携带Decorin基因的质粒DNA构建及其对兔耳增生性瘢痕的治疗作用  

Study on construction of recombinant plasmid carrying the Decorin gene and its effects on hypertrophic scars of rabbit ears

作　　者：杨佳丽 王艳香 杜丽 卢育新 吴家兵 程晓晨 潘自强 赵希荣 张庆林 Yang Jiali;Wang Yanxiang;Du Li;Lu Yuxin;Wu Jiabing;Cheng Xiaochen;Pan Ziqiang;Zhao Xirong;Zhang Qinglin(College of Life Science and Food Engineering,Huaiyin Institute of Technology,Huai′an 223003,China;The Institute of Radiation Medicine,The Academy of Military Medical Science,Beijing 100850,China;Department of Dermatology,Luanzhou People′s Hospital,Luanzhou 063700,China)

机构地区：[1]淮阴工学院生命科学与食品工程学院,淮安223003 [2]军事科学院军事医学研究院辐射医学研究所,北京100850 [3]河北省滦州市人民医院皮肤科,滦州063700

出　　处：《中华整形外科杂志》2020年第10期1121-1127,共7页Chinese Journal of Plastic Surgery

基　　金：江苏省研究生实践创新计划(SJCX17_0699)。

摘　　要：目的探讨携带核心蛋白聚糖(decorin,DCN)基因的重组质粒对兔耳增生性瘢痕的治疗作用。方法将PCR扩增的人Decorin基因片段克隆到质粒载体pUDK上构建重组质粒pDCN,并进行酶切和测序鉴定。重组质粒pDCN转染293T细胞,并检测细胞内DCN及TGF-β1的表达。建立兔耳增生性瘢痕模型,将12只新西兰大白兔采用随机数字表法分为PBS组、空质粒组、rhDCN组、pDCN低剂量(20μg/cm2)组、pDCN中剂量(40μg/cm2)组、pDCN高剂量(60μg/cm2)组,观察肥大指数、瘢痕组织病理改变及DCN表达等,研究pDCN对兔耳增生性瘢痕的治疗作用。结果经过酶切、测序鉴定,Decorin基因片段成功插入质粒载体pUDK上,重组质粒pDCN构建成功。pDCN转染至293T细胞后,DCN的RNA及蛋白表达水平均上调,TGF-β1表达受到抑制。应用不同剂量的pDCN实验性治疗兔耳增生性瘢痕,结果显示pDCN中剂量组兔耳瘢痕的肥大指数(1.61±0.40)显著小于PBS组(2.07±0.55)(P<0.05),而pDCN低剂量和高剂量组肥大指数均与PBS组没有显著差异。免疫组织化学结果显示,pDCN中剂量组中兔耳局部DCN表达显著高于PBS组(P<0.05);同时,病理检测显示瘢痕组织中炎性细胞浸润明显减少,纤维沉积减少,表明中剂量pDCN可有效抑制兔耳增生性瘢痕的增生。结论携带Decorin基因的质粒pDCN通过抑制TGF-β1表达,对兔耳增生性瘢痕有一定的治疗作用。Objective To construct a recombinant plasmid DNA carrying the decorin(DCN)gene and study its therapeutic effect on hypertrophic scars of rabbit ears.Methods The human decorin gene fragment amplified by PCR was cloned into plasmid vector pUDK to construct the recombinant plasmid pDCN,which was identified by enzyme digestion and sequencing.pDCN was transfected into 293T cells,and the expression of DCN and TGF-β1 was detected.The therapeutic effect of pDCN on rabbit ear hypertrophic scar model was observed by hypertrophy index,pathological examination and immunohistochemical staining.Results The decorin gene was successfully inserted into pUDK,which was examined by enzyme digestion and sequencing.The expression level of mRNA and protein of DCN was up-regulated in 293T cells post pDCN transfection,and the expression of TGF-β1 was suppressed.Then the rabbit ear hypertrophic scars were treated with different doses of pDCN,and the results showed that the hypertrophy index of the medium dose(200μg/cm2)pDCN group was significantly lower than that of the PBS group(P<0.05),while there was no significant difference in the hypertrophy index of the low dose and high dose pDCN group compared with the PBS group.The expression of DCN in ears skin in the medium dose pDCN group was significantly higher than that in the PBS group(P<0.05).The pathological examination showed that inflammatory cell infiltration and fibrous deposition in scar tissue were significantly reduced.These results indicated that the medium-dose pDCN could effectively inhibit the hyperplasia of hypertrophic scar in rabbit ears.Conclusions pDCN,the plasmid carrying decorin gene,has therapeutic effects on hypertrophic scars of rabbit ears by inhibiting TGF-β1 expression.

关 键 词：核心蛋白多糖 重组质粒 增生性瘢痕 转化生长因子-Β1 

分 类 号：R622[医药卫生—整形外科]