自噬保护被动Heymann肾炎大鼠足细胞的机制研究

发布时间：2018-05-17 20:30

本文选题：膜性肾病 + 足细胞　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分、PHN大鼠足细胞损伤和凋亡的研究目的制备膜性肾病经典的动物模型被动Heymann肾炎(PHN),观察疾病进程中足细胞的损伤,探讨是否存在足细胞的凋亡。方法采用1月龄SPF级的SD雄性大鼠通过静脉注射兔抗大鼠FX1A抗血清建立大鼠PHN模型并随机分为4组:分别在模型建立后的第2、4、7、21天时处死大鼠并取材,每组8只;同时设立对照组,进行同样的处理。临床生化分析仪检测大鼠的血肌酐、尿素氮以及24小时尿蛋白总量;病理染色和透射电镜观察PHN模型鼠疾病进程中的病理改变和足细胞的损伤;免疫组织化学染色检测PHN大鼠肾小球内C5b-9随时间进展的沉积;足细胞标志蛋白WT1免疫组化染色标记足细胞并通过Weibel-Gomez方法计数单个肾小球足细胞数量,评估足细胞出现明显缺失的时间点;活性caspase3的免疫组化染色和tunel染色评估PHN大鼠肾小球足细胞的凋亡。结果(1)造模后第2天即可观察到C5b-9在肾小球基底膜部分阶段沉积,第4天起沿基底膜呈线状沉积,并随疾病进展沉积逐渐增多;PASM染色显示随疾病进展肾小球基底膜逐渐增厚至21天时呈现明显的局灶性阶段性增厚;透射电镜观察显示逐渐出现上皮下颗粒状电子致密物的沉积及足突融合,至第21天可见典型膜性肾病改变;尿蛋白定量结果显示,造模第3天起尿蛋白排出量即有明显升高,至第21天时可高达50.6±6.0mg/24h; (2)造模后第21天时观察到单位肾小球足细胞绝对数目的显著降低(vs对照组,p0.05); (3)活性Caspase-3和TUNEL染色结果一致显示造模后第21天肾小球内可检出凋亡的足细胞。结论成功制备了膜性肾病的动物模型PHN,观察了膜性肾病典型的病理改变进程,足细胞损伤是其病变的核心环节,在造模后第21天时观察到足细胞的凋亡。第二部分、调控自噬对PHN大鼠足细胞损伤的影响目的 (1)观察不同时间点足细胞自噬激活的改变;(2)通过自噬增强剂雷帕霉素和抑制剂3-甲基腺嘌呤(3-MA)调控自噬,探讨调控足细胞自噬对PHN疾病进展和足细胞损伤的影响。方法 (1)制备PHN模型鼠,并在2、4、7、21天时分别处死;(2)制备PHN模型鼠,随机分为三组,分别给予生理盐水、雷帕霉素溶剂、3-MA溶剂干预,同时设立空白对照组和DMSO溶剂对照组,21天时处死大鼠并取材。透射电镜观察足细胞自噬泡数量的改变,western blot检测自噬标记蛋白的改变;全自动生化分析仪测定24h尿蛋白定量、血清BUN及Cr;免疫荧光染色检测肾小球内C5b-9的沉积;PASM染色和透射电镜观察肾脏病变和足细胞损伤;Weibel-Gomez方法计数单位肾小球足细胞数量;活性caspase-3的免疫组化染色观察凋亡的足细胞,western blot半定量分析肾小球内细胞的凋亡。结果 (1) PHN疾病进程中伴随着足细胞自噬的激活,自噬增强剂雷帕霉素和抑制剂3-MA可以起到调控足细胞自噬的作用。(2)调控自噬不会影响PHN模型鼠的损伤因素C5b-9在肾小球的沉积。(3)雷帕霉素增强自噬可以改善PHN大鼠肾小球基底膜增厚的程度和范围,减少肾小球固有细胞尤其是足细胞的凋亡,改善PHN大鼠足细胞的缺失,进而明显减轻PHN模型鼠蛋白尿的排出。(4) 3-MA抑制自噬明显加重PHN大鼠肾小球基底膜增厚的程度和范围,加重足细胞的损伤,足细胞凋亡明显增加,足细胞缺失更加明显,蛋白尿排除明显增加。结论在PHN的病变过程中,自噬发挥着保护作用,适度增强自噬可以减少足细胞的凋亡,进而减轻肾脏病变,缓解蛋白尿;相反,当自噬被抑制后,足细胞损伤加重,进而加重肾脏病变和蛋白尿程度,自噬可能是足细胞对抗补体介导的细胞损伤的机制之一。第三部分、自噬保护PHN大鼠足细胞的机制研究目的 (1)观察PHN疾病进程中足细胞内质网(ER)的损伤和内质网应激(ERS)激活的变化过程,推测其和足细胞凋亡及自噬的关系;(2)观察增强或者抑制自噬后ER损伤的改变及ERS信号分子的变化,探讨自噬减少PHN大鼠足细胞凋亡的可能机制。方法透射电镜观察足细胞ER的损伤;免疫印迹检测ERS相关蛋白的表达。结果 (1)足细胞的ER在PHN疾病进程中损伤逐渐加重,表现为内质网腔扩张、核糖体脱落等;(2) ERS相关蛋白在PHN疾病早期(第2天)表达上调,早于LC3表达上调的时间;(3)增强足细胞的自噬水平后ER损伤明显缓解并且ERS相关蛋白表达下调,足细胞自噬被抑制后ER的损伤明显加重同时ERS相关蛋白表达上调。结论自噬发挥减缓PHN疾病进程并减轻足细胞损伤的作用可能是通过缓解足细胞内质网应激的强度和持续的时间实现的,进而调控内质网应激的生存前信号和凋亡前信号通路,减少足细胞凋亡。调节“内质网应激一自噬”信号途径可能是治疗膜性肾病的新途径。
[Abstract]:The first part, the study of PHN rat foot cell injury and apoptosis to prepare the classic animal model of membranous nephropathy, passive Heymann nephritis (PHN), observe the injury of foot cells in the course of disease, and explore the existence of the apoptosis of podocyte. Methods 1 month old SPF SD male rats were used to establish rats by intravenous anti rat FX1A antiserum. The PHN model was randomly divided into 4 groups: the rats were killed and selected at the 2,4,7,21 day after the model establishment, and 8 rats in each group were selected. At the same time, the control group was set up to do the same treatment. The serum creatinine, urea nitrogen and the total amount of urine protein in the 24 hours were detected by the clinical biochemical analyzer. The pathological staining and transmission electron microscopy were used to observe the disease process of the PHN model rats. Pathological changes and injury of podoni; immunohistochemical staining was used to detect the deposition of C5b-9 in