin mRNA表达量的变化。【结果】我们利用亲和层析联合分子筛层析的方法,提取了纯度较高的mIgA1,并对其进行了鉴定及热聚合，通过MTT法发现由HSPN患儿血清中mIgA1热聚合后的aIgA1可以引起实验动物系膜细胞和足细胞的损伤；通过ELISA法发现HSPN患儿aIgA1可以刺激系膜细胞及足细胞产生大量的TNF-α；通过清热止血方等四组药物干预HSPN系膜细胞、足细胞损伤模型，发现清热止血方+GTW在抑制系膜细胞增殖中效果明显，清热止血方+GTW、GTW、强的松+肝素三组含药血清抑制系膜细胞、足细胞产生TNF-α中较单用清热止血方效果明显；四组药物均可以上调足细胞损伤模型（HSPN患者IgA1-系膜细胞共培养液上清组、HSPN患者IgA1组）nephrin mRNA的表达，但以清热止血方+GTW、GTW、清热止血方三组效果突出，，强的松+肝素在上调足细胞损伤模型（HSPN患者IgA1组）nephrin mRNA的表达中较空白对照组差异无统计学意义。【结论】aIgA是HSPN肾小球系膜细胞、足细胞损伤的重要因素之一；肾小球系膜细胞异常增殖和系膜细胞、足细胞产生大量TNF-α是HSPN肾脏损伤的重要表现；清热止血方、清热止血方+GTW、GTW、强的松+肝素四组药物均可通过阻断系膜细胞-足细胞轴在HSPN（血尿+蛋白尿型）治疗中起到快速降低蛋白尿的作用；清热止血方+GTW、GTW、清热止血方在直接保护足细胞作用方面优于强的松+肝素，强的松+肝素在阻断系膜细胞-足细胞损伤轴方面优于单用清热止血方；清热止血方联合GTW应用过程中，在阻断系膜细胞-足细胞损伤轴中GTW的作用优于于清热止血方，在直接保护足细胞中二者效果相当，总体而言，二者联合应用优于单用一种。
[Abstract]:[objective] to study the effect of purified HSPN (hematuria proteinuria type) IgA1 on glomerular Mesangial cells and podocytes injury model in vitro. To compare the effects of Qingre Zhixue granule (GTW), Qingre Zhixue granule (GTW) and prednisone heparin (prednisone) on the secretion and expression of TNF- 伪 nephrin in glomerular Mesangial cells and podocytes of experimental animals after aIgA1 stimulation. To understand whether Qingre Zhixue prescription GTW plays a rapid role in reducing proteinuria in the treatment of HSPN by blocking Mesangial cell-podocyte axis and / or protecting podocytes directly, and validating the effect of Qingre Zhixue recipe combined with GTW. The action of GTW of clearing away heat and stopping bleeding is important or the synergistic effect of the two drugs. [methods] in this experiment, four groups of experimental animals containing serum of Qingre Zhixue granule (GTW), Qingre Zhixue granule (GTW) and prednisone heparin (prednisone) were prepared. The model of HSPN Mesangial cell and podocyte injury was established by treating the purified HSPN (hematuria proteinuria type) pathogenic IgA1 on rat Mesangial cells of SD rats in vitro, and the serum of each group was used to interfere with the Mesangial cells. Model of podocyte injury. The effects of serum aIgA1 on the proliferation of Mesangial cells and podocytes were observed in patients with HSPN and healthy controls, and the secretion of TNF-a in Mesangial cells, podocytes injury models and two kinds of cell injury models after drug intervention were detected. The changes of Nephrin mRNA expression in podocyte injury model and podocyte injury model were detected by RT-PCR. [results] the mIgA1 with high purity was extracted by affinity chromatography combined with molecular sieve chromatography, and its identification and thermal polymerization were carried out. By MTT method, it was found that the damage of Mesangial cells and podocytes of experimental animals could be caused by aIgA1 after hot polymerization of mIgA1 in serum of children with HSPN. It was found by ELISA method that aIgA1 could stimulate Mesangial cells and podocytes to produce a large amount of TNF- 伪 in children with HSPN. Four groups of drugs, such as Qingre Zhixue prescription, were used to interfere with HSPN Mesangial cells and podocyte injury model. It was found that Qingre Zhixue recipe (GTW) had obvious effect in inhibiting the proliferation of Mesangial cells. The effect of Qingre Zhixue recipe GTW and prednisone heparin on inhibiting Mesangial cells in three groups was more obvious than that of Qingre Zhixue recipe alone. All the four groups could upregulate the expression of nephrin mRNA in the IgA1 group of the patients with HSPN in the IgA1-Mesangial cell co-culture supernatant group, but the effect of the three groups was remarkable. There was no significant difference between prednisone heparin and control group in upregulating the expression of nephrin mRNA in IgA1 patients with podocyte injury. [conclusion] aIgA is one of the important factors of glomerular Mesangial cell and podocyte injury in HSPN. The abnormal proliferation of Mesangial cells and Mesangial cells in glomerular Mesangial cells and the production of a large amount of TNF- 伪 by podocytes are important manifestations of renal injury in HSPN. GTW and GTW, prednisone heparin could reduce proteinuria rapidly by blocking Mesangial cell-podocyte axis in the treatment of HSPN (hematuria proteinuria type). The effects of GTW and GTW on direct protection of podocytes were superior to those of prednisone heparin, and prednisone heparin was superior to Qingre Zhixue recipe in blocking the injury axis of Mesangial cell-podocyte. During the application of Qingre Zhixue recipe combined with GTW, the effect of GTW in blocking the injury axis of Mesangial cell-podocyte was better than that of Qingre Zhixue recipe, and the effect of both was the same in direct protection of podocyte.
【学位授予单位】：河南中医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R272

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