瘢痕疙瘩成纤维细胞胶原合成的实验研究

目的 探讨瘢痕疙瘩中胶原过度聚集的原因.方法 从正常皮肤与活跃增生瘢痕疙瘩标本分别培养真皮成纤维细胞,采用³H-脯氨酸掺入法分别检测单层培养及胶原凝胶三维培养体系中成纤维细胞的胶原合成量.用斑点杂交法检测单层培养细胞的人前.α1(Ⅰ)型胶原mRNA水平.结果 瘢痕疙瘩成纤维细胞Ⅰ型胶原mRNA水平升高,其胶原合成量也显著高于正常真皮成纤维细胞(P<0.01).结论 在增生活跃瘢痕疙瘩中,成纤维细胞胶原合成功能处于活化状态,胶原合成增加是导致胶原过度积聚的重要原因.     

槲皮素对瘢痕疙瘩成纤维细胞胶原合成的影响

目的研究槲皮素对瘢痕疙瘩成纤维细胞胶原合成的影响,探讨其作用机理。方法体外培养瘢痕疙瘩及正常皮肤成纤维细胞,应用羟脯氨酸比色法对不同药物浓度处理后成纤维细胞胶原合成量进行分析;RT-PCR及Real-timePCR检测Ⅰ、Ⅲ型胶原及TGFβ-1基因表达水平。结果槲皮素可抑制体外培养瘢痕疙瘩成纤维细胞胶原合成,该作用呈剂量依赖效应;槲皮素可降低Ⅰ、Ⅲ型胶原和TGFβ-1基因mRNA水平。结论槲皮素对于瘢痕疙瘩成纤维细胞胶原合成有显著抑制效应,其作用机制之一为通过抑制TGFβ-1基因在转录水平降低了前胶原mRNA水平,因而使成纤维细胞胶原合成减少。     

瘢痕疙瘩成纤维细胞对干扰素α-2b作用的反应

为探讨瘢痕疙瘩成纤维细胞对干扰素α 2b(IFNα 2b)作用的反应性 ,用IFNα 2b分别处理体外培养正常皮肤与瘢痕疙瘩中的成纤维细胞 ,采用3H 脯氨酸掺入、胃蛋白酶消化法检测成纤维细胞胶原合成量。结果发现 ,瘢痕疙瘩成纤维细胞经103U/mL的IFNα 2b作用后的胶原合成量显著降低(P<0.01) ;但与正常皮肤成纤维细胞的反应不同 ,经104U/mLIFNα 2b作用后的胶原合成量却未进一步减少。这提示瘢痕疙瘩成纤维细胞对IFNα 2b下向调节胶原合成作用的反应敏感性在一定程度上有所降低。     

雷公藤甲素对瘢痕疙瘩成纤维细胞增殖和胶原合成的影响

目的探讨雷公藤甲素对瘢痕疙瘩成纤维细胞增殖和胶原合成的影响。方法取手术切除的瘢痕疙瘩组织作成纤维细胞的原代培养,传代培养后加入不同浓度的雷公藤甲素作用,观察细胞的形态学变化,应用MTT法检测细胞的活性,应用免疫组化和Western-blot法检测雷公藤甲素对瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原合成的影响。结果雷公藤甲素能显著抑制瘢痕疙瘩成纤维细胞的增殖,且存在一定的浓度依赖性;雷公藤甲素作用后瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原表达减弱。结论雷公藤甲素能有效抑制瘢痕疙瘩成纤维细胞的增殖和Ⅰ型、Ⅲ型前胶原的合成。     

PDTC对瘢痕疙瘩成纤维细胞增殖和胶原合成的影响

目的:评价核因子κB(NF-κB)抑制剂吡咯烷二硫氨基甲酸(PDTC)对体外培养的人瘢痕疙瘩成纤维细胞增殖及胶原合成的作用.方法:体外培养人瘢痕疙瘩成纤维细胞,分别应用MTT比色法和羟脯氨酸比色法检测PDTC作用后细胞增殖及胶原合成的变化.结果:和对照组相比,PDTC能显著抑制瘢痕疙瘩成纤维细胞的增殖及胶原合成(P＜0.01).结论:PDTC具有体外抗瘢痕疙瘩的作用,有可能成为治疗瘢痕疙瘩的有效药物.     

