α-Actinins对足细胞运动性的影响机制

发布时间：2018-03-22 00:31

本文选题：足细胞　切入点：细胞骨架　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：目的：观察α-actinins基因敲低及突变后足细胞骨架结构的变化及对足细胞运动性和牵拉力的影响。方法：体外培养条件永生化的野生型及α-actinin-4K256E基因杂合和纯合突变型足细胞。利用慢病毒转染RNA干扰技术敲低野生型足细胞中α-actinin-1和α-actinin-4的表达；应用Western Blot检测转染效果；免疫荧光观察细胞形态,α-actinins及细胞骨架结构F-actin的分布变化；伤口愈合实验用于检测细胞的运动性；牵拉力学显微镜用于检测细胞的牵拉力。结果：Western Blot结果显示慢病毒转染小干扰RNA技术可高效敲低α-actinin-1和α-actinin-4基因。α-Actinin-1和α-actinin-4基因敲低后,细胞骨架出现紊乱,细胞的牵拉力增加,而且细胞的运动性也增加。相反,α-actinin-4突变型的足细胞表现为牵拉力下降,运动性降低。结论：α-Actinins参与细胞骨架结构重排和牵拉力形成,可调节足细胞的收缩力和限制细胞的运动性。α-Actinin-4突变的足细胞可获得某些功能而降低了细胞的牵拉力和运动性。这表明α-actinins功能的改变可能通过改变足细胞的牵拉力和运动性造成肾小球损伤。目的：观察体内外α-actinins基因敲低及突变后对细胞间黏附分子vinculin和β3integrin mRNA及蛋白表达水平的影响方法：间接免疫荧光法检测α-actinin-1和α-actinin-4敲低后vinculin, F-actin的表达分布变化,及活性β3integrin在野生型,α-actinin-4基因敲除,及α-actinin-4K256E基因杂合和纯合突变型小鼠中的表达变化。实时定量PCR及Western Blot检测vinculin在α-actinins基因敲低及突变型足细胞中mRNA及蛋白表达水平的变化。另外,实时定量PCR也检测了β3integrin在上述几种细胞中mRNA表达水平的变化。结果：Vinculin在α-actinin-4基因敲低足细胞中mRNA及蛋白表达水平均有增高,但在α-actinin-1基因敲低和α-actinin-4突变足细胞中表达无明显变化。β3Integrin在α-actinin-4突变型足细胞中mRNA表达水平明显下降。在α-actinin-4基因敲除小鼠肾组织中,活性(33integrin表达增加,而在α-actinin-4杂合型和纯合型突变的小鼠肾组织中,活性(33integrin的表达下降。结论：α-Actinin-4敲低可影响vinculin的表达,而α-actinin-4基因敲除和突变可明显影响小鼠肾脏活性β3integrin的表达。这表明α-actinins可影响足细胞黏着斑的表达。目的：观察α-actinins基因敲低及突变后对小GTP酶活性的影响,同时观察野生型,α-actinin-4K256E基因杂合和纯合突变小鼠肾脏中小GTP酶活性的变化。方法：利用小GTP酶活性试剂盒检测α-actinins基因敲低及突变型足细胞中RhoA, Racl和Cdc42的活性,并同时检测野生型,α-actinin-4基因杂合及纯合型突变小鼠肾脏裂解液中上述三种小GTP酶的活性。结果：在α-actinin-4基因敲低细胞中,RhoA活性显著下降,Cdc42的活性明显增高,而Racl活性则无明显变化。在α-actinin-1基因敲低细胞中,RhoA活性也有所增加,Racl和Cdc42活性则无变化。在α-actinin-4基因突变细胞中,杂合型和纯合型α-actinin-4细胞中RhoA舌性均有所增加,而Racl和Cdc42活性则无变化。同时纯合型α-actinin-4突变小鼠肾脏中EhoA活性也有所增加,而Racl和Cdc42舌性则无明显变化。结论：α-Actinins基因敲低及突变可不同程度的影响小GTP酶中各种分子的活性。这些小GTP酶活性的变化与足细胞骨架重排,黏附和迁移都有密切关系。
[Abstract]:Objective: To observe the changes of the cytoskeleton structure of the podocytes after the knockout of the alpha -actinins gene and the effect on the motility and traction of the podocytes.
