ATRQβ-001疫苗改善糖尿病肾损伤及作用机制研究

发布时间：2018-05-24 05:34

本文选题：ATRQβ-001疫苗 + 糖尿病肾病　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分ATRQβ-001疫苗对STZ诱导的糖尿病肾病大鼠有效性研究目的：前期工作中我们研制出一种针对人血管紧张素Ⅱ受体1型的短肽治疗性疫苗ATRQβ-001,本研究旨在验证ATRQβ-001疫苗在糖尿病肾病模型老鼠上是否有效,以链脲佐菌素(STZ)腹腔注射方法建立糖尿病肾病模型。方法：将ATR-001短肽与Qp-2aa病毒样颗粒(VLP)用化学耦联剂Sulfo-SMCC耦联制备ATRQβ-001疫苗。Sprague Dawley大鼠随机分为对照组和糖尿病组,糖尿病组用链脲佐菌素(60mg/kg)腹腔注射,1周后,模型组又随机分为4组,每组15只老鼠：(1)糖尿病肾病(DN)组：等剂量皮下注射生理盐水干预；(2)奥美沙坦(OM)组：按5mg/kg/d给予灌胃；(3) ATRQP-001组：将ATRQβ-001疫苗与氢氧化铝佐剂乳化后,按400μg/只于第0、14、21天皮下多点免疫；(4) Qβ VLP (VLP)组：将QβVLP与氢氧化铝佐剂乳化,按4001μg/只于第0、14、21天皮下多点免疫。干预后继续观察14周。所有大鼠每周称量体重,监测血糖,每两周测定ATR-001特异性抗体滴度,每四周测定血压,于第4、8、14周代谢笼测定老鼠尿量及24小时尿蛋白量。饲养结束后,检测血肌酐、尿素氮水平和肾脏病理结构改变。结果：ATRQP-001疫苗可产生较高滴度抗ATR-001抗体,并可有效降低糖尿病老鼠的血压水平,最高降幅达19.4mmHg。疫苗免疫后不影响血糖血脂水平,但可降低24小时尿蛋白量、血肌酐、尿素氮水平,并减轻肾体比,逆转nephrin、podocin表达下降,减轻足细胞损伤和系膜扩张,且与奥美沙坦治疗组无明显统计学差异。结论：ATRQβ-001疫苗可有效改善链脲佐菌素(STZ)诱导的糖尿病肾病模型大鼠的肾功能和血压水平,并减轻肾脏病理损害,与奥美沙坦治疗组无明显差异。第二部分ATRQP-001疫苗肾脏保护机制研究目的：本研究旨在探讨ATRQβ-001疫苗的作用机制,主要从炎症纤维化和对RAS系统的调控两方面研究,并在体外用抗ATR-001抗体在大鼠肾脏系膜细胞系上进一步评价。方法：检测血浆中肾素活性(PRA)、血管紧张素(Ang)Ⅱ、Ang (1-7)、肾脏皮质AngⅡ和Ang(1-7)含量,其中PRA和Ang Ⅱ用放射免疫分析方法检测,Ang(1-7)用高压液相色谱技术检测。Masson三色染色观察肾脏纤维化,免疫组化检测肾脏TGF-β1表达和单核巨噬细胞浸润情况,蛋白免疫印迹(western blot)检测RAS相关蛋白表达和ERK1/2、p38 MAPK磷酸化水平,实时定量聚合酶链反应(qRT-PCR)检测RAS成分、促炎细胞因子和促纤维化生长因子mRNA表达水平。体外实验评估抗ATR-001抗体(Anti-ATR-001)对高糖诱导的大鼠肾脏系膜细胞影响,western blot检测TGF-β1和磷酸化ERK、Smad3水平,qRT-PCR检测TGF-β1、胶原蛋白(Collagen)Ⅳ和纤连蛋白(Fibronectin)表达。结果：与奥美沙坦相比,ATRQβ-001疫苗并不增加循环中PRA和Ang Ⅱ浓度,二者均可降低肾脏组织中下调肾脏Ang Ⅱ浓度,并增加Ang(1-7)含量,抑制肾脏局部ACE-AngⅡ-ATIR轴表达,上调ACE2-Ang (1-7)-mas轴。ATRQβ-001疫苗可有效减轻肾脏间质纤维化水平,并减少肾小球和间质巨噬细胞浸润数目,TGF-β1的表达和活性氧产生亦明显减少。肾脏组织炎性细胞因子和促纤维生长因子mRNA表达也在经ATRQp-001疫苗和奥美沙坦治疗后有所下降,且与两组之间无统计学差异。体外实验中Anti-ATR-001可有效抑制高糖刺激下肾脏系膜细胞(RMCs) TGF-β1表达和Smad3磷酸化水平,其下游Collagen IV和Fibronectin的表达亦下调结论：ATRQβ-001疫苗主要通过调控RAS双轴和抑制炎症纤维化两方面起到肾脏保护作用。第三部分ATRQβ-001疫苗的安全性研究目的：ATRQβ-001疫苗可减轻STZ诱导的糖尿病肾病模型大鼠肾脏病理损害,将进一步在Sprague Dawley (SD)大鼠上观察疫苗的安全性。方法：将ATR-001短肽与Qβ-2aa病毒样颗粒(VLP)用化学耦联剂Sulfo-SMCC耦联制备ATRQp-001疫苗。Sprague Dawley大鼠随机分为对照组和ATRQβ-001疫苗组,每组10只老鼠,疫苗组按400μg/只于第0、14、21天皮下多点免疫,对照组给予等剂量皮下注射生理盐水。每两周测定抗ATR-001特异性抗体滴度,每四周测定血压。在长达150天的观察期后,ELISA方法检测血清中C3a和C5a含量,并取心脏、肺组织、肾脏、肝脏、脾脏,进行HE染色和CD14、CD19免疫组化染色,观察各组织脏器有无明显炎症反应及炎性细胞浸润情况,取肾脏组织再进行Masson三色、PAS染色和电镜检测,确定有无免疫介导的肾脏病理损害。结果：ATRQβ-001疫苗可产生较高滴度抗ATR-001抗体,对正常老鼠血压水平无影响。血清中C3a和C5a含量与对照组无明显差异。HE染色显示疫苗组各组织未见明显炎症反应,CD14、CD19免疫组化染色亦未见单核细胞和B细胞浸润,肾脏组织未见系膜扩张、间质纤维化等病变,电镜下足突无融合消失,肾小球系膜区无增生,基底膜无增厚,且未见明显免疫复合物沉积。结论：ATRQβ-001疫苗不降低正常老鼠血压,未产生明显免疫介导的组织病理损害,安全性良好。
[Abstract]:The first part of the study on the efficacy of ATRQ beta -001 vaccine on STZ induced diabetic nephropathy rats: We developed a short peptide therapeutic vaccine against the human angiotensin II receptor 1 (ATRQ beta -001) in the early work. The purpose of this study was to verify whether the ATRQ beta -001 vaccine was effective in diabetic nephropathy model mice, with streptozotocin (S). TZ) the diabetic nephropathy model was established by intraperitoneal injection. Methods: the ATRQ beta -001 vaccine.Sprague Dawley rats were randomly divided into the control group and the diabetes group by the coupling of the ATR-001 short peptide and Qp-2aa virus like particles (VLP) with the chemical coupling agent Sulfo-SMCC, and the diabetic group was intraperitoneally injected with streptozotocin (60mg/kg). After 1 weeks, the model group was randomly divided into two groups. 