病理性瘢痕成纤维细胞的研究进展

成纤维细胞是瘢痕形成的效应细胞,瘢痕疙瘩和肥厚性瘢痕成纤维细胞存在着广泛的异质性。成纤维细胞胶原合成增加、降解减少导致病理性瘢痕中胶原过度积聚。成纤维细胞的增殖异常是病理性瘢痕过度增生和持续存在的主要原因。成纤维细胞对细胞因子等因素反应性有明显异常。肥厚性瘢痕的收缩性和成纤维细胞的异质性有直接关系。     

病理性瘢痕成纤维细胞的研究进展

成纤维细胞是瘢痕形成的效应细胞，瘢痕疙瘩和肥厚性瘢痕成纤维细胞存在着广泛的异质性。成纤维细胞原合成增加，降解减少导致病理性瘢痕中胶原过度积聚。成纤维细胞的增殖异常是病理性瘢痕过度增生和持续存在的主要原因。成纤维细胞对细胞因子等因素反应性有明显异常。肥厚性瘢痕的收缩性和成纤维细胞的异质性有直接关系。     

皮肤肿瘤

20 0 3 1 5 62　抑癌基因MTSI对人瘢痕疙瘩成纤维细胞的生物学影响 /韩军涛 (四军大西京医院烧伤科 )…∥中国美容医学 . 2 0 0 3 ,1 2 ( 2 ) . 1 3 2～ 1 3 4将外源性MTSI基因导入人瘢痕疙瘩成纤维细胞后 ,通过MTT法测定细胞生长曲线 ,通过3H 胸腺嘧啶核苷 ( 3H TdR )及3H 脯氨酸掺入的方法测定细胞的DNA合成量及胶原合成量 ,来分别比较转染前后细胞的生物学变化。结果在转染MTSI后瘢痕疙瘩成纤维细胞的生长增殖受到明显的抑制 ,同时其DNA合成量及胶原合成量也明显降低 ,而转染空载体组及正常细胞均未出现上述变化。表明外源性M…     

咪喹莫特对瘢痕疙瘩成纤维细胞增殖和胶原产生的影响

目的 探讨咪喹莫特对瘢痕疙瘩成纤维细胞增殖和胶原产生的影响.方法 从手术切除的瘢痕疙瘩组织中培养成纤维细胞,加入不同浓度的咪喹莫特作用后,观察细胞的形态学变化,MTT法检测细胞的活性.免疫组化和蛋白免疫印迹法检测咪喹莫特对瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原产生的影响.结果 在10～100μg/mL范围内,咪喹莫特能显著抑制瘢痕疙瘩成纤维细胞的增殖,且存在剂量和时间依赖性;进一步检测到咪喹莫特作用后瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原表达减弱.结论 咪喹莫特能有效抑制瘢痕疙瘩成纤维细胞的增殖和Ⅰ型、Ⅲ型前胶原的产生.     

己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子-β1表达的影响北大核心CSCD

目的探讨己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子（TGF）-β1表达的影响。方法取人瘢痕疙瘩及正常皮肤组织培养的第5～8代成纤维细胞,在含有0．1—3g／L己酮可可碱的环境中培养。应用噻唑蓝（M1Tr）法检测成纤维细胞增殖,双抗体夹心-ELISA法测定TGF—β1表达,RT—PCR检测I、Ⅲ型前胶原mRNA表达。结果0．1～2g／L己酮可可碱能明显抑制瘢痕疙瘩和正常皮肤成纤维细胞的增殖,呈明显的量效一时效关系,抑制作用在浓度2g／L时达到最高。浓度为0．5～2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞TGF—B1表达,1或2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞I、Ⅲ型前胶原mRNA表达。结论己酮可可碱对瘢痕疙瘩和正常皮肤成纤维细胞的增殖、TGF—β1以及I、Ⅲ型前胶原表达均有明显的抑制作用。     

