IGF、VEGF、Decorin和Versican在病理性瘢痕中的表达及意义
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摘要
[目的]探讨病理性瘢痕形成中,尤其是瘢痕疙瘩,几种细胞因子(IGF、VEGF)和细胞外基质(decorin、versican)的表达及其临床意义,揭示其对瘢痕的作用及机制,为临床预防和治疗瘢痕提供分子及病理学水平的理论依据。
[方法]1.对瘢痕疙瘩、正常瘢痕和正常皮肤成纤维细胞进行体外培养,采用光镜、透射电镜、流式细胞术观察三种细胞形态、超微结构及细胞活性的差异;2.将石蜡包埋的组织块(瘢痕疙瘩和正常皮肤)进行总RNA提取,对VEGF、decorin、versican进行PCR检测;3.用免疫组化、免疫荧光标记并激光共聚焦显微镜观察、ELISA检测、实时荧光定量PCR和westen blot对IGF、VEGF、decorin、versican四种物质在成纤维细胞中的表达进行检测和分析;4.建立西双版纳近交系猪背部瘢痕模型,对其进行体内实验,采用HE染色、Masson氏三色染色、透射电镜、原位杂交(检测decorin的表达)等检测方法观察滇丹参对瘢痕的作用。通过以上实验观察分析,以研究四种物质在病理性瘢痕中的表达、临床意义及滇丹参对瘢痕的作用。
[结果]1.体外培养的瘢痕疙瘩和正常皮肤成纤维细胞光镜观察显示:三种细胞在形态学上没有明显的差异;透射电镜结果显示,瘢痕疙瘩较正常瘢痕和正常皮肤的成纤维细胞内有更为丰富的粗面内质网和线粒体,流式细胞术结果示,瘢痕疙瘩成纤维细胞S期百分比较高,即表示细胞的活性较好;2.石蜡包埋的组织块提取总RNA的PCR结果示:与正常皮肤相比,瘢痕疙瘩中VEGF的表达明显增高,decorin的表达明显降低;而versican的四种亚型在瘢痕疙瘩中的表达与正常皮肤相比没有明显差异;3.成纤维细胞免疫荧光标记结果显示:在瘢痕疙瘩成纤维细胞中,IGF、VEGF和versican的表达较高,而在正常皮肤中decorin的表达更高;4.成纤维细胞培养上清液ELISA检测结果:细胞培养的上清液中,与正常皮肤相比,瘢痕疙瘩中IGF和VEGF的浓度更高,而与瘢痕疙瘩相比,正常皮肤中decorin的浓度更高;5.成纤维细胞提取RNA实时荧光定量实时荧光定量PCR:与正常皮肤相比,瘢痕疙瘩中VEGF的表达有2倍的增长,decorin的表达有约5倍的降低;而versican的四种亚型在瘢痕疙瘩中均有表达,且都较正常皮肤高,表达量由高到低是V1、V3、V0、V2,分别是10倍、8倍、4倍、3倍的增高;6.westenblot结果示:在蛋白水平,与正常皮肤相比,瘢痕疙瘩中VEGF是明显增高的;而与瘢痕疙瘩相比,decorin则在正常皮肤中有较高的表达;7.动物模型标本病理染色、透射电镜观察:与对照组相比,加入滇丹参的实验组中成纤维细胞较少,纤维细胞较多,粗面内质网不发达,细胞核异染色质丰富,胶原排列较疏松;8.动物模型标本Decorin原位杂交分析:在正常皮肤中Decorin mRNA表达见于真皮层,与对照组相比,实验组注射滇丹参后2个月在真皮层出现弱阳性表达,而对照组中未见表达。
[结论]在皮肤的创伤愈合中,IGF、VEGF和versican的过度表达可能促进了病理性瘢痕的形成,而decorin的低表达可能导致了病理性瘢痕的形成;滇丹参对瘢痕成纤维细胞的生长、增殖及胶原合成有抑制作用。
[Objective]This study was designed to explore the expression and significance of growth factors(IGF,VEGF) and extracellular matrix(decorin,versican) during formation of scars,especially keloids.Reveal the mechanism to provide basic theory for clinical precaution and treatment to scars at molecular and pathological level.
[Method]1.The f ibroblasts of keloid,normal scar and normal skin were cultured in vitro.Their difference of morphology,ultrastructure and cell activity will be observed by light microscopy,transmission electron microscopy,flow cytometry;2.Total RNA Will be extracted from paraffin embedded tissue(keloids and normal skin) and expression of VEGF,decorin and versican will be detected by PCR;3.By immunohistochemistry,immunofluorescence and confocal laser microscopy,ELISA detection,real-time fluorescence quantitative PCR and the westen blot,the expression of IGF,VEGF,decorin and versican will be detected and analyzed in the fibroblasts;4.Hypertrophic scar models were made on pig back in vivo by different concentration Salvia yunnanensis C.H.Wright injection, detected by HE staining,Masson's staining,transmission electron microscope and in situ hybridization.Using these experimental analyse,to study the biological behavior and pathological changes of hypertrophic scar fibroblasts affected by Salvia yunnanensis C.H.Wright.
