Dissertation > Medicine, health > Oncology > Skin tumors

Study on the Differential Proteomic Analysis between HaCaT Cell Line and Cutaneous Squamous Cell Carcinoma Cell Line SCL-1

Author ZhuHong
Tutor HeChunDi
School China Medical University
Course Dermatology and Venereology
Keywords Skin Squamous cell carcinoma HaCaT cell line SCL -1 cell line Proteome Two - dimensional electrophoresis Mass Spectrometry
CLC R739.5
Type PhD thesis
Year 2006
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ObjectiveCarcinogenesis of skin squamous cell carcinoma is a complex process invol-ving multiple events and steps.Though some molecular pathogenesis studies on human skin squamous carcinoma have been undertaken successfully in gene lev-els,the carcinogenic mechanism is still unclear.At present,there no special molecular marker for early-stage diagnosis and prognosis evaluation.The ob-jective of this study was to establish two-dimensional electrophoresis profiles with high resolution and reproducibility from HaCaT cells and SCL-1 cells,and to identify differential expression proteins.MethodsHuman epidermal keratinocyte HaCaT and SCL-1 cell line was cultured in RPMI1640 medium with 10%heat-inactivated fetal calf serum(FCS)at 37℃, under 5%CO2 in air.After cells grew to confluence on a dish,we collected and lysed cells with lysis solution at a volume ratio of 1:5.The solution was left at room temperature for approximately 1 hour and then centrifuged.The superna-tant was retained,with concentration determined.The protein content in the su-pernatant was determined by means of the Bradford method.For 2-D gel electrophoresis,immobilized pH gradient gel(IPG,pH3-10,pH 4-7 and pH 5-8)iso-electric focusing was set as the first dimen-sion,12%SDS polyacrylamide gel electrophoresis was set as the second dimen-sion.For iso-electric focusing,PROTEAN IEF cell was used.Loaded sample sizes were 1.2 mg(350ul)for each one,respectively.Iso-electric focus was performed at 50V for 13 hour,250V for 30 min,1000V for 1 hour,and finally focused under 10000v for 60000 vh.The IPG was bathed in balance solutionⅠandⅡ,respectively,for 15 mi-nutes each,then transferred onto a 12%SDS polyacrylamide gel and electro-phoresed at 30V for 30 min,and then 120V till Bromophenol blue moved to the edge of glass plate.Each sample was electrophoresed three times.Following electrophoresis,the gels were taken out and stained with Coom-assie Brilliant blue staining.The images were scanned and analyzed with a Im-age Master 2D 4.10 software package,determination of the molecular weight, iso- electric points and matching protein spots.Twelve Protein spots were ex-cised from the gels for in -gel tryptic digest and analys of mass spectrum.Data from MALDI-TOFMS/MS were analyzed using MASCOT(Matrix Science, London)search software.ResultsTwo-dimensional gel electrophoresis profiles of proteome from HaCaT cells and SCL-1 cells were established.With 17cm IPG pH4-7L,there were(690±39)protein spots on the 2-DE gels of HaCaT cells and(665±31)protein spots were obtained in the 2-DE profiles of SCL-1 cells.Both Of them,the match rates of protein spots were about 72%.Twelve differential spots were cut off from the Coomasie blue stained gel,and all of the spots were preliminarily i-dentified,which were tropomyosin;heat shock protein 27;prohibitin;dUTP py-rophosphatase;Triosephosphate isomerase;KRTHB1 protein;Chain B,X-ray crystal structure of Human galectin-1;Chain B,the 2.1 a Structure of a tumour suppressing serpin;eukaryotic translation initiation factor 3,subunit 2 beta;Sfrs1 protein.These proteins were associated with cell metabolism and cytoskeleton.ConclusionIn this study,the well-resolved,reproducible 2-DE patterns of HaCaT cells and SCL-1 cells were established and certain differential proteins were characterized.These data will be helpful for screening the biomarker to further study on skin squamous carcinoma.

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