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Knock-down of Cullin-5 Expression on Alteration of Morphology and Proliferation Ability in Glomerular Podocytes

Author: CaoZuo
Tutor: MaoJianHua
School: Zhejiang University
Course: Pediatrics
Keywords: Podocytes Cullin-5 Ubiquitin-proteasome system Protein degradation Podocyte injury RNA interference Gene knockdown RT-PCR Western-blot MTT Immunofluorescence
CLC: R726.9
Type: Master's thesis
Year: 2011
Downloads: 47
Quote: 0
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Abstract


[Background] the glomerular proteinuria fundamental reason is due to damage to the glomerular filtration barrier, glomerular epithelial cells is a key factor to keep the full functionality of glomerular filtration membrane. Over the past decade, many studies found that the incidence of many congenital nephrotic syndrome patients is due to of Nephrin, Podocin, α-Actinin-4, Synaptopodin WT-1 protein encoding gene mutation caused. The above Gene Knock-out animal models of proteinuria and glomerular epithelial cell foot process fusion lesions, indicating that the expression in glomerular epithelial cell foot processes molecules, (such Nephrin Podocin, CD2AP, TRPC6) and cells The skeleton associated protein (e.g. synaptopodin, α-Actinin-4, Podocalyxin etc.) to maintain glomerular filtration membrane integrity to prevent the leakage of plasma proteins play an important role. Recent studies of the research team also found that molecular expression and regulation changes of the Nephrin Podocin, CD2AP foot process does the incidence of children is closely related with some primary nephrotic syndrome. Meanwhile, we also confirmed Myole the (A myosin workers molecules) are also human beings, the skeleton of the mouse glomerular epithelial cells associated protein expression Knock-down zebrafish proteinuria and glomerular epithelial cell structure changes. Cell protein metabolism and updated regulation and degradation of proteins in eukaryotic cells have two main ways: the lysosomal pathway and the ubiquitin - proteasome system (of Ubiquitin in Proteasome System, UPS) UPS by ubiquitin, 26S the proteasome ubiquitin activating enzyme E1s, E2s of Pan-conjugating enzyme ubiquitin ligase E3s composed, is efficient, specific protein degradation system, is the main mechanism of cell regulation of specific proteins, the removal of misfolded proteins. Ubiquitin ligase E3s main role is to identify the specific needs of the target protein to be degraded, and the transfer of ubiquitin to a target protein, the final target protein degradation by protease. Cullin-5 (CUL5) of many E3s-CRL components involved in a variety of target proteins (such as p53) degradation. Encoding gene (located 11q22-23) sequence of arginine vasopressin highly homologous calcium transport is activated receptor -1 (VACM-1), both of which may be the same kind of protein. So far, it is known functionality of CUL5/VACM-1 is: 1, CUL5 as Backbone assembly belonging to the family of E3 Ub ligases, involved in many eukaryotic intracellular protein pan-biotinylated modified and specific degradation; 2, as the main expression in vascular endothelial cells protein, VACM-1 in a plurality of cell lines express the inhibition of cell proliferation activity, having anti-tumor effect. Our previous studies showed that, CUL5 in the human, mouse glomerular epithelial cell expression and its downregulation can lead to model animal zebrafish proteinuria. We analyze the mechanism can lead to proteinuria CUL5 downregulation likely resulting in CUL5 as the cornerstone of UPS function abnormalities, and eventually causing the foot processes molecules and the cytoskeleton-associated protein metabolism, conversion and update mechanism disorders. [Objective] after study CUL5 knock down, changes in morphology and proliferation of mouse glomerular podocytes. Clarify CUL5 may play a key role in maintaining the function of the foot processes of molecular and glomerular filtration barrier in. [] 1. Mice podocyte cell culture. 2. Immunofluorescence method identified podocyte cytoskeletal proteins. 3 miRNAs design, CUL5 four different sequences, respectively, to design four plasmid and a negative control plasmid. Synthesized by Invitrogen Corporation. 4 Use Invitrogen Corp. transfection reagents in the podocytes CUL5 gene transient transfection, knock low CUL5 expression. 5 using RT-PCR method from the mRNA level, the detection of gene knockdown effect to GAPDH as reference gene after Geo-plo-Analyzer 4.0 software, analysis gel electrophoresis after pictures, semi-quantitative analysis of gene expression differences. 6 Western-blot method from the protein level, the detection of gene knockdown effect, as a reference gene (β-actin, after Image J software analysis, semi-quantitative analysis of protein expression differences. 7. The immunofluorescence assay CUL5 knock changes podocyte morphology after low. 8. the immunofluorescence method detect CUL5 knockdown cytoskeletal proteins. 9.MTT assay cell proliferation in each experimental group, respectively, set of four experiments, that is, 24 hours , 48 hours, 72 hours, 96 hours, to detect cell OD value. [results] mouse podocytes at 33 ℃ allow conditions (permissive condition) the the podocyte proliferation state was \The nucleus was round, is located in the cell body central the foot process less shorter; non-permissive conditions (non-permissive condition), incubated for about 14 days, podocyte differentiation and maturation of the cytoplasm to spread out into 37 ℃ irregular shape, there are a large number of long-smaller foot process formation, cells were \nuclear membrane around 37 ° C non-permitted under the conditions of the podocyte, CUL5 distribution in the cell cytoplasm and foot processes. 3. 33 ℃ allows podocytes under the conditions, F-actin distribution: mainly in podocytes main sudden near interference; 37 ° C under non-permissive conditions podocytes radial F-actin, extend to the projections of the newborn after RNAi technology knockdown CUL5, RT-PCR detection of mRNA relative expression changes: group (P = 0.003), two groups (P = 0.042), the three groups (P = 0.000), the four groups (P = 0.000) and the control group difference P lt; 0.05; negative control group, with no significant difference between the control group the negative control group and the control, P = 0.091. 5. knock low CUL5 after, with the changes in the relative expression of the Western-blot detection of protein: an interference one group, two groups, three groups of four groups and the control group difference P lt; 0.05; There was no significant difference, P = 0.051 6 after knockdown CUL5, immunofluorescence assay cell fluorescence change, each the interference group cell fluorescence intensity was significantly weakened, while little difference between the control group and the fluorescence intensity of the negative control group . after knockdown CUL5, changes in podocyte morphology: cell body narrow, irregularly shaped, jagged, foot process Fusion 8. knockdown CUL5, the podocyte proliferation changes: various interference group podocyte proliferation capacity compared with the control group, P lt; 0.01; There are also differences in the negative control group and the control group, but the difference was obvious. [Conclusion] 1.CUL5 in the distribution of the different growth stages of podocytes. maintain podocyte normal morphology 2.CUL5 in play a certain role. hind 3.CUL5 knockdown decreased cell proliferation, also plays an important role CUL5 maintain the normal function of podocytes.

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