摘要
目的研究长链非编码RNA(LncRNA)GAPLINC对瘢痕疙瘩成纤维细胞HKF增殖以及胶原合成的影响与潜在机制。方法瘢痕疙瘩组织(n=15)取自2019年9月至2020年9月开封市中心医院既往未接受过任何抗瘢痕疙瘩治疗的15名患者,从瘢痕疙瘩切除手术中获得。正常皮肤组织(n=21)取自同期在整形手术中捐赠组织的21名健康捐赠者。运用逆转录定量聚合酶链反应(RT-qPCR)检测LncRNA GAPLINC在瘢痕疙瘩组织中的表达。在人瘢痕疙瘩成纤维细胞HKF中转染靶向LncRNA GAPLINC(si-LncRNA GAPLINC)的小干扰RNA(siRNA)、阴性对照siRNA(si-NC)、LncRNA GAPLINC过表达质粒(pcDNA-LncRNA GAPLINC)、阴性对照载体pcDNA。采用RT-qPCR检测LncRNA GAPLINC、Ⅰ型胶原蛋白(collagen TypeⅠ)、Ⅲ型胶原蛋白(colla-gen TypeⅢ)及转化生长因子-β1(TGF-β1)mRNA表达,MTT法检测细胞增殖,克隆形成实验检测细胞克隆形成,Western blot检测Ⅰ型、Ⅲ型胶原蛋白的表达。结果瘢痕疙瘩组织中LncRNA GAPLINC表达高于正常皮肤组织,差异有统计学意义(P<0.05)。si-LncRNA GAPLINC组瘢痕疙瘩成纤维细胞HKF中LncRNA GAPLINC表达量比si-NC组减少,pcDNA-LncRNA GAPLINC组瘢痕疙瘩成纤维细胞HKF中LncRNA GAPLINC表达量比pcDNA组增加(P<0.05)。si-LncRNA GAPLINC组瘢痕疙瘩成纤维细胞HKF的24 h、48 h、72 h增殖能力、克隆形成数、Ⅰ型、Ⅲ型胶原蛋白的蛋白及mRNA表达量、TGF-β1 mRNA表达量均低于si-NC组(P<0.05)。pcDNA-LncRNAGAPLINC组瘢痕疙瘩成纤维细胞HKF的24 h、48 h和72 h增殖能力、克隆形成数、Ⅰ型、Ⅲ型胶原蛋白的蛋白及mRNA表达量、TGF-β1 mRNA表达量比pcDNA组升高(P<0.05)。结论LncRNA GAPLINC具有潜在的促进瘢痕疙瘩成纤维细胞HKF增殖与胶原合成作用,其机制可能与上调TGF-β1的表达有关。
Objective To study the effect and potential mechanism of long non-coding RNA(LncRNA)GAPLINC on HKF proliferation and collagen synthesis in keloid fibroblasts.Methods Keloid tissues(n=15)were obtained from 15 patients who had not previously received any anti-keloid treatment in Kaifeng Central Hospital from September 2019 to September 2020,and were obtained from keloid resection.Normal skin tissue(n=21)was taken from 21 healthy do-nors who had donated tissue during plastic surgery during the same period.The expression of LncRNA GAPLINC in ke-loid tissue was detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR).In human keloid fibroblast HKF cells,small interfering RNA(siRNA)targeting LncRNA GAPLINC(si-LncRNA GAPLINC),negative control siRNA(si-NC),LncRNA GAPLINC overexpression plasmid(pcDNA-LncRNA GAPLINC),Negative control vector pcDNA.RT-qPCR was used to detect the mRNA expressions of LncRNA GAPLINC,Collagen TypeⅠ,Colla-gen TypeⅢ(Collagen TypeⅢ)and transforming growth factor-β1(TGF-β1),MTT assay to detect cell prolifera-tion,clone formation assay to detect cell clone formation,Western blot to detectⅠType and III collagen expression.Results The expression of LncRNAGAPLINC in keloid tissue was about higher than that in normal skin tissue,the difference was statistical significance(P<0.05).The expression of LncRNA GAPLINC in keloid fibroblasts HKF in the si-LncRNA GAPLINC group was lower than that of the si-NC group,and the expression of LncRNA GAPLINC in the keloid fibroblasts HKF in the pcDNA-LncRNA GAPLINC group was higher than that of the pcDNA group(P<0.05).The 24 h,48 h and 72 h proliferation ability,the number of colonies,the protein and mRNA expression of collagen type I and III,and the expression of TGF-β1 mRNA in si-LncRNA GAPLINC group were lower than those of HKF in si-NC group(P<0.05).The 24 h,48 h and 72 h proliferation ability,the number of colonies,the protein and mRNA expression of collagen type I and III,and the expression of TGF-β1 mRNA of keloid fibroblast H
作者
王卫东
王端祥
聂顺义
WANG Wei-dong;WANG Duan-xiang;NIE Shun-yi(Department of burn and plastic surgery,Kaifeng Central Hospital,Kaifeng,Henan 475003,China)
出处
《医药论坛杂志》
2023年第23期27-32,共6页
Journal of Medical Forum
