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SublyticC5b-9 induced Gadd45γ express Thy-1 nephritis lesions of GMCs apoptosis

Author: CheZuo
Tutor: WangYingWei
School: Nanjing Medical University
Course: Immunology
Keywords: Growth arrest and DNA damage inducible gene 45γ (Gadd45γ) Small hairpin RNA (shRNA) Mesangial cells (GMCs) Sub -dissolved C5b-9 (sublytic C5b-9) GMCs Gadd45γ Apoptosis Thy-1 nephritis (Thy-1N) Histopathological changes
CLC: R692.3
Type: Master's thesis
Year: 2009
Downloads: 8
Quote: 0
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Abstract


Objective: To construct the wild-type rat growth arrest and DNA damage inducible gene 45γ (growth arrest and DNA damage-inducible gene 45γ, Gadd45γ) and its specificity small hairpin RNA (short hairpin RNA, shRNA) eukaryotic expression plasmid and observed in rat mesangial cells (glomerular mesangial cells, GMCs) overexpression and gene silencing Gadd45γ situation. METHODS: The recombinant DNA technology in rats Gadd45γ gene sequences cds or against different sites designed four pairs of shRNA sequences were cloned into the eukaryotic expression plasmid pcDNA3.1/HA or pGCsi.U6.neo.GFP in . In the restriction analysis and sequencing correct with GenEscort TM Ⅲ transfection reagent the above two plasmids were transfected into rat GMCs, use immunocytochemistry and Western blot HA-Gadd45γ fusion protein expression and screening the best silencing efficiency of shRNA. Results: The restriction enzyme digestion and DNA sequence analysis shows that two recombinant plasmids were constructed correctly. Immunocytochemistry and Western blot analysis showed that the constructed pcDNA3.1/Gadd45γ plasmid capable of expressing in GMCs in the rat; Gadd45γshRNA-3 has the best silencing efficiency. Conclusion: The successful construction of the wild-type Gadd45γ rat genes and their specific shRNA eukaryotic expression plasmid, the experimental results for the further study of the biological function of genes Gadd45γ foundation. Objective: To investigate the in vitro dissolution Asian type C5b-9 (sublytic C5b-9) stimulation induces apoptosis in GMCs and raised Gadd45γ gene expression, while identifying Gadd45γ overexpression or gene silencing sublytic C5b-9 induced apoptosis in lesions of GMCs . Methods: First, cultured rat GMCs and give sublytic C5b-9 stimulation (experimental set different treatment control), flow cytometry (flow cytometry, FCM) analysis of apoptosis of GMCs and used Real-time PCR and Western blot examination by sublytic C5b-9 stimulated GMCs synthesis Gadd45γmRNA abundance and protein levels. Then, pGadd45γ plasmid or Gadd45γshRNA plasmids were transfected GMCs, and Gadd45γshRNA transfection screening pGadd45γ best time and GMCs for the corresponding packet processing. Regularly check each group Gadd45γmRNA abundance and protein expression and apoptosis rate GMCs quantitative analysis. Results: rat GMCs in sublytic C5b-9 stimulation 3h, apoptosis was significantly higher. Meanwhile, Gadd45γmRNA and protein expression was also increased significantly. Sublytic C5b-9 stimulation group and pGadd45γ plasmid group of GMCs apoptosis rate was significantly higher; plasmid transfection pGadd45γ GMCs and then give sublytic C5b-9 stimulation, the phenomenon is more obvious. In contrast, transfection of plasmid Gadd45γshRNA GMCs and then give sublytic C5b-9 stimulation of apoptosis was significantly lower than the other group. Conclusion: Sublytic C5b-9 stimulation can induce apoptosis in rat GMCs; by the sublytic C5b-9-induced apoptosis and its upregulation Gadd45γ GMCs have a certain relationship. Objective: To investigate renal tissue with Gadd45γshRNA silence genes within Gadd45γ Thy-1 nephritis (Thy-1 nephritis, Thy-1N) lesions in rats GMCs apoptosis inhibition. Methods: First, copy the rat Thy-1N model, regular use of Real-time PCR and Western blot method to check their kidney tissues Gadd45γ expression levels. Then apply the renal artery injection plus electroporation method, Gadd45γshRNA plasmid into rat kidney tissue, and then injected with anti-rat thymocyte serum (ie, anti-Thy-1 Ab) rat Thy-1N. Were taken anti-Thy-1 Ab and 5d 3h after injection when the rat kidney tissue, the application of Real-time PCR was determined Gadd45γmRNA abundance in Western blot and immunofluorescence its Gadd45γ protein levels. In addition, the application of TdT-mediated dUTP nick end labeling (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) technique and electron microscopic examination of renal histopathology, and use automatic biochemical analyzer when the rats 5d 24h urine total protein changes. Results: rat Thy-1N lesions 40min, the expression level of the renal tissue Gadd45γ began to increase when the experiment 3h increased more apparent. The plasmid was introduced Gadd45γshRNA renal tissue and then copy the Thy-1N, regularly inspect the specimens found, Gadd45γshRNA treated Thy-1N rats (Gadd45γshRNA Thy-1N group), and its renal tissue Gadd45γ gene expression was significantly lower than the Thy-1N model group. Morphology (TUNEL and electron microscopy) also found that, Gadd45γshRNA Thy-1N rats, the incidence of apoptosis after glomerular GMCs significantly fewer; 5d when, 24h urine protein secretion also decreased significantly. Conclusion: Silence rat kidney tissue Gadd45γ gene can inhibit Thy-1N rats GMCs apoptosis in renal tissue lesions.

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Nephritis
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