| Objective: To verify whether abnormal expression and natural bindingof calcium/calmodulin dependent serine protein kinase(CASK) andinhibitors of differentiation1(Id1) exists in Fb of keloid, and to observe theeffect of artesunate on the two genes. To provide experimental basis for theproliferation regulation mechanism of keloid Fb.Method（1）Samples of keloid and normal skin tissue during operation werecollected and cultured in vitro. Cells from the third to the eighth generationwere used for test.（2）HE staining was used to identify the cell type of Fb，expressionof CASK and Id1in the two kinds of Fb were observed withimmunofluorescence, to test the natural combination of CASK and Id1protein of Fb by immunoprecipitation.（3）Fb of keloid were stimulated with artesunate in the concentrationof10,30,50,80,120mg/L,and the median inhibitory concentration wasdetermined through the MTT colorimetric assay which served as the intervention concentration of artesunate.（4）Normal control group(NC), scar control group(SC), and scaradministration group(SA) were set up in the experiment, the cycle andapoptosis of Fb before and after being intervented by artesunate weredetected with flowcytometric assay，the nucleic acid and protein expressionof CASK and Id1of Fb in each group were determined with RT-PCR andWestern blotting.Results（1）Respectively6samples of Fb of keloid and that of normal skinwere successfully cultured, expressions of CASK and Id1were detected intwo kinds of Fb, the results of immunoprecipitation showed that Id1can bedetected in the CASK precipitate,CASK can be detected in the Id1precipitate,there was a natural binding of CASK and Id1in Fb.（2）The concentration of75mg/L was selected as the interventionconcentration of artesunate.Among the three groups,there were statisticallyapparent discrepancy in the percentage of cells in G0/G1phase and G2/Mphase (with F figures118.064and163.840,P figures entire less than0.05).The percentage of cells in G0/G1phase of SA group was (91.4±1.4)%,apparently bigger than that of SC and NC sets [(80.7±0.3)%and (82.4±0.6)%, with t values respectively12.740and9.872,P values all below0.05)]. The percentage of cells in G2/M phase of SA group was (6.9± 0.3)%, apparently less than that of SC and NC groups [(13.7±0.3)%and(12.7±0.8)%, with t values respectively43.702and12.276, P values allbelow0.05)]. There were apparently disparitys among the three groups inthe early and late apoptotic rates(with F values respectively61.879and4710.862, P values all below0.05). The early and late apoptotic rates of SAgroup were respectively (7.1±1.0)%and (14.9±0.3)%, significantly higherthan those of SC group and NC group [with early apoptotic raterespectively (2.6±0.4)%and (2.7±0.3)%, t values respectively7.974and7.767, P values all below0.05;with late apoptotic rate respectively (2.3±0.3)%and (2.5±0.4)%, t values respectively72.882and69.792, P valuesall below0.05].（3）The mRNA deliverance of CASK in SC set was0.658±0.024,lessthan NC group (1.076±0.008,t=28.997, P <0.05) and SA group(0.855±0.008,t=13.549, P <0.05).The protein deliverance of CASK inSC set was0.067±0.007, less than NC group (0.179±0.015,,t=12.042, P<0.05) and SA group (0.132±0.010,t=9.498, P <0.05).(4) The mRNAexpression of Id1in SC group was0.416±0.006, higher than that of NCgroup (0.317±0.020,t=8.299, P <0.05) and SA group (0.217±0.009,t=32.417, P <0.05). The protein expression of Id1in SC group was0.789±0.034, higher than that of NC group (0.366±0.029,t=16.341, P <0.05)and SA group (0.114±0.006,t=33.978, P <0.05). Conclusion（1）There was abnormal expression of CASK and Id1in Fb ofkeloid,which indicated that the proliferation of keloid Fb may beassociated with both.（2）The natural combination of CASK and Id1existed in Fb ofkeloid，which showed that there may exist CASK/Id1signaling pathwayinvolved in value-added regulation of Fb（3）The proliferation of keloid Fb was inhibited by artesunate incertain dose and time range，the mechanism of which may be associatedwith the downregulation of the expression of Id1and the upregulation ofthe expression of CASK. |
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