猪脱细胞真皮基质对小鼠创面毛囊再生中基质细胞衍生因子-1及Wnt3a/β-catenin信号通路表达的影响

doi:10.3877/cma.j.issn.1673-9450.2019.01.006

中华损伤与修复杂志(电子版) ›› 2019, Vol. 14 ›› Issue (01) : 26 -33. doi: 10.3877/cma.j.issn.1673-9450.2019.01.006

所属专题：文献；

论著

猪脱细胞真皮基质对小鼠创面毛囊再生中基质细胞衍生因子-1及Wnt3a/β-catenin信号通路表达的影响

杜烨¹, 冯自波¹, 李恭驰², 邹利军¹, 杨鸿¹, 王知¹, 陈江海², 潘银根³, 李炳辉¹^,^†(

)

^1. 430077　武汉，华中科技大学同济医学院附属梨园医院创面修复科
^2. 430022　武汉，华中科技大学同济医学院附属协和医院手外科
^3. 226200　启东市人民医院整形美容科

收稿日期:2018-12-05 出版日期:2019-02-01
通信作者: 李炳辉
基金资助:
湖北省自然科学基金面上项目(2017CKB892)

Effect of porcine acellular dermal matrix on the expression of stromal cell-derived factor-1 and Wnt3a/β-catenin signaling pathway in mice hair follicle regeneration

Ye Du¹, Zibo Feng¹, Gongchi Li², Lijun Zou¹, Hong Yang¹, Zhi Wang¹, Jianghai Chen², Yingen Pan³, Binghui Li¹^,^†()

^1. Department of Wound Repair, Liyuan Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430077, China
^2. Department of Hand Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
^3. Department of Plastic and Beauty, Qidong People′s Hospital, Qidong 226200, China

Received:2018-12-05 Published:2019-02-01
Corresponding author: Binghui Li
About author:
Corresponding author: Li Binghui, Email: libinghui2006@sina.com

全文 ( ) 下载PDF ( ) 导出引用

引用本文：

杜烨, 冯自波, 李恭驰, 邹利军, 杨鸿, 王知, 陈江海, 潘银根, 李炳辉. 猪脱细胞真皮基质对小鼠创面毛囊再生中基质细胞衍生因子-1及Wnt3a/β-catenin信号通路表达的影响[J/OL]. 中华损伤与修复杂志(电子版), 2019, 14(01): 26-33.

Ye Du, Zibo Feng, Gongchi Li, Lijun Zou, Hong Yang, Zhi Wang, Jianghai Chen, Yingen Pan, Binghui Li. Effect of porcine acellular dermal matrix on the expression of stromal cell-derived factor-1 and Wnt3a/β-catenin signaling pathway in mice hair follicle regeneration[J/OL]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2019, 14(01): 26-33.

目的

探讨猪脱细胞真皮基质(ADM)对小鼠创面毛囊再生中基质细胞衍生因子-1(SDF-1)及Wnt3a/β-catenin信号通路表达的影响。

方法

取新鲜猪皮，经脱细胞处理后制成微粒状，灭菌，密封，常温保存，以备后面实验使用。制作18只C57BL/6小鼠背部全层皮肤缺损模型，以脊柱为中线，在左右各制作直径为6 mm的缺损，左右侧分别以纱布和猪ADM覆盖，隔天纱布侧换药，猪ADM侧不做处理，于模型建立第7天，按照所用材料不同分为纱布组与猪ADM组，再按所取部位不同分为纱布窗口组、猪ADM窗口组、纱布创面组、猪ADM创面组、纱布创缘组、猪ADM创缘组，其中9只小鼠组织用于蛋白质印迹法检测，另外9只小鼠组织用于免疫组织化学检测。通过蛋白质印迹法检测肿瘤坏死因子-α(TNF-α)、β-catenin、转化生长因子-β(TGF-β)、Wnt3a、血管内皮生长因子(VEGF)、SDF-1、成纤维细胞生长因子(FGF)2、FGF9、AKT的蛋白表达，免疫组织化学检测Wnt3a、SDF-1、FGF2、FGF9的表达。数据比较采用独立样本t检验。

