Role and mechanism of P311 in the differentiation of mouse skin fibroblasts into myofibroblasts
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摘要:
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。 Abstract: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 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. -
参考文献
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图 1 3组小鼠皮肤Fb培养72 h后的形态 倒置相差显微镜×200。1A.空载体组细胞呈梭形、纺锤状；1B.P311组细胞变得宽大、扁平且伸出伪足；1C.P311+siMRTF-A组细胞呈梭形、纺锤状,未见明显细胞塌陷或伪足形成
注：对空载体组、P311组、P311+心肌素相关转录因子A小干扰RNA（siMRTF-A）组小鼠皮肤成纤维细胞（Fb）分别转染空载腺病毒、P311高表达腺病毒、P311高表达腺病毒+siMRTF-A
图 2 蛋白质印迹法检测的3组小鼠皮肤Fb培养72 h后MRTF-A、SRF及α-SMA的蛋白表达
注：MRTF-A为心肌素相关转录因子A,SRF为血清应答因子,α-SMA为α平滑肌肌动蛋白,GAPDH为3-磷酸甘油醛脱氢酶,siMRTF-A为MRTF-A小干扰RNA,Fb为成纤维细胞；条带上方1、2、3分别指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+siMRTF-A的P311+siMRTF-A组
图 3 胶原凝胶收缩实验检测的3组小鼠皮肤Fb培养72 h后的凝胶收缩面积。3A、3B、3C.分别为空载体组、P311组和P311+siMRTF-A组,图3B较图3A凝胶收缩面积明显增大,图3C较图3B凝胶收缩面积明显减小
注：对空载体组、P311组、P311+心肌素相关转录因子A小干扰RNA（siMRTF-A）组小鼠皮肤成纤维细胞（Fb）分别转染空载腺病毒、P311高表达腺病毒、P311高表达腺病毒+siMRTF-A；图中白圈内为凝胶,白圈外面积为凝胶收缩面积
图 4 蛋白质印迹法检测的2组小鼠皮肤Fb培养72 h后细胞、细胞质和细胞核内MRTF-A和SRF的蛋白表达
注：MRTF-A为心肌素相关转录因子A,SRF为血清应答因子,GAPDH为3-磷酸甘油醛脱氢酶,Fb为成纤维细胞；GAPDH为细胞和细胞质内蛋白表达的内参照,层粘连蛋白β为细胞核内蛋白表达的内参照；对空载体组、P311组细胞分别转染空载腺病毒、P311高表达腺病毒；条带上方1、2、3、4、5、6分别指空载体组细胞总蛋白、P311组细胞总蛋白、空载体组细胞质蛋白、P311组细胞质蛋白、空载体组细胞核蛋白、P311组细胞核蛋白
Table 1. 3组小鼠皮肤Fb培养72 h后MRTF-A、SRF及α-SMA的蛋白表达比较（
组别 样本数 MRTF-A SRF α-SMA 空载体组 3 0.82±0.08 0.97±0.39 0.991±0.016 P311组 3 1.10±0.07 1.50±0.49 1.420±0.237 P311+siMRTF-A组 3 0.43±0.08 0.64±0.23 0.846±0.165 F值 62.92 74.37 9.59 P值 <0.001 <0.001 0.014 P1值 0.009 0.001 0.046 P2值 0.001 0.011 0.570 P3值 <0.001 <0.001 0.013 注：MRTF-A为心肌素相关转录因子A,SRF为血清应答因子,α-SMA为α平滑肌肌动蛋白；对空载体组、P311组、P311+MRTF-A小干扰RNA（siMRTF-A）组小鼠皮肤成纤维细胞（Fb）分别转染空载腺病毒、P311高表达腺病毒、P311高表达腺病毒+siMRTF-A；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值分别为空载体组与P311组、空载体组与P311+siMRTF-A组、P311组与P311+siMRTF-A组比较所得 
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Table 2. 2组小鼠皮肤Fb培养72 h后细胞、细胞质和细胞核内MRTF-A和SRF的蛋白表达比较（
组别 样本数 细胞 细胞质 细胞核 MRTF-A SRF MRTF-A SRF MRTF-A SRF 空载体组 4 0.80±0.08 0.84±0.14 0.86±0.57 0.45±0.18 0.81±0.69 0.91±0.83 P311组 4 1.12±0.08 1.41±0.25 1.18±0.79 0.67±0.13 0.77±0.66 0.93±0.69 t值 5.86 3.95 3.77 3.97 0.53 1.33 P值 0.001 0.008 0.010 0.007 0.617 0.233 注：对空载体组和P311组小鼠皮肤成纤维细胞（Fb）分别转染空载腺病毒和P311高表达腺病毒；MRTF-A为心肌素相关转录因子A,SRF为血清应答因子
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