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摘要:
目的 探讨Wnt9a在慢性创面愈合过程中的表达变化、作用及其可能机制。 方法 该研究为成组设计实验研究。收集8例糖尿病足溃疡患者（男5例、女3例,年龄45~72岁）的慢性创面组织及周围正常皮肤组织,采用酶联免疫吸附测定（ELISA）法和免疫荧光染法检测Wnt9a的表达。取8只6~8周龄C57BL/6雄性小鼠,在背部制作1个全层皮肤缺损创面,并采用随机数字表法（分组方法下同）分为对照组（不进行其他处理）和慢性创面组（在创缘皮下注射M1型巨噬细胞来源外泌体以构建慢性创面模型）,每组4只。于造模后7 d,采用ELISA法检测2组小鼠创面组织中Wnt9a的表达。另取16只6~8周龄C57BL/6雄性小鼠,同前建立慢性创面模型,分为空载对照组和Wnt9a过表达组（每组8只）,分别于创缘皮下注射增强型绿色荧光蛋白空载腺病毒（AV-eGFP）、Wnt9a基因增强型绿色荧光蛋白重组腺病毒（AV-Wnt9a-eGFP）,于造模后3、7、14 d计算残余创面面积百分比,于造模后14 d采用蛋白质印迹法检测创面组织中Ⅰ型和Ⅲ型胶原蛋白的表达。取收集的人正常皮肤组织,提取成纤维细胞（Fb）,分为分别感染AV-eGFP、AV-Wnt9a-eGFP的空载对照组、Wnt9a过表达组,采用蛋白质印迹法检测感染72 h后的Wnt9a的蛋白表达,采用划痕试验检测划痕后48 h细胞迁移率。另取人正常皮肤Fb,分为分别转染相应小干扰RNA（siRNA）的Wnt9a特异性siRNA（siRNA-Wnt9a）组、阴性对照siRNA（siRNA-NC）组,采用划痕试验检测划痕后48 h细胞迁移率。另取人正常皮肤Fb,分为同前处理的空载对照组和Wnt9a过表达组,感染72 h后,行转录组测序,筛选差异表达基因（DEG）并对DEG进行基因本体论（GO）和京都基因与基因组百科全书富集分析。细胞实验样本数均为3。 结果 ELISA法和免疫荧光法结果显示,人慢性创面组织中Wnt9a表达水平显著低于正常皮肤组织（t值分别为7.68、10.25,P<0.05）。造模后7 d,慢性创面组小鼠创面组织中Wnt9a的表达水平显著低于对照组（t=5.12,P<0.05）。Wnt9a过表达组小鼠造模后3、7、14 d残余创面面积百分比均显著低于空载对照组（t值分别为3.90、6.62、5.73,P<0.05）。造模后14 d,Wnt9a过表达组小鼠创面组织中Ⅰ型和Ⅲ型胶原蛋白的表达水平均显著低于空载对照组（t值分别为6.25、5.48,P<0.05）。Wnt9a过表达组Fb中Wnt9a蛋白表达水平显著高于空载对照组（t=6.96,P<0.05）。划痕后48 h,Wnt9a过表达组细胞迁移率为（71.6±6.4）%,显著高于空载对照组的（38.5±2.4）%（t=8.31,P<0.05）。siRNA-Wnt9a组细胞迁移率为（15±3）%,显著低于siRNA-NC组的（32±4）%（t=5.93,P<0.05）。感染72 h后,与空载对照组相比,Wnt9a过表达组Fb中显著下调的DEG包括多个胶原蛋白家族基因,Wnt9a过表达组Fb中DEG显著富集于非经典Wnt信号通路。 结论 Wnt9a在人和小鼠慢性创面组织中表达下调；过表达Wnt9a可能通过非经典Wnt信号通路促进Fb迁移和胶原重塑,从而加速慢性创面愈合。 Abstract:Objective To investigate the expression changes, role, and its possible mechanism of Wnt9a in chronic wound healing. Methods This study was a group-designed experimental research.The chronic wound tissue and adjacent normal skin tissue were collected from 8 patients with diabetic foot ulcers, including 5 males and 3 females, aged 45-72 years. The expression of Wnt9a was detected by enzyme-linked immunosorbent assay (ELISA) method and immunofluorescence method. Eight male C57BL/6 mice aged 6-8 weeks were used to establish a full-thickness skin defect wound on the back and were divided into control group with no additional treatment and chronic wound group subcutaneously injected with M1 macrophage derived exosomes at the wound edge to construct a chronic wound model using a random number table method (the same grouping method below), with 4 mice in each group. At 7 days after modeling, the Wnt9a expressions in the wound tissue of mice in two groups was detected by ELISA method. Additional 16 male C57BL/6 mice aged 6-8 weeks were used to establish the chronic wound model as before and were divided into blank control group and Wnt9a overexpression group, with 8 mice in each group, which were injected