摘要
目的:观察结缔组织生长因子反义寡核苷酸对转化生长因子β1诱导的人增生性瘢痕成纤维细胞结缔组织生长因子mRNA、蛋白的表达和胶原合成的影响。方法:实验于2005-03/12在哈尔滨医科大学免疫教研室完成。选取哈尔滨医科大学附属第二医院整形美容中心手术标本,包括正常皮肤和增生性瘢痕。将手术中切下的正常皮肤、增生性瘢痕组织在无菌条件下去除表皮后采用组织块贴壁法培养于含体积分数为0.1的胎牛血清的RPMI1640培养液中,置37℃,体积分数为0.05的CO2条件下原代培养。经2.5g/L胰蛋白酶消化传代,传代至3～5代时进行实验。实验分4组:①正常皮肤成纤维细胞组。②增生性瘢痕成纤维细胞组。③增生性瘢痕成纤维细胞+5.0mg/L转化生长因子β1组(简称转化生长因子β1组)。④增生性瘢痕成纤维细胞+5.0mg/L转化生长因子β1+结缔组织生长因子反义寡核苷酸组(简称反义寡核苷酸组)。用5.0mg/L转化生长因子β1刺激增生性瘢痕成纤维细胞后,以脂质体介导方法将结缔组织生长因子反义寡核苷酸转染增生性瘢痕成纤维细胞中,用反转录-聚合酶链反应方法和WesternBlot方法检测细胞中结缔组织生长因子mRNA和蛋白质的表达;采用3H-脯氨酸掺入法检测细胞的胶原合成量。结果:①反转录-聚合酶链反应结果:结缔组织生长因子mRNA在正常皮肤成纤维细胞中无表达,在增生性瘢痕成纤维细胞中表达增强,转化生长因子β1刺激后表达量0.64±0.32明显高于增生性瘢痕成纤维细胞0.31±0.14,差异显著(P<0.01);反义寡核苷酸组可以大部分抑制结缔组织生长因子mRNA的表达,表达量0.12±0.62明显低于增生性瘢痕成纤维细胞组和转化生长因子β1组,差异显著(P<0.01)。②Western印迹结果:蛋白水平趋势与mRNA水平相一致。结缔组织生长因子蛋白在正常皮肤成纤维细胞中无表达,在增生性瘢痕成纤维细胞中表达增强84.63±11.49,转化生长因子β1刺激后表达量102.89±14.35明显高于增生性瘢痕成纤维细胞组,差异显著(P<0.01);反义寡核苷酸组可以大部分抑制转化生长因子β1的作用,结缔组织生长因子蛋白表达量41.75±10.56低于增生性瘢痕成纤维细胞组和转化生长因子β1组(P<0.01)。③结缔组织生长因子反义寡核苷酸对增生性瘢痕成纤维细胞胶原合成的作用:增生性瘢痕成纤维细胞组、转化生长因子β1组、反义寡核苷酸组的3H-脯氨酸掺入率分别为5381±185,8273±357,2475±859,转化生长因子β1可以显著增加成纤维细胞胶原的合成(P<0.01);而结缔组织生长因子反义寡核苷酸对转化生长因子β1刺激后的成纤维细胞胶原合成有明显抑制作用,差异显著(P<0.01)。结论:结缔组织生长因子反义寡核苷酸能够抑制转化生长因子β1引起的结缔组织生长因子mRNA、蛋白质表达的增高和胶原合成的增多,表明阻断结缔组织生长因子可能是延缓瘢痕纤维化的有效手段。
AIM： To observe the effects of connective tissue growth factor （CTGF） antisense oligonucleotides （ASODN） on the expression of CTGF mRNA and protein as well as the collagen synthesis in the human hyperplastic scar fibroblast （HSF） induced by transforming growth factor （TGF）-β1.
METHODS： The experiment was finished in the Immunology Institute of Harbin Medical University from March to December 2005. All the operation samples were obtained from the Plastic Surgery Center of Second Affiliated Hospital of Harbin Medical University, including normal skin and hyperplastic scar, which were ablated epidermis in asepsis condition and cultured by tissue mass attached method in the condition of 37 ℃, 0.05 CO2 and RPMI1640 culture medium including 0.1 fetal calf serum. Fibroblasts were passaged by 2.3 g/L trypsin and used for experiments between passages 3 and 5. The study was consisted of four groups： ① normal skin fibroblast group②HSF group③HSF ±5.0 mg/L TGF-β1（TGF- β1 group）④HSF±5.0 mg/L TGF-β1±CTGF ASODN（ASODN group）. CTGF ASODN were transfected by liposome mediation into the HSF induced by 5.0 mg/L TGF-β1. The expressions of CTGF mRNA and protein were assessed by reverse transcription polymerase chain reaction （RT-PCR） and Western Bolt Method, while the collagen synthesis of HSF was assessed by 3H-proline incorporation method.
RESULTS： ①RT-PCR result： The expressions of CTGF mRNA was none in the normal skin fibroblast, increased in HSF, and higher after TGF-β1 induction （0.64±0.32） than HSF （0.31±0.14）, with the significant difference （P 〈 0.01）; ASODN restrained mostly the expression of CTGF mRNA, which were significantly lower in ASODN group （0.12±0.62） than HSF group and TGF-β1 group, with the significant difference （P 〈 0.01）. ② Western Blot result： The expression tendency of protein was accorded to that of mRNA. CTGF protein was not expressed in the normal skin fibroblasts, strongly expressed in HSF （84.63±11.49）, remarkably increased expression after TGF-β1 induction （102.89±14.35） than that of HSF group, with the significant difference （P 〈 0.01）; ASODN restrained mostly the effects of TGF-β1, so the expression of CTGF protein was lower in ASODN group （41.75±10.56） than HSF group and TGF-β1 group, with the significant difference （P 〈 0.01）. ③Effects of CTGF ASODN on collagen synthesis of HSF： The 3H-proline incorporation rates of HSF group, TGF- β1 group and ASODN group were 5 381±185, 8 273±357, 2 475±859 respectively. TGF-β1 remarkably increased the collagen synthesis of HSF （P〈 0.01）, whereas CTGF ASODN obviously inhibited the collagen synthesis of HSF induced by TGF-β1, with the significant difference（P 〈 0.01）.
CONCLUSION： CTGF ASODN can inhibit the increased expression of CTGF mRNA and protein as well as the enhanced collagen synthesis which are all induced by TGF-β1, implying that CTGF block may be an effective therapy for deferring scar fibrosis,
出处
《中国临床康复》
CAS
CSCD
北大核心
2006年第16期92-94,i0004,共4页
Chinese Journal of Clinical Rehabilitation
