兔自体真皮成纤维细胞对增生性瘢痕作用的实验研究  

Experiment study on the effects of rabbit autologous dermal fibroblasts on hypertrophic scar

作　　者：杨晓双 胡鹏[1] 王达利[1] 魏在荣[1] 冀玫宏 罗昭和 Yang Xiaoshuang;Hu Peng;Wang Dali;Wei Zairong;Ji Meihong;Luo Zhaohe(Department of Plastic Surgery Affiliated Hospital Zunyi Medical College,Zunyi 563000,China)

机构地区：[1]遵义医学院附属医院烧伤整形外科,563000

出　　处：《中华整形外科杂志》2018年第9期758-768,共11页Chinese Journal of Plastic Surgery

基　　金：国家自然科学基金（81060157,81560313）

摘　　要：目的 探讨兔耳瘢痕形成早期瘢痕内局部移植自体真皮成纤维细胞（Fbs）对增生性瘢痕形成及创面愈合质量的影响,以探讨Fbs用于增生性瘢痕的防治的可行性.方法 运用机械法联合酶消化法从新西兰大白兔背部正中正常皮肤组织中提取真皮Fbs,取第3代生长良好的细胞,向成骨细胞、成脂肪细胞诱导分化.取2只实验兔作为预实验观察对象（即未干预组）,于兔双耳腹侧手术形成全层皮肤缺损创面,创面不做任何处理,待其自然愈合,记录完全上皮化时间及增生性瘢痕形成的情况;另取6只实验用兔设立兔左耳为实验组,右耳为对照组,于创面上皮化后第2天（术后20 d）、术后30 d（瘢痕增生最明显时）,采用体外培养的经BrdU标记后的第4代自体真皮Fbs于实验组创面及增生性瘢痕周围的皮下至软骨上层之间进行环形注射,对照组在上述相应时间点注射相同剂量的生理盐水.第2次细胞移植后7d（术后37 d左右）切取增生性瘢痕组织,记录3组（未干预组、实验组、对照组）增生性瘢痕的大体外观及其组织学变化;实验组术后37 d增生性瘢痕组织行免疫荧光染色对移植细胞进行示踪;实时荧光-多聚合酶链反应（Real Time-PCR,RT-PCR）检测对照组及实验组术后37 d增生性瘢痕组织中转化生长因子-β1（Transforming growth factor-β1,TGF-β1）和核心蛋白多糖（decorn,DCN）的mRNA表达变化情况,酶联免疫吸附法（ELISA）检测增生性瘢痕组织中DCN、TGF-β1、Ⅰ型胶原及Ⅲ型胶原的蛋白表达情况.结果 术后37 d实验组瘢痕较对照组平、软,色泽轻度变浅,体积缩小.组织学结果提示实验组与对照组相比真皮浅层Fbs、炎性细胞数量减少,结缔组织的增生及胶原沉积减少,基底层细胞、胶原纤维排列整齐;RT-PCR结果表明,实验组增生性瘢痕组织中TGF-β1 mRNA表达量较对照组明显降低,DCN mRNA表达量明显增高,差异均有统计�Objective To investigate the effect of local autologous dermal fibroblasts transplantation on hypertrophic scar formation and wound healing quality in early scar formation.To explore the feasibility of fibroblasts for prevention and treatment of hypertrophic scar.Methods Dermal fibroblasts were isolated from the dorsal skin tissue of New Zealand white rabbits by mechanical method combined with enzyme digestion.Passage 3 cells were induced to differentiate into osteoblasts and adipocytes.The complete epithelialization time and hypertrophic scar formation after full-thickness skin defect were confirmed by pre-experiment study.In the experiment,6 rabbits were used,left ear asexperimental group and right ear as control group.In the experimental group,the passage 4 dermal fibroblasts labeled with 5-bromodeoxyuridine （5-BrdU） were injected subcutaneously around the wound and hypertrophic scar on 20 d （day 2 after epithelialization） and 30 d （most obvious scar hyperplasia） after surgery.As a control group,physiological saline was injected following the same protocol.On 37 days after surgery,the hypertrophic scar tissue were harvested and assessed by gross view and histological examination.The transplanted cells were detected by immunofluorescence staining,transforming growth factor beta 1 （TGF-β1） and decorin （DCN） mRNA expression were assayed by real-time fluorescence polymerase chain reaction （RT-PCR）,and the protein expression of TGF-β1 、DCN、Collagen type Ⅰ and type Ⅲ were tested by enzyme linked immunosorbent assay（ELISA）.Results Compared with the control group,the scar in the experimental group was flatter and softer,the color was slightly lighter,and the volume was reduced.The histological results showed that compared with the control group,the number of fibroblasts and inflammatory cells in the superficial dermis was reduced,the proliferation of connective tissue and collagen deposition were reduced,and the basal cells and collagen fibers were arranged in order in the experim

关 键 词：增生性瘢痕 成纤维细胞 细胞移植 兔耳瘢痕模型 

分 类 号：R622[医药卫生—整形外科]