双重因素sC5b-9和HBx对裂孔膜蛋白在足细胞表达的影响

发布时间：2018-03-21 22:10

本文选题：足细胞　切入点：乙型肝炎病毒X蛋白　出处：《华中科技大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的肾小球足细胞是一种高度分化的终末细胞，相邻两足突之间相互连接形成的裂孔隔膜构成了肾小球最后一道滤过屏障。对乙型肝炎病毒相关性膜性肾病（HBV-MN）的临床病理研究显示患者均存在不同程度的足突融合，提示HBV-MN发生过程中存在裂孔隔膜的异常。与原发性膜性肾病相比，HBV-MN不仅存在补体攻膜复合物对足细胞的损伤，还存在HBV蛋白对足细胞的作用，因此本研究通过构建体外足细胞的亚溶量C5b-9（sC5b-9）模型，利用携带HBx基因的复制缺陷型腺病毒转染足细胞，观察sC5b-9和HBx双重损伤因素对培养足细胞裂孔膜蛋白表达和分布有何影响。方法通过检测细胞培养上清液中的LDH活性，采用“棋盘实验”的方法确定sC5b-9攻击足细胞的抗体和补体浓度，瑞士-吉姆萨染色观察sC5b-9对足细胞形态的影响，间接免疫荧光染色与激光共聚焦显微镜观察足细胞裂孔膜蛋白分布的变化，western-blot检测足细胞裂孔膜蛋白表达量的变化。结果通过LDH的测定确定了sC5b-9模型的抗体浓度为1%，补体浓度为1%。细胞形态学观察显示sC5b-9可以造成足细胞突触减少，胞质轻微空泡变性，胞核肿胀呈颗粒样改变，核膜界限模糊等变化。免疫荧光染色显示sC5b-9和HBx双重作用下裂孔隔膜蛋白分布异常较单因素更显著：nephrin主要朝核周分布聚集，胞质和突起的表达急剧减少；空白组podocin和CD2AP在胞质均匀分布，膜周呈小颗粒状，而双重损伤组呈粗丝状或者大颗粒样分布于膜周和突起。western blot检测显示双重损伤组较sC5b-9单因素损伤组CD2AP、nephrin的表达显著减少，podocin的表达反而升高；与HBx单因素损伤组比较，CD2AP、nephrin和podocin的表达均显著降低。结论sC5b-9和HBx蛋白双重因素可以加重裂孔膜蛋白的损伤。这揭示了在HBV-MN疾病进程中补体攻膜复合物和HBx蛋白可能共同发挥损伤足细胞的作用，损伤的靶点为裂孔膜蛋白，将为HBV-MN的诊疗提供新的思路。
[Abstract]:Objective glomerular podocytes are highly differentiated terminal cells. The fissure membrane formed by the connection between the adjacent two podocytes forms the last filtration barrier of the glomeruli. The clinicopathological studies of HBV-MNs in patients with hepatitis B virus associated membranous nephropathy (HBV-MN) show that there are various degrees of foot process fusion in all patients. Compared with primary membranous nephropathy, HBV-MN has not only the damage of complement attack membrane complex on podocyte, but also the effect of HBV protein on podocyte. In this study, we constructed the model of subsolubilization of podocyte C5b-9sC5b-9 in vitro, and transfected podocyte into podocyte by replication-deficient adenovirus carrying HBx gene. To observe the effect of sC5b-9 and HBx on the expression and distribution of lobes membrane protein in cultured podocytes. Methods the activity of LDH in the supernatants of cultured podocytes was detected. The concentration of antibody and complement of sC5b-9 attack podocyte was determined by "chessboard experiment", and the effect of sC5b-9 on podocyte morphology was observed by Swiss-Gimsa staining. Indirect immunofluorescence staining and laser confocal microscopy observed the changes of protein distribution in podocytes by Western blot. Results the antibody concentration of sC5b-9 model was determined by LDH analysis. (1) the complement concentration is 1. Morphological observation shows that sC5b-9 can cause synapse reduction in podocytes. The cytoplasm showed slight vacuolar degeneration, nuclear swelling showed granular changes, and the nuclear membrane boundaries were blurred. Immunofluorescence staining showed that the abnormal distribution of membrane proteins in the split cell membrane under the action of sC5b-9 and HBx was significantly higher than that of single factor. The expression of podocin and CD2AP in the cytoplasm of the blank group was evenly distributed, and the membrane around the membrane was small granular, while the expression of cytoplasm and protuberance decreased sharply. In the double injury group, the expression of CD2APPnphrin was significantly decreased in the double injury group compared with that in the univariate sC5b-9 injury group, and the expression of ppodocin was significantly increased in the double injury group. The results showed that the expression of CD2APPnphrin in the double injury group was significantly lower than that in the sC5b-9 univariate injury group. Compared with the univariate HBx injury group, the expression of CD2APPnphrin and podocin decreased significantly. Conclusion the double factors of sC5b-9 and HBx protein can aggravate the damage of the membrane protein. This suggests that the complement attack membrane complex and the HBx protein may play an important role in the process of HBV-MN disease. To work together to damage podocytes, The target of injury is hiatus membrane protein, which will provide a new idea for the diagnosis and treatment of HBV-MN.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R726.9

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