Effect of activated human mast cells and mast cell-derived mediators on proliferation,type I collagen production and glycosaminoglycans synthesis by human dermal fibroblasts

Although an increased number of mast cells in fibrotic tissues such as scleroderma, keloid or healing wound has been highlighted, it is still unclear whether or not mast cells are fibrogenic. The aim of the present study is to determine whether functionally active human mast cells can provide human dermal fibroblasts directly with fibrogenic properties. In order to examine the effects of IgE-mediated mast cell activation on fibroblast proliferation and synthesis of type I collagen, we utilized an in vitro defined system in which cultured human mast cells were co-cultured with human dermal fibroblasts. We also employed a three-dimensional fibroblast culture system using supplementation of L-ascorbic acid as an assay system to investigate the effects of mast cell-derived mediators on synthesis of glycosaminoglycans by human fibroblast. Fibroblast proliferation was actively stimulated with IgE-activated mast cells. However, this stimulatory effect was canceled in co-cultures with a higher number of IgE-activated mast cells. In the presence of a higher number of activated mast cells, the fibroblast cell layer was destroyed, in contrast to an intact cell layer in the presence of same number of the mast cells without activation. Type I collagen synthesis was unchanged in fibroblasts co-cultured with mast cells. The total amount of main disaccharide units, particularly DELTADi-HA, was increased when fibroblasts were exposed to histamine. Thus, we conclude that other factors, in addition to mast cells, are important in the development of human tissue fibrosis or sclerosis.     

Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures

Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in keloid tissue was not different from that in normal skin. The activities of 2 enzymes catalyzing intracellular collagen biosynthesis, prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT) were significantly elevated in the keloids, the mean increase in the former enzyme being 5-fold and in the latter 3-fold with respect to the controls. The mean procollagen production rate in the keloid fibroblasts was at the control level, with only 1 keloid cell line showing a procollagen synthesis rate higher than the mean value + 2 SD of the controls. The mean PH and GGT activities of the keloid fibroblasts were not elevated, but PH activity in 2 cell lines and GGT activity in 1 cell line were higher than the mean + 2 SD for the controls. Cellular type I, III, IV, and V procollagen mRNAs were measured by slot blot hybridization using specific human cDNA clones for the various collagen types. The amounts of type I, III, and V procollagen mRNAs corresponded to the ratios in which these collagen types are produced by fibroblasts. No synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. The total amount of type I and III procollagen mRNAs correlated significantly (p less than 0.01) with the procollagen synthesis rate measured after radioactive labeling of the cells in the keloid and control fibroblasts, indicating that collagen production in these cells is mainly controlled by regulating the final steady state levels of collagen mRNA. The results suggest that fibroblasts isolated from keloids often synthesize normal amounts of collagen.     

Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/AkT signaling pathways

Keloid, a chronic fibro-proliferative disease, exhibits distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells (MCs), and a milieu of enriched cytokines. Previous studies have demonstrated that co-culture with MCs stimulate type I collagen synthesis in fibroblasts, but the signaling mechanisms remain largely unknown. In this study, we investigated the signaling pathways involved in MC-stimulated type I collagen synthesis and the effects of green tea extract (GTE) and its major catechin, (-)-epigallocatechin-3-gallate (EGCG), on collagen homeostasis in keloid fibroblasts. Our results showed that MCs significantly stimulated type I collagen expression in keloid fibroblasts, and the upregulation of type I collagen was significantly attenuated by blockade of phosphatidylinositol-3-kinase (PI-3K), mammalian target of rapamycin (mTOR), and p38 MAPK signaling pathways, but not by blockade of ERK1/2 pathway. Furthermore, GTE and EGCG dramatically inhibited type I collagen production possibly by interfering with the PI-3K/Akt/mTOR signaling pathway. Our findings suggest that interaction between MCs and keloid fibroblasts may contribute to excessive collagen accumulation in keloids and imply a therapeutic potential of green tea for the intervention and prevention of keloids and other fibrotic diseases.     

