| Objective: To investigate the influence of PAI-1 on proliferation of fibroblast from SD rat skin,RNA interference technology was applied to downregulate PAI-1 expression.At the cellular level,the effect of PAI-1 on fibroblast proliferation and transformation were explored through vitro experiment.Further more,a theoretical basis was provided for PAI-1 siRNA which can improve the keloid model of SD rat from the early experiments on animals and a new idea was explored for gene treatment of keloid.Method:1 Cultured SD rat skin fibroblasts though explant cultureThe explant culture was adopted to obtain the original generation of SD rat skin fibroblasts,and the single fibroblasts were gained by subculture which removed impurities such as epithelial cells,red blood cells,etc.Afterwards,cells growth and morphologic changes were observed under inverted microscope.2 Identified cells by immunocytochemistryThe vimentin expression was detected by immunocytochemistry,according to this,we knew whether the cells cultured were fibroblasts.3 Transfected PAI-1 into fibroblasts cultured though vitro experiment,and divided into groupsDifferent siRNAs were respectively transfected by lipofectamine 2000 into the sixth generation of subcultured fibroblasts through the vitro experiment and divided into three groups.The experimental group was transfected PAI-1,while the negative control group and blank control group was respectively transfected nonspecific PAI-1 and none.4 The expression of PAI-1 mRNA and TGF-?1 mRNA on 24 h was determined by Real time PT-PCR.5 The protein expression of PAI-1,AKT,ERK,p-AKT,p-ERK on 48 h was determined by Western blot.6 Counted the total number of cells and plotted growth tendency curve and survival rate curve after Trypan blue stained.Respectively transfected for 24 hours,48 hours and 72 hours,cell suspension were prepared for Trypan blue staining.The total number of cells were counted under the microscope,as well as the total number of cells not been dyed(or the total number of cells stained).Then,growth curve and survival rate curve of fibroblasts were plotted.The abscissa denoted the foster time while the ordinate denoted the cell count.7 Plotted viability curve of fibroblasts by CCK-8Respectively transfected for 24 hours,48 hours and 72 hours,all the cell suspension was injected CCK-8.After 4 hours,Optical density(OD)was evaluated by enzyme linked immunoassay.The OD value and the proliferation inhibition rate(%)of the culture fibroblasts were calculated from the formula.The cell growth curve was drew by foster time as the abscissa and the cells OD value as the ordinate.The proliferation inhibition rate curve was drew by foster time as the abscissa and the proliferation inhibition rate as the ordinate.8 Statistical analysisStatistical analysis of all the experiment data were performed with SPSS21.0 software.All the data were measurement data,and they were described by mean and standard deviation.P<0.05 was considered statistically significant.Normality test and homogeneity test of variance were analyzed before data analysis.If the data obeyed the normal distribution and equal variance assumed,one-way ANOVA was applied to multi-sample analysis and SNK-q test was applied to comparision among groups.If not,Kruskal-Wallis H test was applied to multi-sample analysis and Mann-Whitney U test was applied to comparision among groups.Results:1 SD rat skin fibroblasts were successfully obtained through explant cultures,the single component of fibroblasts were gained by subculture to 5-6generation;2 The vimentin was detected by immunocytochemistry staining.Under the microscope,the cells intracytoplasmic showed brown or claybank particles,and the cells nucleus showed no changes.The vimentin expression was positive,which meaned that the culture cells were fibroblasts;3 PAI-1 was successfully transfected into fibroblasts detected by Real time PCR,and the expression of PAI-1 mRNA and TGF-?1 mRNA was obviously downregulation.The population mean of RQ values was evaluated different or not identical(P < 0.05).The difference between experimental group and negative control group?experimental group and blank control group showed statistical significance(P < 0.05),while the difference between negative control group and blank control group did not show statistical significance(P>0.05);4 PAI-1 siRNA was transfected into fibroblasts successfully detected by Western blot,and the protein expression of PAI-1,p-AKT and p-ERK was obviously downregulation.The difference between experimental group and negative control group,experimental group and blank control group showed statistical significance(P < 0.05),while the difference between negative control group and blank control group did not show statistical significance(P>0.05);Statistical results of the dephosphorylation protein expression of AKT and ERK in experimental group did not show statistical significance compared with negative control group and blank control group;5 According to the trends of growth tendency curve and survival rate curve drew after Trypan blue stained,the growth rate of fibroblasts which were transfected PAI-1 siRNA compared with negative control group and blank control group obviously slowed down over time,and the survival rate decreased significantly;6 According to the trends of growth tendency curve and the proliferation inhibition rate curve drew after injected CCK-8,proliferation activity of fibroblasts which were transfected PAI-1 siRNA showed lower compared with negative control group and blank control group,and the proliferation inhibition rate showed higher.Conclusisons:Skin fibroblasts of SD rat were successfully obtained.PAI-1 siRNA was successfully transfected into fibroblasts.Through a series of experiment,downregulation the expression of PAI-1 could inhibit proliferation and transformation of SD rat skin fibroblasts,inhibit expression of TGF-? 1,and restrain signal pathway of AKT and ERK.Thus the proliferation rate of fibroblasts showed a slower pace,meanwhile,the proliferation activity and the survival rate showed decreased. |
PDF Full Text Request