一氧化氮合酶对增生性瘢痕影响意义的研究

The Expression of the NOS Gene in Hypertrophic Scar

目的观察一氧化氮合酶(Nitrogen oxide synthetase,NOS)在增生性瘢痕与正常皮肤组织及细胞中的表达特征及其差别,并通过应用NO供体或NOS抑制剂对培养的瘢痕成纤维细胞进行体外药物干扰,探索NO、NOS在增生性瘢痕形成中的作用。方法取临床增生性瘢痕手术病人的瘢痕组织及周围少许正常皮肤,体外培养获得皮肤和瘢痕成纤维细胞,通过免疫细胞化学及RT-PCR方法比较两种组织及细胞中NOS的表达及分布的差异。分别以100～500μM的NO供体硝普钠(Sodiu m nitroprusside,SNP)、NOS抑制剂N-硝基-L-精氨酸甲酯(L-Arginine methyl,L-NAME)作用于瘢痕成纤维细胞,观察细胞增殖的变化;并用免疫细胞化学和RT-PCR方法比较作用前后成纤维细胞中I、III型胶原表达的变化。结果免疫组化检测及RT-PCR方法检测显示增生性瘢痕组织及细胞中NOS表达相对于皮肤组织及成纤维细胞中明显减少(p<0.05)。瘢痕细胞经SNP作用后,细胞增殖显著降低。免疫细胞化学方法和RT-PCR方法检测Ⅰ、Ⅲ型胶原结果显示,从SNP200μM组开始Ⅰ、Ⅲ型胶原表达显著降低(p<0.05)。经L-NAME200μM作用后,Ⅰ、Ⅲ型胶原表达显著增加(p<0.05)。结论增生性瘢痕组织及细胞中NOS含量较正常皮肤组织及细胞明显减少,NO供体SNP对体外培养的增生性瘢痕成纤维细胞增殖以及胶原表达起到抑制作用,NOS抑制剂L-NAME对其胶原表达则起到促进作用,结果提示NOS表达降低可能与瘢痕增生密切相关,因此改变NO的产生及NOS的活性可望调控瘢痕成纤维细胞的生物学行为。

Objective Hypertrophic scar （Hs） remains the most disabling sequela for bum survivors. Little is known about its pathogenesis. Tbis study through observing the NOS expression in Hs fibroblasts and the influence of the NO donor SNP and the NOS inhibitor L-NAME to the collagen expression in Hs and normal fibroblasts, explore the relation of NO and forming mechanism of Hs. Methods Reconstructive surgery was performed to remove Hs from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient＇s normal skin around the scar. We have studied NOS and collagen expression in Hs and normal fibroblasts and tissues using immunocytochemistry and RT-PCR. Then, Hs fibroblasts were cultured in the presence of 100 to 500 μM or absence of the SNP and 200 μM L-NAME. And then measure the cellular proliferation of HS and their collagen type Ⅰ and type Ⅲ synthesis studied on the transcriptional as well as translational level using RT-PCR and immunocytochemistry respectively. Results Hs tissue and fibroblasts expresses less cNOS than normal skin and fibroblasts detecting by immunocytochemistru and RT-PCR （p〈0.05）. Cellular proliferation of HS fibroblasts was significantly decreased in the presence of SNP. Fibroblast collagen type Ⅰ and type Ⅲ synthesis was enhanced in the presence of 200 and 500 μM SNP, and it was decreased in the presence of 200 μM L-NAME respectively by RT-PCR and immunocytochemistry （p 〈0.05）. Conclusion Hs fibroblasts and tissues express less cNOS than normal fibroblasts and ti ssues. NO donor SNP reduce the collagen synthesis and Cellular proliferation of Hs fibroblasts; and the NOS inhibitor L-NAME increase the collagen synthesis. It indicates that NO level and the activity of NOS may have a significant inverse correlation with the forming of hypertrophic scar.