4 groups, 15 rats in each group: (1) diabetic nephropathy (DN) group: equal dose of subcutaneous injection of saline; (2) O Mei Chatain (OM) group: 5mg/kg/d was given to the stomach; (3) group ATRQP-001: after the emulsification of ATRQ beta -001 vaccine and aluminum hydroxide adjuvant, subcutaneous immunization with 400 mu g/ only on 0,14,21 days; (4) Q beta VLP (VLP) group: Q beta P was emulsified with Al2O3 adjuvant and subcutaneous immunization at 4001 u g/ only on day 0,14,21. After intervention for 14 weeks, all rats were weighed and monitored every week, blood sugar was monitored, the titer of ATR-001 specific antibody was measured every two weeks, blood pressure was measured every week, and the urine volume and protein amount of 24 hours in the 4,8,14 Zhou Dai cage were measured. After the end of feeding, The serum creatinine, urea nitrogen level and renal pathological structure were detected. Results: ATRQP-001 vaccine can produce high titer and anti ATR-001 antibody, and can effectively reduce the blood pressure level of diabetic mice. The maximum decrease of 19.4mmHg. vaccine does not affect blood glucose and blood lipid level, but it can lower the urine protein, serum creatinine and urea nitrogen level by 24 hours. Reducing renal body ratio, reversing nephrin, podocin expression, alleviating foot cell injury and mesangial dilatation, and no significant difference with olmesartan treatment group. Conclusion: ATRQ beta -001 vaccine can effectively improve the renal function and blood pressure level of streptozotocin (STZ) induced diabetic nephropathy model rats, and reduce renal pathological damage, and Austria There is no significant difference in the treatment group of the mesarartan. Second part of the study of the renal protection mechanism of ATRQP-001 vaccine: the purpose of this study is to explore the mechanism of the ATRQ beta -001 vaccine, mainly from two aspects of inflammatory fibrosis and the regulation of the RAS system, and the further evaluation of the anti ATR-001 antibody in the mesangial cell line of the rat kidney in vitro. The contents of renin activity (PRA), angiotensin (Ang) II, Ang (1-7), renal cortex Ang II and Ang (1-7) were detected, of which PRA and Ang II were detected by radioimmunoassay. Ang (1-7) was detected by high pressure liquid chromatography with.Masson trichromatic staining to observe renal fibrosis and immunohistochemical detection of renal TGF- beta 1 and mononuclear macrophages Infiltration, protein immunoblotting (Western blot) detection of RAS related protein expression and ERK1/2, p38 MAPK phosphorylation level, real-time quantitative polymerase chain reaction (qRT-PCR) detection of RAS components, proinflammatory cytokines and fibrotic growth factor mRNA expression level. In vitro test and evaluation of anti ATR-001 antibody (Anti-ATR-001) on high glucose induced rat kidney Western blot detected TGF- beta 1 and phosphorylated ERK, Smad3 level, qRT-PCR detection of TGF- beta 1, collagen (Collagen) IV and fibronectin (Fibronectin) expression. Results: compared with olmesartan, ATRQ beta -001 vaccine did not increase the concentration of PRA in the circulation, and the two could lower the kidneys in the