Role and mechanism of P311 in the differentiation of mouse skin fibroblasts into myofibroblasts
Objective To explore the role and mechanism of P311 in the differentiation of mouse skin fibroblasts(Fbs)into myofibroblasts.Methods The study was an experimental research.Six 2-day-old male C57BL/6 mouse were used to extract skin Fbs by enzymatic hydrolysis method and routinely cultured.The 1st to 3rd passage cells were taken and divided into empty vector group transfected with empty adenovirus and P311 group transfected with P311 high expression adenovirus,and P311+myocardin-related transcription factor A(MRTF-A)small interfering RNA(siMRTF-A)group transfected with P311 high expression adenovirus and siMRTF-A according to the random number table.After 72 h of culture,the cell proliferation vitality of cells in 3 groups was detected by cell counting kit 8,the protein expressions of MRTF-A,α-smooth muscle actin(α-SMA),and serum response factor(SRF)in cells in 3 groups were detected by Western blotting,the collagen gel contraction assay was performed and the 72 h gel contraction rates in 3 groups were calculated.The sample numbers in the above experiments were all 3.The protein expressions of MRTF-A and SRF in cells,cytoplasm,and nucleus in cells in empty vector group and P311 group were detected by Western blotting,with sample number of 4.Results After 72 h of culture,the cell proliferation vitality of cells in empty vector group,P311 group,and P311+siMRTF-A group was similar(P＞0.05).After 72 h of culture,compared with those in empty vector group,the protein expressions of MRTF-A,α-SMA,and SRF in cells in P311 group were significantly increased(P＜0.05),while the protein expressions of MRTF-A and SRF in cells in P311+siMRTF-A group were significantly decreased(P＜0.05).Compared with those in P311 group,the protein expressions of MRTF-A,SRF,and α-SMA in cells in P311+siMRTF-A group were significantly decreased(P＜0.05).The 72 h gel contraction rate showing cell contractility in P311 group was(84.8±6.2)％ ,which was significantly higher than(27.8±2.6)％ in empty vector group and(24.7±3.2)％ in P311+siMRTF-A group(with P values all＜0.05).The 72 h gel contraction rates in empty vector group and P311+siMRTF-A group were similar(P＞0.05).After 72 hours of culture,the protein expressions of MRTF-A(with tvalues of 5.86 and 3.77,respectively,P＜0.05)and SRF(with t values of 3.95 and 3.97,respectively,P＜0.05)in cells and cytoplasm in P311 group were significantly higher than those in empty vector group,while the protein expressions of MRTF-A and SRF in the nucleus of cells were similar between the two groups(P＞0.05).Conclusions P311 can promote the differentiation of fibroblasts into myofibroblasts through MRTF-A,and then participate in scar formation.
CicatrixSkinFibroblastsMyofibroblastsNeuronal regeneration related proteinMyocardin-related transcription factor A
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