[Result]1.The result of Light microscopy showed:there are no significant differences in morphology of fibroblasts of Keloids,scars and nomal skin.The result of transmission electron microscopy showed there are more rich with rough endoplasmic reticulum and mitochondria in fibroblasts of keloids than that of scars and nomal skin.The result of flow cytometry indicated that fibroblasts of keloid was higher in S phase percentage,that means the better the activity of cells;2.The result of PCR with total RNA from paraffin-embedded tissue show:compared with normal skin,keloid were significantly higher in VEGF expression,decorin expression was significantly reduced;and the four subtypes of versican expression in keloids compared with normal skin,there is no significant difference;3.fibroblasts immunofluorescence showed that:in keloid fibroblasts,the expression of IGF,VEGF and versican were higher,while in normal skin in a higher decorin expression;4.The result of ELISA with fibroblasts culture supernatant show:compared with normal skin,keloid higher concentrations of IGF and VEGF,compared with keloids,normal skin decorin concentrations higher;5.The result of Real-time fluorescence quantitative real-time quantitative PCR with RNA from fibroblast show:compared with normal skin,keloid in the expression of VEGF and 2-fold increase,decorin expression decreased about 5 times;and versican of the four subtypes were expressed in keloid and have higher than normal skin,expression from high to low is the V1, V3,V0,V2,respectively,10 times,8 times,4 times,3 times higher;6.The result of westen blot show:in the protein level,compared with normal skin, VEGF in keloid is significantly elevated;compared with keloids,expression of decorin was higher in normal skin;7.In vito(pig back scar model):Compared with the control group,by pathological staining,the experiment group were less fibroblasts and the collagen density reduced and arranged in regulation.The spaces between fiber bundles were increased.By TEM,compared with the control group,there were less fibroblast than fibro and undeveloped rough endoplasmic reticulum.Heterochranatins were very abundance and an arrangement of collagen fibers which were orderly.In part rough endoplasmic reticulum were over expand and have the characteristic of posterior fibroblast.The expression of decorin mRNA was detected by in situ hybridization:The expression of decorin mRNA was rich in the normal skin dermis.The lower expression of decorin mRNA was found after one more months with Salvia yunnanensis C.H.Wright.injection,but the control group were not found.
[Conclusion]In the skin wound healing,overexpression of IGF,VEGF,and versican may promote scars formation,while low expression of decorin leads to scar formation;Salvia yunnanensis C.H.Wright can decrease fibroblasts growth, proliferation and collagen synthesis.
[方法]1.对瘢痕疙瘩、正常瘢痕和正常皮肤成纤维细胞进行体外培养,采用光镜、透射电镜、流式细胞术观察三种细胞形态、超微结构及细胞活性的差异;2.将石蜡包埋的组织块(瘢痕疙瘩和正常皮肤)进行总RNA提取,对VEGF、decorin、versican进行PCR检测;3.用免疫组化、免疫荧光标记并激光共聚焦显微镜观察、ELISA检测、实时荧光定量PCR和westen blot对IGF、VEGF、decorin、versican四种物质在成纤维细胞中的表达进行检测和分析;4.建立西双版纳近交系猪背部瘢痕模型,对其进行体内实验,采用HE染色、Masson氏三色染色、透射电镜、原位杂交(检测decorin的表达)等检测方法观察滇丹参对瘢痕的作用。通过以上实验观察分析,以研究四种物质在病理性瘢痕中的表达、临床意义及滇丹参对瘢痕的作用。
[结果]1.体外培养的瘢痕疙瘩和正常皮肤成纤维细胞光镜观察显示:三种细胞在形态学上没有明显的差异;透射电镜结果显示,瘢痕疙瘩较正常瘢痕和正常皮肤的成纤维细胞内有更为丰富的粗面内质网和线粒体,流式细胞术结果示,瘢痕疙瘩成纤维细胞S期百分比较高,即表示细胞的活性较好;2.石蜡包埋的组织块提取总RNA的PCR结果示:与正常皮肤相比,瘢痕疙瘩中VEGF的表达明显增高,decorin的表达明显降低;而versican的四种亚型在瘢痕疙瘩中的表达与正常皮肤相比没有明显差异;3.成纤维细胞免疫荧光标记结果显示:在瘢痕疙瘩成纤维细胞中,IGF、VEGF和versican的表达较高,而在正常皮肤中decorin的表达更高;4.成纤维细胞培养上清液ELISA检测结果:细胞培养的上清液中,与正常皮肤相比,瘢痕疙瘩中IGF和VEGF的浓度更高,而与瘢痕疙瘩相比,正常皮肤中decorin的浓度更高;5.成纤维细胞提取RNA实时荧光定量实时荧光定量PCR:与正常皮肤相比,瘢痕疙瘩中VEGF的表达有2倍的增长,decorin的表达有约5倍的降低;而versican的四种亚型在瘢痕疙瘩中均有表达,且都较正常皮肤高,表达量由高到低是V1、V3、V0、V2,分别是10倍、8倍、4倍、3倍的增高;6.westenblot结果示:在蛋白水平,与正常皮肤相比,瘢痕疙瘩中VEGF是明显增高的;而与瘢痕疙瘩相比,decorin则在正常皮肤中有较高的表达;7.动物模型标本病理染色、透射电镜观察:与对照组相比,加入滇丹参的实验组中成纤维细胞较少,纤维细胞较多,粗面内质网不发达,细胞核异染色质丰富,胶原排列较疏松;8.动物模型标本Decorin原位杂交分析:在正常皮肤中Decorin mRNA表达见于真皮层,与对照组相比,实验组注射滇丹参后2个月在真皮层出现弱阳性表达,而对照组中未见表达。
[结论]在皮肤的创伤愈合中,IGF、VEGF和versican的过度表达可能促进了病理性瘢痕的形成,而decorin的低表达可能导致了病理性瘢痕的形成;滇丹参对瘢痕成纤维细胞的生长、增殖及胶原合成有抑制作用。