subcutaneously at the wound edge with enhanced green fluorescent protein empty adenovirus (AV-eGFP) and Wnt9a gene recombinant adenovirus expressing enhanced green fluorescent protein (AV-Wnt9a-eGFP), respectively. At 3, 7, and 14 days after modeling, the percentages of residual wound area were calculated; at 14 days after modeling, the expressions of type Ⅰ and type Ⅲ collagen protein were detected by Western blotting. The collected human normal skin tissue was used to extract fibroblasts (Fbs), which were divided into empty control group infected with AV-eGFP and Wnt9a overexpression group infected with AV-Wnt9a-eGFP. The protein expression of Wnt9a at 72 h after infection was detected by Western blotting, and the cell migration rate at 48 h after scratching was detected by scratch test. Additional human normal skin Fbs were collected and divided into Wnt9a specific small interfering RNA (siRNA-Wnt9a) group and negative control small interfering RNA (siRNA-NC) group, which were transfected with corresponding small interfering RNA (siRNA). Scratch test was conducted to detect the cell migration rate at 48 h after scratching. Additional human normal skin Fbs were taken and divided into empty control group and Wnt9a overexpression group treated as before. At 72 h after infection, transcriptome sequencing was performed to screen for differentially expressed genes (DEGs); the gene ontology (GO) and Kyoto encyclopedia of genes and genomes enrichment analysis were performed on DEGs. The sample number in all cell experiments was 3. Results The results of ELISA method and immunofluorescence method showed that the expression level of Wnt9a in human chronic wound tissue was significantly lower than that in normal skin tissue (with t values of 7.68 and 10.25, respectively, P<0.05). At 7 days after modeling, the expression level of Wnt9a in the wound tissue of mice in chronic wound group was significantly lower than that in control group (t=5.12, P<0.05). The percentages of residual wound area of mice in Wnt9a overexpression group were significantly lower than those in empty control group at 3, 7, and 14 days after modeling (with t values of 3.90, 6.62, and 5.73, respectively, P<0.05). At 14 days after modeling, the protein expression levels of type Ⅰ and type Ⅲ collagen in the wound tissue of mice in Wnt9a overexpression group were significantly lower than those in empty control group (with t values of 6.25 and 5.48, respectively, P<0.05). The Wnt9a protein expression level in Fbs in Wnt9a overexpression group was significantly higher than that in empty control group (t=6.96, P<0.05). At 48 h after scratching, the cell migration rate