结果

在小鼠模型建立第7天创面中，蛋白质印迹法检测β-catenin、Wnt3a、SDF-1的蛋白表达量均是猪ADM窗口组(0.533±0.058、0.446±0.039、0.972±0.048)高于纱布窗口组(0.401±0.005、0.132±0.022、0.175±0.036)，差异均有统计学意义(t＝3.996、12.230、23.130，P值均小于0.05)。β-catenin、Wnt3a、SDF-1的蛋白表达量均是猪ADM创面组(0.557±0.009、0.626±0.066、0.868±0.102)高于纱布创面组(0.302±0.010、0.109±0.019、0.036±0.009)，差异均有统计学意义(t＝32.830、13.020、14.130，P值均小于0.05)。Wnt3a、SDF-1的蛋白表达量均是猪ADM创缘组(0.419±0.014、0.370±0.069)高于纱布创缘组(0.115±0.020、0.056±0.007)，差异均有统计学意义(t＝21.460、7.825，P值均小于0.05)。免疫组织化学检测结果显示Wnt3a、SDF-1、FGF2、FGF9在猪ADM组中的表达高于纱布组，且阳性细胞主要分布于毛囊细胞周围。

结论

猪ADM在小鼠创面中可能通过上调SDF-1及Wnt3a/β-catenin信号通路的表达而促进毛囊的再生。

关键词: 创伤和损伤, 毛囊, 猪, 小鼠, 脱细胞真皮基质, 基质细胞衍生因子-1, Wnt3a/β-catenin信号通路

Objective

To investigate the effects of porcine acellular dermal matrix (ADM) on the expression of stromal cell-derived factor-1 (SDF-1) and Wnt3a/β-catenin signaling pathway in mouse hair follicle regeneration.

Methods

Fresh pig skin was taken for dermal matrix, and after decellularization treatment was sterilized, sealed, and stored at room temperature for later use. Eighteen full-thickness skin defect models of C57BL/6 mice were made with the spine as the midline, 6 mm diameter defects were made on the left and right sides. Left and right sides were covered with gauze and porcine ADM, respectively. The gauze side dressing changed the next day, but the porcine ADM side did nothing. On the 7th day after the model was established, according to different materials, it was divided into gauze group and porcine ADM group, and then according to the different parts taken divided into gauze window group, porcine ADM window group, gauze wound group, porcine ADM wound group, gauze wound edge group and porcine ADM wound edge group. Tissue of nine mice were taken for Western blotting test protein expressions of tumor necrosis factor-α(TNF-α), β-catenin, transforming growth factor-β(TGF-β), Wnt3a, vascular endothelial growth factor(VEGF), stromalcell derived factor-1(SDF-1), fibroblast growth factor2(FGF2), fibroblast growth factor9(FGF9), AKT. The others were taken for immunohistochemistry for detection of Wnt3a, SDF-1, FGF2, FGF9.Data comparisons were performed using independent sample t test.

Results

In the 7th day wound of mice, Western blotting showed that the protein expression levels of β-catenin, Wnt3a, SDF-1 were higher in porcine ADM window group(0.533±0.058, 0.446±0.039, 0.972±0.048) than the gauze window group (0.401±0.005, 0.132±0.022, 0.175±0.036), and the differences were statistically significant (t=3.996, 12.230, 23.130; with P values below 0.05). The protein expression levels of β-catenin, Wnt3a, SDF-1 were higher in the porcine ADM wound group(0.557±0.009, 0.626±0.066, 0.868±0.102) than in the gauze wound group (0.302±0.010, 0.109±0.019, 0.036±0.009), and the differences were statistically significant (t=32.830, 13.020, 14.130; with P values below 0.05). And the protein expression levels of Wnt3a, SDF-1 in the porcine ADM wound margin group(0.419±0.014, 0.370±0.069) were higher than those in the gauze margin group(0.115±0.020, 0.056±0.007), and the differences were statistically significant (t=21.460, 7.825; with P values below 0.05). Immunohistochemistry results showed that Wnt3a, SDF-1, FGF2 and FGF9 in the porcine ADM group were higher than those in the gauze group, and the positive cells were mainly distributed around hair follicle cells.

