脂肪干细胞基质胶修复增生性瘢痕的作用机制

The mechanism of stromal vascular fraction-gel in repairing hyperplastic scar

目的 探讨脂肪干细胞基质胶(SVF-gel)在治疗大鼠增生性瘢痕中的作用及机制.方法 分离提取SVF-gel,并建立大鼠增生性瘢痕模型.瘢痕模型大鼠共12只,根据体重及瘢痕大小采取完全随机对照原则分为实验组和对照组(每组6只),SVF-gel 0.2 ml均匀注射至瘢痕内,对照组注射等量生理盐水.连续注射6周,观察大鼠瘢痕的面积变化情况.术后对大鼠瘢痕组织取材,进行蛋白水平检测.制备SVF-gel条件培养基(SVF-CM),利用细胞计数试剂盒(CCK-8)实验观察SVF-CM对成纤维细胞(Fb)增殖的影响并确定最适浓度及时间.利用基因敲低试剂盒,获得敲低转化生长因子-β1(TGF-β1)基因的成纤维细胞并分组,(1)Fb+NC siRNA,(2)SVF-CM+NC siRNA,(3)SVF-CM+NC siRNA+骨膜素(Periostin),(4)SVF-CM+TGF-β1 siRNA,(5)SVF-CM+TGF-β1 siRNA+Periostin.通过蛋白质印迹法(Western blot),观察 Periostin、TGF-β1、Ⅰ 型及Ⅲ型胶原的表达.通过Graphpad进行统计学结果分析t检验.结果 体内实验,对照组瘢痕面积高于实验组[瘢痕表面面积:(21.18±2.53)mm^(2) 比(7.49±1.31)mm^(2),t=10.75,P<0.01;苏木精-伊红(HE)染色真皮层瘢痕面积:(17.69±3.22)mm^(2) 比(2.43±1.37)mm^(2),t=9.75,P<0.01].Western blot显示,Periostin以及TGF-β1的表达,对照组高于实验组(Western blot蛋白条带灰度值,Periostin:0.91±0.06 比 0.52±0.02,t=14.16,P<0.01;TGF-β1:1.00±0.03 比 0.52±0.06,t=15.46,P<0.01).体外实验,CCK-8实验表明40%SVF-CM处理成纤维细胞48 h,成纤维细胞增殖能力,对照组高于实验组[细胞存活率:(100.00±6.95)%比(79.89±2.51)%,t=7.11,P<0.01].通过Western blot观察,在TGF-β1被敲减后,Periostin、TGF-β1蛋白以及Ⅰ型、Ⅲ型胶原蛋白表达,B 组高于 D 组(Periostin:0.763±0.005 比 0.602±0.004,t=44.24,P<0.01;TGF-β1:0.694±0.026 比 0.588±0.033,t=4.381,P<0.05;Ⅰ 型胶原:0.749±0.002 比 0.472±0.007,t=63.51,P<0.01;Ⅲ型胶原:0.752±0.019 比 0.593±0.009,t=13.34,P<0.01);当加入外源性 Periostin 后(E组),Periostin以及Ⅰ型、Ⅲ型胶原蛋白表达,E组高于D组(Periostin:0.826±0.010比0.602±0.004,t=44.24,P<0.01;Ⅰ 型胶原:0.947±0.002 比 0.472±0.007,t=109.40,P<0.01;Ⅲ型胶原:0.924±0.019 比 0.593±0.009,t=27.33,P<0.01).结论 脂肪细胞基质胶可抑制 TGF-β1/Smad信号通路,从而降低Periostin表达,抑制成纤维细胞增殖,减少Ⅰ、Ⅲ型胶原蛋白产生从而防止增生性瘢痕的形成.

Objective To investigate the mechanism of stromal vascular fraction-gel(SVF-gel)in the treatment of hypertrophic scar in rats.Methods SVF-gel was isolated and extracted from adipose tis-sue,and the rat hypertrophic scar model was established.A total of 12 scar model rats were divided into experimental group and control group(6 rats in each group)according to the principle of complete random control according to body weight and scar size,and 0.2 ml of SVF-gel prepared in advance was evenly in-jected into the scar,and the control group was injected with the same amount of normal saline.After 6 weeks of continuous injection,the area of the scar in the rats was observed.The scar tissue of rats was collected after operation,and the level of scar protein was detected.SVF-gel conditioned medium(SVF-CM)was prepared,and the effects of different concentrations of SVF-CM on fibroblast proliferation were observed by cell counting kit-8(CCK-8)experiment,and the optimal concentration and time were de-termined.Using the gene knockdown kit,the fibroblasts with transforming growth factor-β1(TGF-β1)gene knockdown were obtained,and following experimental groups were set up:group A[fibroblast(Fb)+NC small interfering RNA(siRNA)],group B(SVF-CM+NC siRNA),group C(SVF-CM+NC siRNA+periostin),group D(SVF-CM+TGF-β1 siRNA),group E(SVF-CM+TGF-β1 siRNA+Periostin).Western blotting was used to observe the expression of Periostin,TGF-β1,typeⅠand typeⅢcollagen.Graphpad software was used to analyze the statistical results by t test.Results In vivo experiments,the scar area in the control group was greater than that in the experimental group[scar area:(21.18±2.53)mm^(2) vs.(7.49±1.31)mm^(2),t=10.75,P<0.01;hematoxylin and eosin(HE)staining of the dermal scar area:(17.69±3.22)mm^(2) vs.(2.43±1.37)mm^(2),t=9.75,P<0.01).Western blotting showed that the expression of Periostin and TGF-β1 was higher in the control group than in the experimental group(Periostin:0.91±0.06 vs.0.52±0.02,t=14.16,P<0.01;TGF-β1:1.00±0.03 vs.0.52±0.06,t=15.46,P<0.01).In vitro experiments,CCK-8 experiments showed that the proliferation ability of fibroblasts treated with 40%SVF-CM for 48 h was higher in the control group than in the experimental group[(100.00±6.95)%vs.(79.89±2.51)%,t=7.11,P<0.01].Western blotting showed that af-ter TGF-β1 knockdown,the expression of Periostin,TGF-β1 protein,typeⅠand typeⅢcollagen was higher in group B than in group D(Periostin:0.763±0.005 vs.0.602±0.004,t=44.24,P<0.01;TGF-β1:0.694±0.026 vs.0.588±0.033,t=4.381,P<0.05;CollagenⅠ:0.749±0.002 vs.0.472±0.007,t=63.51,P<0.01;CollagenⅢ:0.752±0.019 vs.0.593±0.009,t=13.34,P<0.01).When exogenous Periostin was added(group E),the expression of periostin and typeⅠand typeⅢcollagen was higher in group E than in group D(Periostin:0.826±0.010 vs.0.602±0.004,t=44.24,P<0.01;CollagenⅠ:0.947±0.002 vs.0.472±0.007,t=109.40,P<0.01;CollagenⅢ:0.924±0.019 vs.0.593±0.009,t=27.33,P<0.01).Conclusion SVF-gel can inhibit TGF-β1/Smad signaling pathway,thereby reducing the expression of Periostin,inhibiting fibroblast proliferation,and reducing the production of collagenⅠandⅢto prevent the formation of hypertrophic scars.