P311在小鼠皮肤成纤维细胞向肌成纤维细胞分化中的作用及其机制

Role and mechanism of P311 in the differentiation of mouse skin fibroblasts into myofibroblasts

目的探讨P311在小鼠皮肤成纤维细胞(Fb)向肌Fb分化中的作用及机制。方法该研究为实验研究。取6只2 d龄雄性C57BL/6小鼠,采用酶解法提取皮肤Fb,并常规培养传代。取第1~3代细胞,按随机数字表法分为转染空载腺病毒的空载体组、转染P311高表达腺病毒的P311组、转染P311高表达腺病毒和心肌素相关转录因子A(MRTF-A)小干扰RNA(siMRTF-A)的P311+siMRTF-A组。培养72 h后,对3组细胞采用细胞计数试剂盒8检测细胞增殖活力,采用蛋白质印迹法检测MRTF-A、α平滑肌肌动蛋白(α-SMA)、血清应答因子(SRF)的蛋白表达,行胶原凝胶收缩实验并计算72 h凝胶收缩率,以上实验样本数均为3;取空载体组和P311组细胞,采用蛋白质印迹法检测细胞、细胞质及细胞核内MRTF-A和SRF的蛋白表达,样本数为4。结果培养72 h后,空载体组、P311组、P311+siMRTF-A组细胞增殖活力相近(P>0.05)。培养72 h后,与空载体组比较,P311组细胞中MRTF-A、α-SMA和SRF的蛋白表达均明显升高(P<0.05),P311+siMRTF-A组细胞中MRTF-A和SRF的蛋白表达均明显降低(P<0.05);与P311组比较,P311+siMRTF-A组细胞中MRTF-A、SRF和α-SMA的蛋白表达均明显降低(P<0.05)。展示细胞收缩能力的P311组的72 h凝胶收缩率为(84.8±6.2)%,明显高于空载体组的(27.8±2.6)%和P311+siMRTF-A组的(24.7±3.2)%(P值均<0.05);空载体组和P311+siMRTF-A组的72 h凝胶收缩率相近(P>0.05)。培养72 h后,P311组细胞内、细胞质内MRTF-A(t值分别为5.86、3.77,P<0.05)和SRF的蛋白表达(t值分别为3.95、3.97,P<0.05)均明显高于空载体组,2组细胞核内MRTF-A和SRF的蛋白表达均相近(P>0.05)。结论P311可通过MRTF-A促进小鼠皮肤Fb向肌Fb分化,进而参与瘢痕形成。

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 1 st to 3 rd 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,andα-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 t values 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.