α-平滑肌肌动融合蛋白抑制成纤维细胞收缩的实验研究

Experimental study on the inhibition of fibroblast contraction by α smooth muscle actin fusion protein

目的 观察α 平滑肌肌动融合蛋白 (α SMA FP)对成纤维细胞收缩功能的影响。方法建立Wistar大鼠皮肤成纤维细胞三维立体凝胶培养模型。成纤维细胞在胶原凝胶中培养 5d后 ,实验组分别用 5、10、5 0、10 0、2 5 0mg/L的α SMA FP进行处理 ,根据α SMA FP剂量不同分为E1、E2 、E3 、E4和E5组。对照组分为空白对照 (C1)组和 2 5 0mg/Lα 骨骼肌肌动融合蛋白 (α SKA FP)处理(C2 )组。测量处理前后凝胶的直径 ,计算收缩率。检测E5组冲洗掉α SMA FP后 (wash组 )凝胶收缩率的变化情况。对传代后的成纤维细胞用α SMA FP或α SKA FP处理后进行α SMA免疫荧光染色。 结果 C1和C2 组凝胶收缩率为 (5 8.6± 3.1) %和 (5 6 .2± 4 .9) % ,二者差异无显著性意义 (P>0.0 5)。E1～E5组的收缩率分别为 (4 5 .5 6± 4 .1) %、(4 2 .3± 4 .2 ) %、(4 1.8± 3.6 ) %、(37.6±5 8) %、(2 6 .4± 4 .7) % ,wash组收缩率为 (4 8.2± 4 .9) %。E1～E5组与C1、C2 组比较 ,差异均有显著性意义 (P <0.0 5 ),与wash组比较 ,差异有非常显著性意义 (P <0.0 1)。α SMA FP着色于成纤维细胞纤维丝上 ,转染α SMA时着色不明显 ,而用α SMA FP处理后 ,α SMA着色明显。 结论 α SMA FP可特异性抑制成纤维细胞收缩 。

Objective To investigate the influence of α smooth muscle actin fusion protein ( α-SMA-FP) on fibroblast contraction, in order to find a new way to control scar contracture. Methods Three dimensional gel culture model of fibroblasts populated collagen lattices (FPCLs) was employed in the study. The fibroblasts were cultured in gel for 5 days. The cells in experimental group were processed by α-SMA-FP in dose of 5, 10, 50, 100 and 250 mg/L, respectively. The cells were therefore divided into E 1, E 2, E 3, E 4 and E 5 groups.Blank control was set to be C 1 group, and the cells processed by 250 mg/L of α-SMA-FP be C 2 group. The contraction rate was calculated by measuring the diameters of the gel before and after the procession. The change of contraction rate in E5 group was observed after the α-SMA-FP being rinsed out. Immunofluorescent staining of α-SMA-FP was carried out in fibroblasts. Results The contraction rate in C1 and C2 groups showed no difference,being (58.6±3.1)% and(56.2±4.9)% respectively, while that in E1 to E5 groups was (45.56±4.1)%,(42.3±4.2)%,(41.8±3.6)%,(37.6±5.8)% and (26.4±4.7)%, respectively. However, the contraction rate in E5 was (53.3±5.6)% after the α-SMA-FP had been rinsed out. The difference of the rates among control group and experimental groups, especially in E5 after α-SMA-FP being rinsed out, was significant (P<0.05 or 0.01). α-SMA-FP was located on the fibers of the fibroblasts as shown by staining, while the α-SMA was not stained.Nevertheless,the staining was obvious in control group. Conclusion α-SMA-FP could inhibit the contraction of fibroblasts specifically with dose dependent effect.