the glomeruli of PHN rats; podocyte WT1 immunohistochemical staining was used to mark the podocyte and count the number of individual glomerular podocytes by Weibel-Gomez method. Immunohistochemical staining and TUNEL staining were used to evaluate the apoptosis of glomerular podocytes in PHN rats. Results (1) the deposition of C5b-9 in the part of the glomerular basement membrane was observed on the second day after modeling, and the basement membrane was linearly deposited on the fourth day and gradually increased with the progression of the disease. PASM staining showed that the glomerular basement membrane gradually thickened with the disease progression. 21 days showed a significant focal stage thickening, and the transmission electron microscopy showed that the deposition of granular electronic dense substance and the fusion of the podocyte process gradually appeared on the twenty-first day, and the urine protein quantitative results showed that the excretion of proteinuria was significantly higher in third days and up to 50.6 + 6.0mg to twenty-first days. /24h; (2) the absolute number of glomerular podocytes was significantly reduced at twenty-first days after modeling (vs control group, P0.05), and (3) active Caspase-3 and TUNEL staining results showed that the apoptotic podonus could be detected in the glomeruli after twenty-first days of modeling. Conclusion the animal model of membranous nephropathy was successfully prepared in animal model, and the typical membranous nephropathy was observed. Histopathological process, podocyte injury is the core of the disease. The apoptosis of podropodal was observed twenty-first days after the model. The second part was to regulate the effect of autophagy on the foot cell injury in PHN rats (1) to observe the changes of autophagy at different time points, and (2) the autophagic enhancer rapamycin and the inhibitor 3- methyl adenine. Methotrexate (3-MA) regulates autophagy and regulates the effect of autophagy on the progression of PHN disease and podocyte injury. Methods (1) PHN model rats were prepared and killed at 2,4,7,21 days. (2) PHN model rats were prepared and randomly divided into three groups, which were given saline, rapamycin solvent and 3-MA solvent, and blank control group and DMSO solvent were set up at the same time. In the control group, rats were killed and selected at 21 days. The changes in the number of autophagic vesicles were observed by transmission electron microscopy. Western blot was used to detect the changes in the autophagic markers. The automatic biochemical analyzer was used to determine the quantity of 24h urine protein, the serum BUN and Cr; the immunofluorescence staining was used to detect the deposition of C5b-9 in the glomeruli; PASM staining and transmission electron microscopy were used to observe the renal lesions and the renal pathological changes. Foot cell injury; Weibel-Gomez method counts the number of unit glomerular podocytes; active caspase-3 immunohistochemical staining observation of apoptotic podocytes, Western blot semi quantitative analysis of the apoptosis of glomerular cells. Results (1) the process of PHN disease accompanied by the activation of autophagy, autophagic enhancer rapamycin and inhibitor 3-MA Play the role of regulating autophagy of podropodal. (2) Regulation of autophagy does not affect the damage factor of PHN model rats C5b-9 in the glomerular deposition. (3) the enhancement of autophagy by rapamycin can improve the extent and scope of the thickening of the glomerular basement membrane in PHN rats, reduce the apoptosis of the glomerular innate cells, especially podropodal, and improve the deletion of the podroblast in the PHN rats. And then obviously alleviated the excretion of proteinuria in the PHN model rats. (4) the inhibition of autophagy by 3-MA significantly aggravated the extent and scope of the thickening of the glomerular basement membrane in PHN rats, aggravated the injury of the Poddar cells, the apoptosis of the Poddar increased obviously, the deletion of podocytes was more obvious, and the exclusion of proteinuria increased obviously. Conclusion autophagy plays a protective role in the process of PHN disease. Appropriate enhancement of autophagy can reduce the apoptosis of the poddate, and then reduce the renal disease and alleviate the proteinuria. On the contrary, when the autophagy is suppressed, the injury of the Poda is aggravated, and then the renal disease and the degree of proteinuria are aggravated. Autophagy may be one of the mechanisms of the cell damage mediated by the podocyte. The third part, autophagy protects the PHN rat foot. The mechanism of cell study (1) observe the changes in the injury of the endoplasmic reticulum (ER) and the activation of endoplasmic reticulum stress (ERS) during the process of PHN disease, speculate on the relationship with the apoptosis of podocytes and autophagy, and (2) to observe the changes of ER damage and the change of ERS signal molecules after autophagy, and to explore the autophagy to reduce the foot cell withering in PHN rats. The possible mechanism of ER was observed by transmission electron microscopy. The expression of ERS related proteins was detected by Western blot. Results (1) the ER of podocytes increased gradually in the process of PHN disease, manifested as endoplasmic reticulum dilation, ribosome exfoliation and so on. (2) the expression of ERS related protein was up regulation in the early stage of PHN disease (second days), earlier than LC3 expression. (3) (3) after the enhancement of autophagy, the damage of the ERS related protein was obviously reduced and the expression of ERS related protein was down. The damage of ER was obviously aggravated and the expression of ERS related protein was up regulated after the autophagy was inhibited. It is possible to regulate the signal pathway of the pre subsistence signal and pre apoptotic signal pathway for endoplasmic reticulum stress and reduce the apoptosis of the Poda. The regulation of "endoplasmic reticulum stress a autophagy" signal pathway may be a new approach to the treatment of membranous nephropathy.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R726.9

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