Conclusion

In the wound repair process, porcine ADM may promotes the regeneration of hair follicles by up-regulating the expression of SDF-1 and Wnt3a/β-catenin signaling pathway.

Key words: Wounds and injuries, Hair follicle, Swine, Mice, Acellular dermal matrix, Stromal cell-derived factor-1, Wnt3a/beta-catenin signaling pathway

图1 小鼠模型建立第7天创面情况。A示硅胶支撑架被缝于创面上；B示去掉硅胶支撑架可见窗口处(箭头示)有肉芽组织

图2 各组全层皮肤缺损创面小鼠在模型建立第7天TNF-α、β-catenin、TGF-β、Wnt3a、VEGF、SDF-1、FGF2、FGF9、AKT的蛋白质分析结果。ADM为脱细胞真皮基质；TNF-α为肿瘤坏死因子-α；TGF-β为转化生长因子-β；VEGF为血管内皮生长因子；SDF-1为基质细胞衍生因子-1；FGF为成纤维细胞生长因子；GAPDH为甘油醛-3-磷酸脱氢酶

表1 纱布窗口组和猪ADM窗口组全层皮肤缺损创面小鼠在模型建立第7天各指标表达量的比较(±s)

组别	鼠数(只)	TNF-α	β-catenin	TGF-β	Wnt3a	VEGF
纱布窗口组	9	0.754±0.033	0.401±0.005	0.869±0.121	0.132±0.022	0.219±0.063
猪ADM窗口组	9	0.348±0.033	0.533±0.058	0.301±0.088	0.446±0.039	0.563±0.011
t值	?	15.170	3.996	6.580	12.230	9.350
P值	?	<0.05	<0.05	<0.05	<0.05	<0.05
组别	鼠数(只)	SDF-1	FGF2	FGF9	AKT
纱布窗口组	9	0.175±0.036	0.372±0.113	0.177±0.027	0.162±0.029
猪ADM窗口组	9	0.972±0.048	0.568±0.129	0.415±0.051	0.256±0.017
t值	?	23.130	1.977	7.087	4.931
P值	?	<0.05	0.119	<0.05	<0.05

表1 纱布窗口组和猪ADM窗口组全层皮肤缺损创面小鼠在模型建立第7天各指标表达量的比较(±s)

组别	鼠数(只)	TNF-α	β-catenin	TGF-β	Wnt3a	VEGF
纱布窗口组	9	0.754±0.033	0.401±0.005	0.869±0.121	0.132±0.022	0.219±0.063
猪ADM窗口组	9	0.348±0.033	0.533±0.058	0.301±0.088	0.446±0.039	0.563±0.011
t值	?	15.170	3.996	6.580	12.230	9.350
P值	?	<0.05	<0.05	<0.05	<0.05	<0.05
组别	鼠数(只)	SDF-1	FGF2	FGF9	AKT
纱布窗口组	9	0.175±0.036	0.372±0.113	0.177±0.027	0.162±0.029
猪ADM窗口组	9	0.972±0.048	0.568±0.129	0.415±0.051	0.256±0.017
t值	?	23.130	1.977	7.087	4.931
P值	?	<0.05	0.119	<0.05	<0.05

表2 纱布创面组和猪ADM创面组全层皮肤缺损创面小鼠在模型建立第7天各指标表达量的比较(±s)

组别	鼠数(只)	TNF-α	β-catenin	TGF-β	Wnt3a	VEGF
纱布创面组	9	0.537±0.079	0.302±0.010	0.712±0.087	0.109±0.019	0.179±0.007
猪ADM创面组	9	0.479±0.021	0.557±0.009	0.674±0.017	0.626±0.066	0.754±0.037
t值	?	1.227	32.830	0.743	13.020	26.870
P值	?	0.287	<0.05	0.499	<0.05	<0.05
组别	鼠数(只)	SDF-1	FGF2	FGF9	AKT
纱布创面组	9	0.036±0.009	0.126±0.025	0.065±0.024	0.091±0.005
猪ADM创面组	9	0.868±0.102	0.679±0.072	0.382±0.029	0.281±0.041
t值	?	14.130	12.580	14.690	8.011
P值	?	<0.05	<0.05	<0.05	<0.05