in Wnt9a overexpression group was (71.6±6.4)%, which were significantly higher than (38.5±2.4)% in empty control group (t=8.31, P<0.05). The cell migration rate in siRNA-Wnt9a group was (15±3)%, which were significantly lower than (32±4)% in siRNA-NC group (t=5.93, P<0.05). At 72 h after infection, compared with that in empty control group, the significantly downregulated DEGs in Fbs in Wnt9a overexpression group included multiple collagen family genes, and DEGs in Fbs in Wnt9a overexpression group were significantly enriched in the non-classical Wnt signaling pathway. Conclusions Wnt9a expression is downregulated in chronic wound tissue of human and mice, and the overexpressed Wnt9a may promote migration of Fbs and collagen remodeling through non-classical Wnt signaling pathway, thereby accelerating chronic wound healing. -
Key words:
- Fibroblasts /
- Wnt signaling pathway /
- Chronic wounds /
- Wound healing, Wnt9a /
- Cell migration /
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参考文献
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图 2 2组慢性创面小鼠造模后各时间点创面愈合情况。2A、2B、2C、2D.分别为空载对照组小鼠造模后0（即刻）、3、7、14 d创面；2E、2F、2G、2H.分别为Wnt9a过表达组小鼠造模后 0、3、7、14 d创面,图2F、2G、2H残余创面面积分别小于图2B、2C、2D
注：分别于空载对照组、Wnt9a过表达组小鼠创缘皮下注射增强型绿色荧光蛋白空载腺病毒、Wnt9a基因增强型绿色荧光蛋白重组腺病毒
图 3 2组小鼠造模后14 d慢性创面组织中Ⅰ型和Ⅲ型胶原蛋白的表达及胶原纤维排列情况。3A.蛋白条带图；3B、3C.分别为空载对照组、Wnt9a过表达组,Wnt9a过表达组创面组织中胶原纤维排列更为规则 Masson ×200
注：分别于空载对照组、Wnt9a过表达组小鼠创缘皮下注射增强型绿色荧光蛋白空载腺病毒、Wnt9a基因增强型绿色荧光蛋白重组腺病毒
图 4 蛋白质印迹法检测的2组人皮肤成纤维细胞感染72 h后Wnt9a的蛋白表达
注：空载对照组、Wnt9a过表达组细胞分别感染增强型绿色荧光蛋白空载腺病毒、Wnt9a基因增强型绿色荧光蛋白重组腺病毒；条带上方1表示空载对照组,2表示Wnt9a过表达组
图 5 4组人正常皮肤Fb划痕后不同时间点的迁移情况。5A、5B.分别为空载对照组、Wnt9a过表达组划痕后0 h；5C、5D.分别为空载对照组、Wnt9a过表达组划痕后48 h,图5D残余划痕面积明显小于图5C；5E、5F.分别为对照siRNA组、siRNA-Wnt9a组划痕后0 h；5G、5H.分别为对照siRNA组、siRNA-Wnt9a组划痕后48 h,图5H残余划痕面积明显小于图5G
注：空载对照组、Wnt9a过表达组成纤维细胞（Fb）分别感染增强型绿色荧光蛋白空载腺病毒（AV-eGFP）、Wnt9a基因增强型绿色荧光蛋白重组腺病毒（AV-Wnt9a-eGFP）,阴性对照siRNA（SiRNA-NC）组、Wnt9a特异性siRNA（siRNA-Wnt9a）组分别转染阴性对照小干扰RNA、siRNA-Wnt9a
图 6 2组人正常皮肤Fb感染72 h的差异表达基因。5A.火山图；5B.基因本体论富集分析
注：图6B上方1表示感染Wnt9a基因增强型绿色荧光蛋白重组腺病毒的Wnt9a过表达组,2表示感染增强型绿色荧光蛋白空载腺病毒的空载对照组；红色、蓝色、灰色分别表示与空载对照组相比,Wnt9a过表达组成纤维细胞（Fb）中显著上调差异表达基因、显著下调差异表达基因、无显著差异基因
图 7 2组人正常皮肤Fb感染72 h差异表达基因的京都基因与基因组百科全书富集分析
注：图上方1表示感染Wnt9a基因增强型绿色荧光蛋白重组腺病毒的Wnt9a过表达组,2表示感染增强型绿色荧光蛋白空载腺病毒的空载对照组；红色、蓝色、灰色分别表示与空载对照组相比,Wnt9a过表达组成纤维细胞（Fb）中显著上调差异表达基因、显著下调差异表达基因、无显著差异基因
Table 1. 2组慢性创面小鼠造模后各时间点残余创面面积百分比比较（%,
组别 鼠数 3 d 7 d 14 d 空载对照组 8 78.3±2.6 60.3±1.8 48.0±3.6 Wnt9a过表达组 8 70.6±2.2 78.3±2.7 16.3±4.1 t值 3.90 6.62 5.73 P值 <0.001 <0.001 <0.001 注：分别于空载对照组、Wnt9a过表达组小鼠创缘皮下注射增强型绿色荧光蛋白空载腺病毒、Wnt9a基因增强型绿色荧光蛋白重组腺病毒；处理因素主效应,F=607.52,P<0.001；时间因素主效应,F=273.72,P<0.001；两者交互作用,F=79.74,P<0.001 
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