雷公藤多苷对贝伐珠单抗诱导的小鼠蛋白尿的影响及其相关机制的研究

发布时间：2018-05-18 18:25

本文选题：雷公藤多苷片 + 蛋白尿　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景在过去十年中,抗血管形成治疗已广泛应用于临床,贝伐珠单抗(bevacizumab,BEV)为新型抗血管内皮生长因子(vascular endothelial growth factor,VEGF)的人源化单克隆抗体,通过选择性与VEGF结合,阻止VEGF与其受体的结合,阻断下游信号通道,起到抗新生血管形成的作用,进而抑制肿瘤生长。蛋白尿是贝伐珠单抗不良反应之一,可导致抗血管形成治疗中断或延迟,严重蛋白尿患者则需要永久停止抗血管生成治疗,严重影响临床疗效。多项研究表明雷公藤多苷片(Tripterysium wilfordii polyglucoside,TWP)可以通过保护足细胞改善糖尿病肾病的蛋白尿,在中-重度蛋白尿的治疗中,发挥独特优势,具有作用时间短、起效速度快、持续有效等特点。本研究观察TWP对贝伐珠单抗诱导小鼠肾损伤的保护作用并探讨其作用机制。目的研究血管形成抑制剂之一贝伐珠单抗诱导小鼠蛋白尿的形成机制,探索雷公藤对贝伐珠单抗诱导的小鼠蛋白尿的干预作用及可能作用机制。方法30只健康清洁级小鼠分为5组:Control组、BEV组(贝伐珠单抗60mg/kg·w-1i.v)、BEV+TWP1(BEV 60mg/kg·w-1 i.v+TWP 4mg/kg/d-1)、BEV+TWP2(BEV60mg/kg·w-1 i.v+TWP 8mg/kg/d-1)、BEV+TWP3(BEV 60mg/kg·w-1 i.v+TWP16mg/kg/d-1),Control组给予尾静脉注射相同体积的生理盐水。4周末收集小鼠24-h尿液,检测尿蛋白总量;收集小鼠血液标本,检测血液中血肌酐(CREA)、尿素氮(BUN)、谷丙转氨酶(ALT)、谷草转氨酶(AST)等生化指标。最后处死小鼠,留取肾组织,HE染色方法检测肾组织病理变化,电镜方法检测足细胞超微结构,免疫组化、Western blot方法检测肾组织VEGF、podocin、nephrin蛋白表达,quantitative PCR方法检测VEGF m RNA、podocin m RNA、nephrin m RNA表达;结果BEV组24-h尿蛋白量显著高于Control组;与BEV组比较,BEV+TWP2、BEV-TWP3组24-h尿蛋白量明显减少,差异有统计学意义(P0.01);BEV+TWP1 24-h尿蛋白量无明显差异。5组AST、ALT、BUN、GREA无明显差异(P0.05)。肾组织病理变化:Control组小鼠肾组织结构正常;BEV+TWP1组未见明显病理改变;BEV组小鼠肾小球内皮细胞萎缩,呈空泡状结构改变;BEV+TWP2、BEV-TWP3组肾小球结构较BEV组明显改善。电镜下观察到Control组小鼠肾组织呈正常结构足细胞,而BEV组足细胞广泛融合;BEV+TWP2、BEV-TWP3组小鼠足细胞较BEV组有明显改善。免疫组化结果显示Control组、BEV+TWP2、BEV-TWP3组小鼠肾组织VEGF的表达呈中、强阳性,BEV组小鼠呈弱阳性或阴性;在VEGF、nephrin、podocin蛋白及m RNA表达量方面,BEV组较Control组显著下降,BEV+TWP2、BEV-TWP3较BEV组明显升高,差异有统计学意义(P0.01);BEV+TWP1组无明显差异(P0.05)。结论贝伐珠单抗下调VEGF、nephrin、podocin蛋白及m RNA表达,从而损伤肾小球滤过膜,导致蛋白尿。而雷公藤多苷片具有促进足细胞修复、减少蛋白尿的作用,其部分机制可能与提高VEGF、nephrin、podocin蛋白及m RNA表达量有关。
[Abstract]:Background in the past decade, anti-angiogenesis therapy has been widely used in clinical practice. Bevacizumab Bev) is a novel humanized monoclonal antibody against vascular endothelial growth factor (VEGF), which binds selectively to VEGF. Blocking the binding of VEGF to its receptors, blocking downstream signal channels, and inhibiting angiogenesis, thus inhibiting tumor growth. Proteinuria is one of the adverse reactions of bevacizumab, which can lead to the interruption or delay of anti-angiogenesis therapy, while the severe proteinuria patients need to stop anti-angiogenesis therapy permanently, which seriously affects the clinical efficacy. A number of studies have shown that Tripterysium wilfordii polyglucoside TWP) can improve proteinuria in diabetic nephropathy by protecting podocytes. Tripterysium wilfordii polyglucoside can play a unique role in the treatment of moderate to severe proteinuria. Sustained effectiveness and so on. The aim of this study was to observe the protective effect of TWP on the renal injury induced by bevacizumab in mice and to explore its mechanism. Objective to study the mechanism of mouse proteinuria induced