[Objective]This study was designed to explore the expression and significance of growth factors(IGF,VEGF) and extracellular matrix(decorin,versican) during formation of scars,especially keloids.Reveal the mechanism to provide basic theory for clinical precaution and treatment to scars at molecular and pathological level.
[Method]1.The f ibroblasts of keloid,normal scar and normal skin were cultured in vitro.Their difference of morphology,ultrastructure and cell activity will be observed by light microscopy,transmission electron microscopy,flow cytometry;2.Total RNA Will be extracted from paraffin embedded tissue(keloids and normal skin) and expression of VEGF,decorin and versican will be detected by PCR;3.By immunohistochemistry,immunofluorescence and confocal laser microscopy,ELISA detection,real-time fluorescence quantitative PCR and the westen blot,the expression of IGF,VEGF,decorin and versican will be detected and analyzed in the fibroblasts;4.Hypertrophic scar models were made on pig back in vivo by different concentration Salvia yunnanensis C.H.Wright injection, detected by HE staining,Masson's staining,transmission electron microscope and in situ hybridization.Using these experimental analyse,to study the biological behavior and pathological changes of hypertrophic scar fibroblasts affected by Salvia yunnanensis C.H.Wright.
[Result]1.The result of Light microscopy showed:there are no significant differences in morphology of fibroblasts of Keloids,scars and nomal skin.The result of transmission electron microscopy showed there are more rich with rough endoplasmic reticulum and mitochondria in fibroblasts of keloids than that of scars and nomal skin.The result of flow cytometry indicated that fibroblasts of keloid was higher in S phase percentage,that means the better the activity of cells;2.The result of PCR with total RNA from paraffin-embedded tissue show:compared with normal skin,keloid were significantly higher in VEGF expression,decorin expression was significantly reduced;and the four subtypes of versican expression in keloids compared with normal skin,there is no significant difference;3.fibroblasts immunofluorescence showed that:in keloid fibroblasts,the expression of IGF,VEGF and versican were higher,while in normal skin in a higher decorin expression;4.The result of ELISA with fibroblasts culture supernatant show:compared with normal skin,keloid higher concentrations of IGF and VEGF,compared with keloids,normal skin decorin concentrations higher;5.The result of Real-time fluorescence quantitative real-time quantitative PCR with RNA from fibroblast show:compared with normal skin,keloid in the expression of VEGF and 2-fold increase,decorin expression decreased about 5 times;and versican of the four subtypes were expressed in keloid and have higher than normal skin,expression from high to low is the V1, V3,V0,V2,respectively,10 times,8 times,4 times,3 times higher;6.The result of westen blot show:in the protein level,compared with normal skin, VEGF in keloid is significantly elevated;compared with keloids,expression of decorin was higher in normal skin;7.In vito(pig back scar model):Compared with the control group,by pathological staining,the experiment group were less fibroblasts and the collagen density reduced and arranged in regulation.The spaces between fiber bundles were increased.By TEM,compared with the control group,there were less fibroblast than fibro and undeveloped rough endoplasmic reticulum.Heterochranatins were very abundance and an arrangement of collagen fibers which were orderly.In part rough endoplasmic reticulum were over expand and have the characteristic of posterior fibroblast.The expression of decorin mRNA was detected by in situ hybridization:The expression of decorin mRNA was rich in the normal skin dermis.The lower expression of decorin mRNA was found after one more months with Salvia yunnanensis C.H.Wright.injection,but the control group were not found.
[Conclusion]In the skin wound healing,overexpression of IGF,VEGF,and versican may promote scars formation,while low expression of decorin leads to scar formation;Salvia yunnanensis C.H.Wright can decrease fibroblasts growth, proliferation and collagen synthesis.
引文
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