| The regulation of proliferation of endothelial cell plays an important role in angiogenesis, tumor development and tissue repair after trauma. Id (inhibitor of differentiation) proteins play a key role in the regulation of proliferation of endothelial cell. The heterodimers between Id proteins and bHLH(basic helix-loop-helix) transcription factors have no function to bind E-box,and inhibit transcriptional activity of bHLH, so Id proteins suppress cell differentiation and promote cell proliferation. Nature 1999 evaluated that Id proteins are as brake of cell differentiation; Investigating signal transduction related to Id protein has an important significance.The gene of hCASK(human calcium/calmodulin-dependent serine protein kinase) had been found in 1997,CASK belongs to a member of MAGUK(membrane-associated guanylate kinase). In 2000,our lab confirmed the GUK domain of CASK can bind to Id1 in yeast,this finding provides a new clue to further investigate the functions and signal pathway of CASK. OBJECTIVE: 1)to further confirm the interaction between CASK and Id1 in human endothelial cell, to explore signal pathway of CASK and Id1. 2) To study the biological functions of CASK-Id1 pathway. 3) To explore bFGF effecting on CASK-Id1 pathwayMOTHODs:1) culture of primary human umbilical vein endothelial cell(HUVEC):after fetal umbilical vein endothelial cells had been digested by 0.25％ trypsogen for 10 mins, HUVECs were collected by centrifugation. HUVECs were cultured at 37℃, 5% CO2 in M199 medium with 10% FCS. 2) ECV304 cell strain and ECV304 were stable transfected by human CASK (IPTG induced expression) cultured in the same way as above. 3) immune coprecipitation:the lysate of ECV304 and primary HUVEC was incubated with CASK antibody so the complex of IgG-CASK and other protein that could bind with CASK was coprecipitated with Agarose, the precipitation was analysed by Western Blotting to determine the existence of Id1. 4) laser scanning confocal microscope: with different fluorescence labelled antibody to detect the sublocalizations of CASK and Id1 in primary HUVEC. 5) Western Blotting: serum starvation for 6h followed by stimulation with bFGF at different concentration for 8h ,the expressions of CASK and Id1 were observed. 6) RT-PCR: the changes of the expression of p53 mRNA were observed after overexpression of CASK was induced by IPTG in ECV304. 7)Treatment with Id1 and CASK antisense oligonucleotide: The expressions of Id1 and CASK after treatment with theirs antisense oligonucleotide were analysed through Western Blotting . 8) Electrophoretic Mobility Shift Assay: after the overexpression of CASK was induced by IPTG in ECV304, E-box of p53 promotor binding to related bHLH transcription factor was observed at different time points; Super shift experiment was used to analyse whether bHLH transcription factor E12/E47 was involved in the binding.; after the overexpression of CASK or(and) Id1 antisense oligonucleotide treatment, the binding of E-box to related bHLH transcription factor was observed;after ECV304 were stimulated by different concentration of bFGF, the binding of E-box to related bHLH transcription factor was observed.RESULTs: 1. The specific bindings between CASK and Id1 exist in human endothelial cell1)With immune coprecipitation we confirmed that endogenous CASK can interact with Id1 in ECV304 and primary HUVEC. 2)　The results of confocal indicated the complex of CASK and Id1 localized in as granule in primary HUVEC. 2. The expressions of CASK and Id1 in ECV304 were effected by bFGF and treatment of antisense oligonucleotideWestern Blotting: the expression of CASK increased obviously after induced by IPTG; the expression of Id1 decreased obviously after treatment with Id1 antisense oligonucleotide,but treatment with CASK antisense oligonucleotide could not block the endogenous CASK expression; bFGF could effect on the expressions of CASK showing a manner of dosage and effect. The expressions of CASK had no difference when the concentration of bFG...
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