表2 纱布创面组和猪ADM创面组全层皮肤缺损创面小鼠在模型建立第7天各指标表达量的比较(±s)

组别	鼠数(只)	TNF-α	β-catenin	TGF-β	Wnt3a	VEGF
纱布创面组	9	0.537±0.079	0.302±0.010	0.712±0.087	0.109±0.019	0.179±0.007
猪ADM创面组	9	0.479±0.021	0.557±0.009	0.674±0.017	0.626±0.066	0.754±0.037
t值	?	1.227	32.830	0.743	13.020	26.870
P值	?	0.287	<0.05	0.499	<0.05	<0.05
组别	鼠数(只)	SDF-1	FGF2	FGF9	AKT
纱布创面组	9	0.036±0.009	0.126±0.025	0.065±0.024	0.091±0.005
猪ADM创面组	9	0.868±0.102	0.679±0.072	0.382±0.029	0.281±0.041
t值	?	14.130	12.580	14.690	8.011
P值	?	<0.05	<0.05	<0.05	<0.05

表3 纱布创缘组和猪ADM创缘组全层皮肤缺损创面小鼠在模型建立第7天各指标表达量的比较(±s)

组别	鼠数(只)	TNF-α	β-catenin	TGF-β	Wnt3a	VEGF
纱布创缘组	9	0.680±0.026	0.395±0.051	0.690±0.044	0.115±0.020	0.197±0.028
猪ADM创缘组	9	0.184±0.001	0.447±0.016	0.216±0.021	0.419±0.014	0.633±0.048
t值	?	34.150	1.708	16.680	21.460	13.600
P值	?	<0.05	0.163	<0.05	<0.05	<0.05
组别	鼠数(只)	SDF-1	FGF2	FGF9	AKT
纱布创缘组	9	0.056±0.007	0.153±0.007	0.053±0.018	0.058±0.023
猪ADM创缘组	9	0.370±0.069	0.502±0.108	0.258±0.031	0.207±0.016
t值	?	7.825	5.595	9.801	9.271
P值	?	<0.05	<0.05	<0.05	<0.05

表3 纱布创缘组和猪ADM创缘组全层皮肤缺损创面小鼠在模型建立第7天各指标表达量的比较(±s)

组别	鼠数(只)	TNF-α	β-catenin	TGF-β	Wnt3a	VEGF
纱布创缘组	9	0.680±0.026	0.395±0.051	0.690±0.044	0.115±0.020	0.197±0.028
猪ADM创缘组	9	0.184±0.001	0.447±0.016	0.216±0.021	0.419±0.014	0.633±0.048
t值	?	34.150	1.708	16.680	21.460	13.600
P值	?	<0.05	0.163	<0.05	<0.05	<0.05
组别	鼠数(只)	SDF-1	FGF2	FGF9	AKT
纱布创缘组	9	0.056±0.007	0.153±0.007	0.053±0.018	0.058±0.023
猪ADM创缘组	9	0.370±0.069	0.502±0.108	0.258±0.031	0.207±0.016
t值	?	7.825	5.595	9.801	9.271
P值	?	<0.05	<0.05	<0.05	<0.05

图3 小鼠模型建立第7天，猪ADM组与纱布组创面组织的Wnt3a、SDF-1、FGF2、FGF9免疫组织化学检测结果显示猪ADM组的阳性细胞(箭头示)多于纱布组，且主要分布在毛囊细胞周围(免疫组织化学染色，×200)。ADM为脱细胞真皮基质，SDF-1为基质细胞衍生因子-1，FGF为成纤维细胞生长因子

