Effects and mechanism of age on the stiffness and the fibrotic phenotype of fibroblasts of human hypertrophic scar
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摘要:
目的 探讨年龄对人增生性瘢痕硬度和成纤维细胞（Fb）纤维化表型的影响及其可能的分子机制。 方法 采用实验研究方法。收集2020年1—6月解放军总医院第四医学中心烧伤整形外科收治的10例瘢痕患者（男4例、女6例）手术切除的增生性瘢痕组织和10例患者（男5例、女5例，年龄7~41岁）手术后剩余的正常全层皮肤组织。根据患者年龄，将6例患者［（10.7±1.6）岁］瘢痕组织纳入年轻组，将4例患者［（40.0±2.2）岁］瘢痕组织纳入年长组。对正常皮肤和2组瘢痕组织，行苏木精-伊红（HE）染色观察组织形态，行Masson染色观察胶原形态、排列并测定胶原含量，冻干及金属镀膜后在扫描电子显微镜下观察真皮层胶原纤维微观形态。采用原子力显微镜在液相下测量2组瘢痕组织硬度。取2组瘢痕组织，分离和培养Fb，采用倒置相差显微镜观察其形态，并采用细胞免疫荧光法检测桩蛋白的表达以反映细胞形态，采用细胞免疫荧光法检测促纤维化蛋白α平滑肌肌动蛋白（α-SMA）、转化生长因子β1（TGF-β1）和Ⅰ型胶原表达及机械力转导相关蛋白Yes相关蛋白（YAP）和增殖相关蛋白Ki67的表达，采用实时荧光定量反转录PCR法检测促纤维化基因TGF-β1、α-SMA和Ⅰ型胶原，抑制纤维化基因TGF-β3及机械力转导相关基因Rho相关激酶1（ROCK1）和YAP mRNA表达。对数据行单因素方差分析、LSD-t检验。 结果 HE染色可见，正常皮肤表皮层凹凸不平，真皮层可见血管和汗腺等附属器；年轻组、年长组瘢痕组织表皮层均较为扁平，真皮层血管和汗腺等附属器罕见。Masson染色和扫描电子显微镜下可见，正常皮肤胶原纤维排列松散、无序，而2组瘢痕组织胶原纤维排列均较为致密、整齐，且年轻组瘢痕组织胶原纤维较年长组更为致密。年轻组、年长组瘢痕组织胶原含量明显高于正常皮肤组织（t=8.02、3.15，P<0.05或P<0.01），年长组瘢痕组织胶原含量明显低于年轻组（t=4.84，P<0.05）。年长组瘢痕组织真皮层硬度为（50.3±1.1）kPa，明显高于年轻组的（35.2±0.8）kPa（t=11.43，P<0.05）。2组瘢痕Fb在倒置相差显微镜下及经细胞免疫荧光法观察，在形态上无明显差异。年长组瘢痕Fb细胞质中Ⅰ型胶原和TGF-β1表达较年轻组明显升高，2组瘢痕Fb细胞质中α-SMA表达相近。年长组瘢痕Fb细胞质和细胞核中YAP表达较年轻组明显增多，2组瘢痕Fb细胞核中Ki67表达无明显差异。年长组瘢痕Fb中TGF-β1和Ⅰ型胶原mRNA表达量明显高于年轻组（t=2.87、4.85，P<0.05或P<0.01），TGF-β3 mRNA表达量明显低于年轻组（t=3.36，P<0.05），α-SMA mRNA表达量与年轻组无明显差异（t=1.14，P>0.05）。年长组瘢痕Fb中ROCK1和YAP mRNA表达量明显高于年轻组（t=2.98、7.60，P<0.05或P<0.01）。 结论 年长者皮肤损伤后更容易发生瘢痕愈合，其分子机制可能是由于创面愈合过程中会产生硬度较高的细胞外基质成分使得组织硬度增加，从而激活ROCK和YAP/转录共激活因子PDZ结合基序基因的表达，进而促进促纤维化基因和蛋白的表达。 Abstract:Objective To explore the effects and potential molecular mechanism of age on the stiffness and the fibrotic phenotype of fibroblasts (Fbs) of human hypertrophic scar. Methods The experimental research method was used. From January to June 2020, the surgically removed hypertrophic scar tissue of 10 scar patients (4 males and 6 females) and residual full-thickness normal skin tissue of 10 cases (5 males and 5 females, aged 7-41 years) were collected after operation in Department of Burns and Plastic Surgery of the Fourth Medical Center of the PLA General Hospital. The hypertrophic scar tissue of 6 patients aged (10.7±1.6) years was included into the young group and the hypertrophic scar tissue of 4 patients aged (40.0±2.2) years was included into the elderly group according to the age of patients. For the normal skin tissue and scar tissue in the two groups, hematoxylin eosin (HE) staining was performed to observe the tissue morphology, Masson staining was performed to observe the morphology and arrangement of collagen and quantify the content of collagen, and scanning electron microscope was used to observe the microscopic difference of dermal collagen fibers after the samples were freeze-dried and metal coated. The stiffness of scar tissue in the two groups was measured by