Methods: in vitro immortalized wild-type and alpha -actinin-4K256E gene heterozygous and homozygous mutant podocytes. Knockdown of wild-type podocytes and alpha -actinin-1 alpha -actinin-4 using lentiviral transfection of RNA interference technology; application of Western Blot to detect the transfection effect; the cell morphology was observed by immunofluorescence, -actinins and alpha distribution the cytoskeletal structure of F-actin; wound healing experiment for motion detection of cells; mechanical stretch microscope for tension detection cells.
Results: Western Blot showed that lentiviral transfection of small interfering RNA can efficiently knockdown of alpha -actinin-1 and alpha -actinin-4 alpha and alpha -Actinin-1 gene. -actinin-4 gene knockdown, cytoskeleton disorder, tension cells increased, and the movement of cells also increased. On the contrary, the podocyte alpha -actinin-4 mutant showed pulling down, moving down.
Conclusion: alpha -Actinins involved in cytoskeletal rearrangement and stretch formation movement can regulate podocyte contractility and limit cell. Alpha -Actinin-4 mutant podocytes can obtain certain functions and reduce the cell traction force and motion. This shows that changing the alpha -actinins function may cause glomerular injury by changing the foot cell traction force and motion.
Objective: To observe the effect of alpha -actinins gene knockout and mutation on the expression of vinculin and beta 3integrin mRNA and protein expression of intercellular adhesion molecules in vivo and in vitro
Methods: indirect immunofluorescence detection of alpha -actinin-1 alpha and -actinin-4 knockdown of vinculin expression in the distribution of F-actin, and the activity of 3integrin in wild-type alpha beta, -actinin-4 gene knockout, and alpha -actinin-4K256E gene heterozygous and homozygous mutant mice the expression of PCR and Western. Real time quantitative Blot detection in vinculin alpha -actinins gene knockdown the expression and mutation of mRNA and protein changes in podocytes. In addition, quantitative real-time PCR to detect the expression changes of mRNA in these cells in beta 3integrin.
Results: Vinculin -actinin-4 gene knock in alpha levels were increased mRNA and protein expression of low enough cells, but in the alpha -actinin-1 gene knockdown and alpha -actinin-4 mutation in podocytes showed no significant change. 3Integrin mRNA in beta alpha -actinin-4 mutant podocytes expression level decreased significantly. In alpha knockout of -actinin-4 gene in renal tissue in mice, the activity (increase the expression of 33integrin, while in alpha -actinin-4 heterozygous and homozygous mutant mice kidney tissue, activity (decreased the expression of 33integrin.
Conclusion: the knockdown of alpha -Actinin-4 can affect the expression of vinculin, while the knockout and mutation of alpha -actinin-4 can significantly affect the expression of beta 3integrin in mice kidney activity. This indicates that alpha -actinins can affect the expression of adhesion cells in podocyte.
Objective: To observe the effect of alpha -actinins knockdown and mutation on the activity of small GTP enzyme, and observe the change of small GTP enzyme activity in kidney of wild type, -actinin-4K256E gene heterozygous and homozygous mutant mice.
Methods: the activity of RhoA, Racl and Cdc42 in alpha -actinins knockdown and mutant podocytes was detected by small GTP enzyme activity kit, and the activities of the above three small GTP enzymes in the kidney lysate of wild type, alpha -actinin-4 gene heterozygous and homozygous mutant mice were detected simultaneously.
Results: in the alpha -actinin-4 gene knockdown cells, RhoA activity decreased significantly, the activity of Cdc42 increased significantly, while the Racl activity did not change significantly. In the alpha -actinin-1 gene knockdown cells, RhoA activity also increased, no change of Racl and the activity of Cdc42. -actinin-4 gene mutation in alpha cells, increased RhoA the tongue of both heterozygous and homozygous alpha -actinin-4 cells, while Racl and Cdc42 activity did not change. At the same time the homozygous alpha -actinin-4 mutant mice kidney EhoA activity also increased, while the Racl and Cdc42 of the tongue had no obvious change.
Conclusion: the knockdown and mutation of -Actinins gene can influence the activities of various molecules in small GTP enzymes to varying degrees. These small GTP activities are closely related to the rearrangement, adhesion and migration of podocyte.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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