Study Of The Effect Of C/EBPd On The Proliferation And Differentiation Of SDHCE Cells

Objective To explore the mechanism of proliferation and differentiation and expression of specific protein markers of SDHCEC by studying the expression and regulation of C/EBPd in the proliferation and differentiation of SDHCE cells.Methods 1.The expression and location of CK3,C/EBPdand P27 were studied by immunofluorescence of the SDHCE cells and frozen tissue sections of central cornea and limbus.2.rhTGF-β1 was added into the culture medium of SDHCE cells with the final concentration of 0ng/ml,0.5ng/ml,1ng/ml,3ng/ml,5ng/ml and cultured for 12h,24h and 48h in order to detect the effect of different concentration of TGF-βlon proliferation and differentiation of SDHCE cells by MTT and analysis of cell cycle through flow cytometry.3.The recombined eukaryotic expression vector,pEGFP-C1-C/EBPd plasmid,was constructed and transfected into SDHCE cells mediated by lipofectamine at different ratio.The transfection efficiency and cytotoxicity of transfection mixtures were detected with inverted microscope to optimize the transfection condition.4.SDHCE cells were transfected with pEGFP-C1-C/EBPd plasmid under optimized transfection condition, and rhTGF-β1 was added after 24 hours.The content of C/EBPdmRNA was detected by real-time PCR.Results 1.Immunofluorescent histochemical staining showed CK3 stained only the superficial cells of cornea and limbal epithelia,while no stainings was found on limbal basal cells;C/EBPd stained only on nucleus of limbal basal cells;P27 was found stained on nucleus or cytoplasm of limbal basal cells.2.The effect of TGF-βlon proliferation and differentiation of SDHCE cells:rhTGF-β1 was added into the culture medium of SDHCE cells with the final concentration of 0ng/ml,0.5ng/ml,1ng/ml,3ng/ml,5ng/ml and cultured for 12h,24hand48h,detected absorbance of each well under the enzyme-labelling meter with the wave length of 490nm,and drew the growth curve.The results showed:the proliferation was inhibited markedly 48h after 1ng/ml of TGF-β1was added;SDHCE cells were collected at 12h,24h and 48h after 1ng/ml of TGF-β1 was added into the culture medium,and analysed cell cycle through flow cytometry.After 24h, SDHCE cells differentiated greatly.3.The transfection efficiency was elevated with the increasing of DNA.When the amount of Lipofectamine was no more than 15.0ul,the transfection mixture was of little cytotoxicity.When the amount of Lipofectamine was up to 17.5ul,the cytotoxicity was significantly increased.4.In C/EBPd and TGF-β1+ C/EBPdgroup,the expression of C/EBPdmRNA increased in SDHCE cells at 24h after transfection(i.e before stimulation)than that in normal control group.In TGF-β1+ C/EBPdgroup,the expression of C/EBPdmRNA decreased after stimulation but was still higher than that in control group.Conclusion 1.C/EBPd can be used as a differentiated marker of limbal epithelial cell,and it can identify limbal epithelial cell combined with other markers.2.TGF-β1 can inhibit the proliferation and stimulate differentiation of SDHCE cells.We can study the mechanism of proliferation and differentiation of SDHCE cells.3.By optimizing the parameter,Lipofectamine transfection reagent could transfect pEGFP-C1-C/EBPd plasmid into SDHCE cells with high efficiency.4.The recombined eukaryotic expression vector,pEGFP-C1-C/EBPd plasmid,is constructed and we study the mechanism of proliferation and differentiation of SDHCE cells by detecting the content of C/EBPdmRNA by real-time PCR.It will provide a basis for clinical application of SDHCE cells.