atomic force microscope under the liquid phase. The scar tissue in the two groups was collected and the Fbs were isolated and cultured. The morphological differences of the Fbs were observed under the inverted phase contrast microscope, and the protein expression of paxillin was detected with cellular immunofluorescence to reflect the morphology of the Fbs. Cellular immunofluorescence was used to detect the expressions of pro-fibrosis protein α-smooth actin (α-SMA), transforming growth factor-β1 (TGF-β1), and type Ⅰ collagen, mechanotransduction-related protein Yes-associated protein (YAP), and the proliferation-related protein Ki67. Real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of pro-fibrosis genes of TGF-β1, α-SMA, and type Ⅰ collagen, fibrosis inhibiting gene of TGF-β3, and mechanotransduction-related genes of Rho-associated protein 1 (ROCK1) and YAP. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results HE staining showed that the epidermal layer of normal skin was uneven, and blood vessels and sweat glands could be seen in the dermal layer; the epidermal layer of the scar tissue in the two groups was relatively flat, and blood vessels and sweat glands were rare. Masson staining and scanning electron microscopy showed that the collagen fibers in normal skin arranged loosely and disorderly, while the collagen fibers in scar tissue of the two groups arranged densely and orderly, and the collagen fibers in scar tissue of the young group were denser than those of the elderly group. The collagen content in scar tissue of the young group and the elderly group was significantly higher than that of the normal skin tissue (t=8.02, 3.15, P<0.05 or P<0.01), and the collagen content in scar tissue of the elderly group was significantly lower than that of the young group (t=4.84, P<0.05). The dermal stiffness of scar tissue in the elderly group was (50.3±1.1) kPa, significantly higher than (35.2±0.8) kPa in the young group (t=11.43, P<0.05). There were no obvious differences in the morphology of scar Fbs in the two groups observed under inverted phase contrast microscope and by cellular immunofluorescence. The expressions of type Ⅰ collagen and TGF-β1 in scar Fbs cytoplasm of the elderly group were significantly higher than those in the young group, while the expressions of α-SMA in scar Fbs cytoplasm were close in the two groups. The expressions of YAP in cytoplasm and nucleus of scar Fbs in the elderly group were significantly higher than those in the young group, while the expressions of Ki67 in scar Fbs nucleus of the two groups were close. The mRNA expressions of TGF-β1 and type Ⅰ collagen in scar Fbs of the elderly group were significantly higher than those in the young group (t=2.87, 4.85, P<0.05 or P<0.01), the mRNA expression of TGF-β3 in scar Fbs of the