重组人内皮型一氧化氮合酶基因转染对增生性瘢痕成纤维细胞的影响

目的 了解重组人内皮型一氧化氮合酶(eNOS)基因转染人增生性瘢痕成纤维细胞(HSFb)的可行性,以及转染后一氧化氮(NO)的生成和Ⅰ、Ⅲ型胶原的合成情况. 方法体外构建人eNOS真核表达载体.取体外培养的人HSFb进行转染实验,根据细胞培养液中所加质粒不同分为pcDNA3.0空载体组、pcDNA3.0-eNOS转染组.另设未转染组,细胞按常规培养.采用实时荧光定量PCR法检测各组细胞eNOS及Ⅰ、Ⅲ型胶原mRNA表达.硝酸还原酶法测定NO含量. 结果细胞转染后eNOS显著表达,pcDNA3.0-eNOS转染组eNOS mRNA相对表达量为5.92±0.21,明显高于pcDNA3.0空载体组(0.98±0.13,P<0.05);pcDNA3.0-eNOS转染组Ⅰ型胶原mRNA相对表达量为0.76±0.15,Ⅲ型胶原mRNA相对表达量为0.79±0.08,均明显低于pcDNA3.0空载体组(0.98±0.15、1.02±0.12,P<0.05).pcDNA3.0-eNOS转染组NO含量为(36.1±0.8)μmol/L,明显高于pcDNA3.0空载体组(28.4±1.0)μmol/L和未转染组(27.7±1.3)μmol/L,P<0.01]. 结论 HSFb可作为eNOS基因转染的靶细胞,转染的细胞能表达eNOS并产生NO,并且对细胞的胶原合成功能有抑制作用.

Effect of transfection of recombinant human endothelial nitric oxide synthase gene on hypertrophic scar fibroblasts in vitro

Objective To investigate the feasibility of transfection of recombinant human endothelial nitric oxide synthase(eNOS) into human hypertrophic scar fibroblasts (HSFbs) ,and to observe NO secretion and the synthesis of collagen Ⅰ and Ⅲ. Methods Recombinant human eNOS with karyocyte expressive vector was constructed in vitro, then was transfccted into HSFbs which was isolated from hypertrophic scar tis- sues and cultured in vitro(T group). The HSFbs untransfected (normal culture)or transfected with empty- vector was used as control group and empty-vector group respectively. The mRNA expression of eNOS, colla- gen Ⅰ and Ⅲ was determined by Reahime PCR. The content of NO was determined by NO assay kit. Re- suits The expression of eNOS mRNA in T group was 5.92 ± 0.21 ,which was obviously higher than that in empty-vector group (0.98 ± 0.13, P < 0.05 ). The expression of collagen Ⅰ mRNA (0.76 ± 0.15 ), and collagen Ⅲ (0.79 ± 0.08 ) in T group was significantly lower than those in empty-vector group (0.98 ± 0. 15,1.02±0.12, P <0.05 ,respectively). The content of NO in T group (36.1±0.8μmol/L) was obvi- ously higher than that in empty-vector group (28.4v1.0 μmol/L, P < 0.01 ) and control group ( 27.7 ± 1.3 μmol/L, P < 0.01 ). Conclusion HSFbs can be the target cells for eNOS gene transfection. The transfected cells can express eNOS and produce NO ,which inhibit the synthesis of collagen.