Elevated prolidase activity in keloids: correlation with type I collagen turnover

BACKGROUND: Keloid pathogenesis involves an altered balance of extracellular matrix metabolism, mainly accumulation of type I collagen. This could be due to excessive synthesis or decreased degradation of matrix, or a combination of both processes. Prolidase, an imidodipeptide-cleaving cytosolic enzyme, plays an important role in the collagen catabolic process by recycling proline for collagen synthesis. Collagen accumulation in keloids is due to an imbalance in the steady state of collagen turnover. OBJECTIVES: To investigate prolidase activity and its role in the steady state of collagen turnover between normal skin and keloid tissue and their derived fibroblasts. METHODS: Ten sets of keloid and normal skin tissues and their derived fibroblasts were employed. Measurements were made of tissue prolidase activity, free proline level, and concentrations of the collagen synthesis product aminoterminal propeptide of type I procollagen (PINP) and the collagen degradative product carboxyterminal telopeptide of type I collagen (ICTP). Also, synthesis of collagens type I and III and matrix metalloproteinases 1 and 2 was investigated using Western blot analysis. RESULTS: Keloid tissues had a significant increase in prolidase activity, up to fourfold that in normal skin. The elevated prolidase activity was accompanied by an increase in tissue PINP and ICTP concentrations in keloid; in addition, the collagen turnover index (PINP/ICTP) was higher in keloids. CONCLUSIONS: The combination of elevated prolidase activity and associated higher collagen synthesis to degradation ratio in keloids suggests a possible metabolic process for the excessive accumulation of type I collagen in keloids.     

Collagen mRNA expression detected by in situ hybridization in keloid tissue

The keloid fibroblasts exhibited increased extracellular matrix gene expression, and prominent elevated type I procollagen mRNA when compared to control fibroblasts cultured from the uninvolved skin of normal people. It also showed markedly elevated type I/III procollagen mRNA ratios, but no synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. By in situ hybridization in keloid tissue, high levels of type I and type III procollagen mRNAs were detected in most of the fibroblasts, suggesting the presence of a subpopulation responsible for the increased collagen production. The levels of type I and type III procollagen mRNAs in these fibroblasts were clearly elevated compared to control skin specimens. And concentration of type I procollagen mRNA was found more predominantly than was type III. These results suggest that deposition of collagen in keloid could result from activation of certain fibroblasts responsible for type I procollagen production.     

SB-431542 inhibits TGF-beta-induced contraction of collagen gel by normal and keloid fibroblasts

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction, which is implicated in wound healing and keloid formation. SB-431542 is a novel specific inhibitor of TGF-beta type I receptor kinase activity. OBJECTIVE: We sought to determine whether SB-431542 inhibited TGF-beta-induced fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with normal or keloid dermal fibroblasts incorporated. RESULTS: TGF-beta induced contraction of collagen gels with normal dermal fibroblasts incorporated, which was efficiently suppressed by SB-431542. Keloid fibroblasts showed higher basal contraction of collagen gels in the absence of TGF-beta than normal fibroblasts, which was enhanced by addition of TGF-beta. SB-431542 suppressed both the basal and TGF-beta-enhanced contraction of collagen gels by keloid fibroblasts. These inhibitory effects of SB-431542 were associated with suppression of TGF-beta-induced alpha-smooth muscle actin (alpha-SMA) expression and phosphorylation of Smad2 in normal and keloid fibroblasts. CONCLUSION: SB-431542 can suppress TGF-beta-induced contraction of collagen gel by normal and keloid dermal fibroblasts. Importantly, SB-431542 can inhibit basal contraction of collagen gel by keloid fibroblasts. These results suggest that an inhibitor of TGF-beta type I receptor kinase activity may have therapeutic potential for excessive skin contraction as observed in keloid.