elderly group was significantly lower than that in the young group (t=3.36, P<0.05), and the mRNA expressions of α-SMA in scar Fbs of the two groups were close (t=1.14, P>0.05). The mRNA expressions of ROCK1 and YAP in scar Fbs of the elderly group were significantly higher than those in the young group (t=2.98, 7.60, P<0.05 or P<0.01). Conclusions The elderly are prone to scar healing after skin injury. The molecular mechanism may be attributed to the production of extracellular matrix components with higher stiffness, which increases tissue stiffness and thereby activates the expressions of ROCK and YAP/transcriptional co-activator with PDZ-binding motif genes, promoting pro-fibrosis gene and protein expression. -
Key words:
- Cicatrix /
- Hardness /
- Fibroblast /
- Age /
- Gene and protein of fibrosis /
- Gene and protein of mechanotransduction
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1 植皮术患者正常皮肤和2组增生性瘢痕患者瘢痕组织形态 苏木精-伊红×100，图中标尺为500 μm。1A.正常皮肤组织褶皱较为明显，表皮基底层凹凸不平，真皮层可见血管和汗腺等附属器；1B.年轻组瘢痕组织表皮层扁平，表皮基底层正常棘突消失，真皮层血管和汗腺等附属器罕见；1C.年长组瘢痕组织结构与图1B相似
2 植皮术正常皮肤和2组增生性瘢痕患者瘢痕组织真皮层胶原排列和分布 Masson×100，图中标尺为500 μm。2A.正常皮肤组织胶原排列松散、无序；2B.年轻组瘢痕组织胶原排列致密且有序；2C.年长组瘢痕组织皮肤胶原排列有序，但较图2B稀疏且胶原含量减少
4 植皮术患者正常皮肤和2组增生性瘢痕患者瘢痕组织真皮层胶原纤维微观形态 扫描电子显微镜×500，图中标尺为200 μm。4A.正常皮肤组织胶原纤维排列松散、无序并相互交织；4B.年轻组瘢痕组织真皮层胶原纤维致密且排列有序；4C.年长组瘢痕组织皮肤胶原纤维聚集成束，且排列有序，但较图4B稀疏
5 2组人瘢痕成纤维细胞形态学观察。5A、5B.分别为年轻组和年长组细胞形态，2组细胞均呈梭形并聚集呈巢状 倒置相差显微镜×50，图中标尺为500 μm；5C、5D.分别为年轻组和年长组细胞形态，2组细胞均呈多边形，相互重叠，并向周围伸展，细胞核呈圆形或椭圆形 Alexa Fluor 594-4'，6-二脒基-2-苯基吲哚×400，图中标尺为100 μm
注：细胞核阳性染色为蓝色，桩蛋白阳性染色为红色
6 2组人瘢痕成纤维细胞中α-SMA、Ⅰ型胶原和TGF-β1蛋白的表达 Alexa Fluor 488-4'，6-二脒基-2-苯基吲哚×100，图中标尺为100 μm。6A、6B、6C.分别为年轻组细胞质中α-SMA、Ⅰ型胶原和TGF-β1的表达；6D、6E、6F.分别为年长组细胞质中α-SMA、Ⅰ型胶原和TGF-β1的表达，α-SMA的表达与图6A相近，Ⅰ型胶原的表达较图6B明显增加，TGF-β1的表达较图6C明显增加
注：细胞核阳性染色为蓝色，α平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原和转化生长因子β1（TGF-β1）阳性染色均为绿色
7 2组人瘢痕成纤维细胞中YAP和Ki67蛋白的表达 Alexa Fluor 488-4'，6-二脒基-2-苯基吲哚×400，图中标尺为100 μm。7A、7B.分别为年轻组和年长组，YAP蛋白广泛分布于细胞质和细胞核，且图7B中YAP蛋白较图7A增加；7C、7D.分别为年轻组和年长组，Ki67蛋白分布于细胞核，且图7D中Ki67蛋白表达与图7C无明显差异
注：细胞核阳性染色为蓝色，Yes相关蛋白（YAP）和Ki67阳性染色均为绿色
8 实时荧光定量反转录PCR法检测2组人瘢痕成纤维细胞中纤维化相关基因和机械力转导相关基因的表达（
注：TGF为转化生长因子，α-SMA为α平滑肌肌动蛋白，ROCK1为Rho相关激酶1，YAP为Yes相关蛋白；与年轻组比较，aP<0.05，bP<0.01
表1 实时荧光定量反转录PCR检测2组患者瘢痕成纤维细胞中纤维化相关基因和机械力转导相关基因的表达
基因名称 引物序列（5’→3’） 产物大小（bp） GAPDH 上游：GGAGCGAGATCCCTCCAAAAT 197 下游：GGCTGTTGTCATACTTCTCATGG TGF-β1 上游：CAATTCCTGGCGATACCTCAG 86 下游：GCACAACTCCGGTGACATCAA TGF-β3 上游：ACTTGCACCACCTTGGACTTC 114 下游：GGTCATCACCGTTGGCTCA α-SMA 上游：GTGTTGCCCCTGAAGAGCAT 116 下游：GCTGGGACATTGAAAGTCTCA Ⅰ型胶原 上游：GAGGGCCAAGACGAAGACATC 140 下游：CAGATCACGTCATCGCACAAC YAP 上游：TAGCCCTGCGTAGCCAGTTA 177 下游：TCATGCTTAGTCCACTGTCTGT ROCK1 上游：AACATGCTGCTGGATAAATCTGG 93 下游：TGTATCACATCGTACCATGCCT 注：GAPDH为3-磷酸甘油醛脱氢酶，TGF为转化生长因子，α-SMA为α平滑肌肌动蛋白，YAP为Yes相关蛋白，ROCK1为Rho相关激酶1 
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