[1]	Chua AW, Khoo YC, Tan BK, et al. Skin tissue engineering advances in severe burns: review and therapeutic applications[J]. Burns Trauma, 2016, 4: 3.
[2]	Bondioli E, Fini M, Veronesi F, et al. Development and evaluation of decellularized membrane from human dermis[J]. J Tissue Eng Regen Med, 2014, 8(4): 325-336.
[3]	Eming SA, Hubbell JA. Extracellular matrix in angiogenesis: dynamic structures with translational potential[J]. Exp Dermatol, 2011, 20(7): 605-613.
[4]	Yan W, Liu H, Deng X, et al. Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway[J]. J Tissue Eng Regen Med, 2018, 12(3): e1461-e1473.
[5]	Nie C, Yang D, Morris SF. Local delivery of adipose-derived stem cells via acellular dermal matrix as a scaffold: a new promising strategy to accelerate wound healing[J]. Med Hypotheses, 2009, 72(6): 679-682.
[6]	Sigalove S. Options in Acellular Dermal Matrix-Device Assembly[J]. Plast Reconstr Surg, 2017, 140(6S Prepectoral Breast Reconstruction): 39S-42S.
[7]	Hughes OB, Rakosi A, Macquhae F, et al. A Review of Cellular and Acellular Matrix Products: Indications, Techniques, and Outcomes[J]. Plast Reconstr Surg, 2016, 138(3 Suppl): 138S-147S.
[8]	Zhang Z, Lv L, Mamat M, et al. Xenogenic (porcine) acellular dermal matrix is useful for the wound healing of severely damaged extremities[J]. Exp Therapeutic Med, 2014, 7(3): 621-624.
[9]	王永飞, 程勇, 许喜生. 毛囊干细胞在创面愈合中的应用进展[J]. 现代医药卫生, 2015, 31(4): 548-551.
[10]	Bermudez DM, Xu J, Herdrich BJ, et al. Inhibition of stromal cell-derived factor-1α further impairs diabetic wound healing[J]. J Vasc Surg, 2011, 53(3): 774-784.
[11]	Nusse R. Wnt signaling and stem cell control[J]. Cell Res, 2008, 18(5): 523-527.
[12]	Clevers H, Nusse R. Wnt/β-catenin signaling and disease[J]. Cell, 2012, 149(6): 1192-1205.
[13]	Kishimoto J, Burgeson RE, Morgan1 BA. Wnt signaling maint ains t he hai r-inducing act ivi ty of t he dermal papilla[J]. Genes Dev, 2000, 14(10): 1181-1185.
[14]	Carre AL, James AW, MacLeod L, et al. Interaction of wingless protein (Wnt), transforming growth factor-beta1, and hyaluronan production in fetal and postnatal fibroblasts[J]. Plast Reconstr Surg, 2010, 25(1): 74-88.
[15]	Ito M, Yang Z, Andl T, et al. Wnt-dependent de novo hair follele regeneration in adult mouse skin wound after wounding[J]. Nature, 2007, 447(7142): 316-320.
[16]	Amini-Nik S, Glancy D, Boimer C, et al. Pax7 expressing cells contribute to dermal wound repair, regulating scar size through a β-catenin mediated process[J]. Stem Cells, 2011, 29(9): 1371-1379.
[17]	Kanda S, Miyata Y, Kanetake H. Fibroblast growth factor-2-mediated capillary morphogenesis of endothelial cells requires signals via Flt-1/vascular endothelial growth factor receptor-1: possible involvement of c-Akt[J]. J Biol Chem, 2004, 279(6): 4007-4016.
[18]	Fei Y, Xiao L, Doetschman T, et al. Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway[J]. J Biol Chem, 2011, 286(47): 40575-40583.
[19]	Hung IH, Yu K, Lavine KJ, et al. FGF9 regulates early hypertrophic chondrocyte differentiation and skeletal vascularization in the developing stylopod[J]. Dev Biol, 2007, 307(2): 300-313.
[20]	Giannouli CC, Kletsas D. TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2[J]. Cell Signal, 2006, 18(9): 1417-1429.
[21]	Cheon SS, Wei Q, Gurung A, et al. Beta-catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing[J]. FASEB J, 2006, 20(6): 692-701.
[22]	Wang X, Chen H, Tian R, et al. Macrophages induce AKT/β-catenin-dependent Lgr5＋ stem cell activation and hair follicle regeneration through TNF[J]. Nat Commun, 2017, 8: 1409.