by bevacizumab, one of the angiogenesis inhibitors, and to explore the effect of Tripterygium wilfordii on mouse proteinuria induced by bevacizumab. Methods Thirty healthy clean mice were divided into 5 groups: the control group (60mg/kg w-1i.v TWP) TWP1(BEV 60mg/kg w-1 i.v TWP 4mg / kg / d -1 TWP 8mg / kg TWP3(BEV 60mg/kg w-1 i.v TWP 8mg / kg TWP3(BEV 60mg/kg w-1 i.v TWP16mgkgkg-1 + control group. The urine samples were collected for 24-h at the end of the week by injecting the same volume of physiological saline into the caudal vein. The serum creatinine (creatinine), urea nitrogen (bun), alanine aminotransferase (alt) and aspartate aminotransferase (AST) in blood of mice were detected. Finally, the mice were killed, the pathological changes of renal tissue were detected by HE staining, the ultrastructure of podocin was detected by electron microscopy, and the expression of VEGFpodocin nephrin protein was detected by immunohistochemical Western blot method. The expression of VEGF m RNApodocin m RNA-nephrin RNA was detected by quantitative PCR method. Results compared with BEV group, the urine protein content of BEV group was significantly higher than that of Control group, and that of BEV WP2BEV-TWP3 group was significantly lower than that of BEV group, and there was no significant difference in 24 h urinary protein content between BEV group and BEV group (P 0.05). There were no obvious pathological changes in the glomerular endothelial cells of the mice in the control group, and the glomerular structure in the BEV TWP2BEV-TWP3 group was significantly improved than that in the BEV group. The normal structure of podocyte was observed in the kidney of Control group under electron microscope, while the podocyte in BEV group was significantly improved compared with that in BEV group, and the podocyte fusion of BEV TWP2BEV-TWP3 group was better than that of BEV group. The results of immunohistochemistry showed that the expression of VEGF in the kidney tissue of the Control group was slightly positive or negative, and the expression of VEGF nephrinpodocin protein and m RNA in the BEV group was significantly lower than that in the Control group, and the expression of TWP2BEV-TWP3 in the BEV group was significantly higher than that in the BEV group. There was no significant difference in P0.01BV TWP1 group (P 0.05). Conclusion bevacizumab can down-regulate the expression of VEGF nephrinpodocin protein and m RNA, which may damage the glomerular filtration membrane and lead to proteinuria. However, Tripterygium wilfordii polyglycoside can promote podocyte repair and reduce proteinuria, which may be related to the increase of VEGF nephrinpodocin protein and m RNA expression.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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