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β_(1)(TGF-β_(1))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-β_(1)、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�
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 proteinα-smooth actin(α-SMA),transforming growth factor-β_(1)(TGF-β_(1)),and typeⅠcollagen,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-fibro
作者
朱冬振
姚斌
崔晓丽
黄沙
付小兵
Zhu Dongzhen;Yao Bin;Cui Xiaoli;Huang Sha;Fu Xiaobing(Research Center for Wound Repair and Regeneration,Medical Innovation Research Department,the PLA General Hospital,Beijing 100048,China;Key Laboratory of Tissue Repair and Regeneration of PLA,Beijing Key Research Laboratory of Skin Injury,Repair and Regeneration,the Fourth Medical Center,the PLA General Hospital,Beijing 100048,China)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2021年第10期937-945,共9页
Chinese Journal of Burns
基金
国家自然科学基金面上项目(32000969,82002056)
解放军总医院军事医学创新研究项目(CX19026)
王正国创伤医学发展基金会生长因子复兴计划(SZYZ-TR-03)。