[1]	周容, 张亚萍, 廖宇, 程晓萍, 管玉龙, 潘广玉, 闫杰, 王贤芝, 苟中山, 潘登科, 李巅远. 超声在基因编辑猪-猴异种并联式心脏移植术中的应用价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(06): 617-623.
[2]	李璐璐, 马利红, 金佳佳, 谷伟. 干扰素基因刺激因子通过肺巨噬细胞胞葬功能调控急性肺损伤小鼠修复的研究[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(02): 97-103.
[3]	唐亦骁, 陈峻, 连正星, 胡海涛, 鲁迪, 徐骁, 卫强. 白果内酯对小鼠肝缺血再灌注损伤保护作用研究[J/OL]. 中华移植杂志(电子版), 2024, 18(05): 278-282.
[4]	张子旭, 郑俊炯, 罗云, 林天歆. 腹腔镜肾部分切除术离体猪肾培训模型的构建[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(03): 277-283.
[5]	李绍杰, 谢奇峰, 李绍春, 杨子昂, 黄永刚, 陈吉彩, 杜舟, 王平, 张剑, 唐健雄. 复合基底膜生物补片应用于腹股沟疝Lichtenstein修补术的随机、对照、多中心临床研究[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(02): 132-138.
[6]	杨阳, 王琤, 周文土, 周冰. Caveolae/Caveolin-1与膜胆固醇共同调控小鼠BMSCs成骨分化[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(03): 137-142.
[7]	王冰倩, 汪振星, 夏芸. 利用微小毛发模型验证α-倒捻子素在毛囊中的抗氧化特性的研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(02): 83-89.
[8]	黄建玲, 王丽. 猪链球菌脑膜炎一例报道[J/OL]. 中华脑科疾病与康复杂志(电子版), 2024, 14(03): 187-189.
[9]	蒋心怡, 顾丹丹, 叶艳, 缪佳蓉. RNA测序研究抗菌肽KT2治疗溃疡性结肠炎的作用机制[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(01): 8-15.
[10]	王瑛, 朱名超, 尹琼. 以贫血乏力为首发症状的人感染猪链球菌病一例[J/OL]. 中华临床实验室管理电子杂志, 2024, 12(01): 43-47.
[11]	临床多学科协作专家组. 腹盆部创伤急诊CT 专家推荐指南（2024）[J/OL]. 中华诊断学电子杂志, 2024, 12(04): 222-229.
[12]	田峰瑞, 蒋锦源, 李阳, 张连阳. Morel-Lavallée 损伤继发血清肿一例[J/OL]. 中华诊断学电子杂志, 2024, 12(04): 245-248.
[13]	陈念, 张连阳. 严重创伤救治中全血输注进展[J/OL]. 中华诊断学电子杂志, 2024, 12(03): 145-148.
[14]	翟亚奇, 刘震宇, 刘圣圳, 冯建聪, 高飞, 宁波, 冯秀雪, 李惠凯, 李明阳, 令狐恩强. 急性上消化道出血活体猪模型在超级微创内镜下急诊止血培训中的应用[J/OL]. 中华胃肠内镜电子杂志, 2024, 11(02): 127-131.
[15]	陈立如, 李志刚, 李春光. 一个标准的动物肺叶切除模型——猪肺的基本解剖[J/OL]. 中华胸部外科电子杂志, 2024, 11(01): 16-22.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Effect of porcine acellular dermal matrix on the expression of stromal cell-derived factor-1 and Wnt3a/β-catenin signaling pathway in mice hair follicle regeneration

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Effect of porcine acellular dermal matrix on the expression of stromal cell-derived factor-1 and Wnt3a/β-catenin signaling pathway in mice hair follicle regeneration