kidneys. Degree, and increase the content of Ang (1-7), inhibit the expression of local ACE-Ang II -ATIR axis of the kidney, up the ACE2-Ang (1-7) -mas axis.ATRQ beta -001 vaccine can effectively reduce the level of renal interstitial fibrosis, and reduce the number of glomerular and interstitial macrophage infiltration, the expression of TGF- beta 1 and the production of reactive oxygen species are also significantly reduced. The expression of VGF mRNA was also decreased after ATRQp-001 vaccine and olmesartan, and there was no statistical difference between the two groups. In vitro, Anti-ATR-001 could effectively inhibit the expression of TGF- beta 1 and Smad3 phosphorylation in renal mesangial cells (RMCs) under high glucose stimulation, and the expression of Collagen IV and Fibronectin downstream in the downstream was also down regulated. The ATRQ beta -001 vaccine plays the role of renal protection mainly through two aspects of regulating RAS biaxe and inhibiting inflammatory fibrosis. The safety of the third part of the ATRQ beta -001 vaccine: the ATRQ beta -001 vaccine can reduce the renal pathological damage in the diabetic nephropathy model rats induced by STZ, and will observe the epidemic in Sprague Dawley (SD) rats. Method: the ATRQp-001 vaccine.Sprague Dawley rats were randomly divided into the control group and the ATRQ beta -001 vaccine group by coupling the ATR-001 short peptide and the Q beta -2aa virus like granule (VLP) with the chemical coupling agent Sulfo-SMCC, each group of 10 mice in each group. The vaccine group was immunized at 400 mu g/ only on the second day of the 0,14,21, and the control group was given the equal dose of the subcutaneous injection. The anti ATR-001 specific antibody titer was measured every two weeks and blood pressure was measured every week. After the observation period of 150 days, the content of C3a and C5a in the serum was detected by ELISA method, and the heart, lung tissue, kidney, liver, spleen, HE staining and CD14, CD19 immunohistochemical staining were used to observe the obvious inflammatory reaction and inflammation in the organs and organs. Masson trichrome, PAS staining and electron microscopy were used to determine the renal pathological damage of renal tissue. Results: the ATRQ beta -001 vaccine could produce high titer and anti ATR-001 antibody and had no effect on the blood pressure level of normal mice. There was no significant difference in serum C3a and C5a content from the control group and the.HE staining showed that the serum content of C3a and C5a in the serum was not significantly different from that of the control group. No obvious inflammatory reaction was found in the tissue of the group. No mononuclear and B cell infiltration was found in CD14, CD19 immunohistochemical staining, no mesangial dilatation, interstitial fibrosis, no fusion disappeared, no hyperplasia of the glomerular mesangial region, no thickening of the basement membrane and no obvious immune complex deposition. Conclusion: ATRQ beta -001 pestilence The seedlings did not reduce the blood pressure of normal mice, and did not produce obvious immune mediated histopathological damage.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R692.9

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