转化生长因子1致成纤维细胞thy-1,Caveolin-1表达的改变及其在肺纤维化中的意义
摘要
前言
小窝蛋白1,即caveolin-1,是细胞表面的穴样内陷(caveolae)家族的最重要的一员,具有多种生物学功能。最近研究提示caveolin-1与肺纤维化关系密切。成纤维细胞是肺纤维化主要的生物合成细胞,近年来的研究表明,成纤维细胞是一个异质性的群体,根据细胞表面蛋白thy-1可以将成纤维细胞分为thy-1+和thy-1-两个亚组,两亚组在诸多方面具有不同的特性,但是两个亚组caveolin-1蛋白的表达是否相同目前国内外尚无报导。本研究目的是从体外方法研究NIH/3T3成纤维细胞在致纤维化因子的刺激下caveolin-1表达水平的变化以及不同亚组caveolin-1表达的差异。博来霉素是肺纤维化模型是经典的模型,我们同时检测了博来霉素致纤维化不同时期caveolin-1表达的变化,初步探讨了caveolin-1在肺纤维化动物模型中的变化意义。
材料与方法
一、材料
1、NIH3T3成纤维细胞株
2、转化生长因子β1
3、caveolin-1抗体
4、thy-1抗体
5、DMEM培养基
6、胎牛血清。
7、wistar大鼠
二、方法
1、流式细胞术
2、荧光激活细胞分选技术
3、免疫印迹技术
4、免疫组化技术
实验结果
1、TGF-betal能够导致成纤维细胞caveolin-1蛋白表达水平下降,并呈时间和剂量依赖性。
2、TGF-beta 1并不能导致NIH3T3成纤维细胞thy-1表型改变。
3、Thy-1+亚组较thy-1-亚组caveolin-1表达水平明显升高。
4、随着纤维化进展,caveolin-1表达水平呈下降趋势。
讨论
肺纤维化是一种进展性、致死性的疾病。成纤维细胞是主要的生物合成细胞,具有异质性。根据表面蛋白thy-1利用荧光激活细胞分选技术将成纤维细胞分为thy-1+和thy-1-亚组,可以更深入的了解成纤维细胞的功能和特性。Cav-1是细胞表面穴样内陷的重要组成部分,参与了多个系统、多种疾病的发生发展。我们首次探讨了两亚组cav-1表达水平,发现thy-1-亚组高于thy-1+亚组,初步推测thy-1-亚组对于致纤维化因子的高反应性可能与cav-1的低表达有关,进一步的验证可以通过siRNA或者腺病毒转染的方法验证。成纤维细胞的异质性不仅表现在不同亚组对不同刺激反应性不同,而且不同部位、不同疾病的成纤维细胞thy-1表达本身也有所不同。气管内滴注博来霉素是肺纤维化的经典模型,通过免疫组化对肺纤维化模型不同时期cav-1的变化做了初步的探讨,表明肺纤维化的进展中同样伴随了cav-1的表达变化,进一步的研究会对疾病的发病机制和治疗提供新的思路。
结论
基于thy-1蛋白的两亚组成纤维细胞cav-1基础表达水平不同,thy-1-亚组对于致纤维化因子的高反应性可能与cav-1的低表达有关;不同的成纤维细胞thy-1阳性率不同,对于致纤维化因子的反应性亦有差异,进一步说明了成纤维细胞的异质性;Cav-1参与了肺纤维化的发展过程,恢复或增加cav-1生物活性的治疗措施可能有助于恢复上皮细胞的正常再生过程和延缓纤维化的进展。
Preface
Caveolin-1 is one of the most important member of a family of membrane proteins that are the major coating proteins of caveolae which are 50～100nm flask-shaped invaginations that represent a morphologically identifiable subset of lipid raft.Recent findings indicate that there is a close relationship between caveolin-1 and pulmonary fibrosis.Fibroblasts which are major biosynthetic cells in pulmonary fibrosis were shown in recent studies that they are not homogeneous subsets.Based on cell membrane glycoprotein thy-1,fibroblasts are divided into two characteristically subsets ie,thy-1+ subset and thy-1- subset.However,expression of caveolin-1 in the two subsets was not explored currently.This article also sought to determine the expression of caveolin-1 in NIH3T3 fibroblasts under the influence of profibroblastic factor TGF-beta1.Further,we also replicated rat fibrosis model by bleomycin instillation intratracheally.And,the caveolin-1 level was also investigated.
Materials and Methods
一、Materials
1、NIH3T3 cell line
2、TGF-β1
3、Caveolin-1 antibody
4、Thy-1 antibody
5、DMEM culture medium
6、Fetal bovine serum
7、wistar rats
二、Methods
1、Flow cytometry
2、fluorescent activated cell sorting
3、immunoblotting
4、immunohistochemistry
Results
1、TGF-βlean decrease the level of caveolin-1 in fibroblast in a time and dose dependent manner.
2、TGF-β1 has little influence on the expression of thy-1 in NIH3T3 fibroblast.
3、Thy-1+ fibroblast subset has a higher baseline level of caveolin-1 than thy-1-subsets.
4、The level of caveolin-1 decreases as fibrosis progress.
Discussion
Idiopathic pulmonary fibrosis is a progressive and lethal disease,whose main biosynthetic cell is fibroblasts.Based on cell membrane glycoprotein thy-1,fibroblasts can be divided into two characteristically subsets.caveolin-1,the most important component of eaveolae,plays a role in multiple systems and diseases.To our knowledge,we are the first to explore the level of caveolin-1 of the two subsets and find thy-1- subset has a higher baseline level.We presume that the decrease level of cavolin-1 was responsible for the high responsibility of thy-1- fibroblast to profibroblastic factors.So far bleomycin is the standard agent for induction of experimental pulmonary fibrosis in animals.In this experiment we also replicated this model in wistar rats and investigated the expression of caveolin-1.Further research on caveolin-1 may provide a new way for the pathogenesis and therapy of idiopathic pulmonary fibrosis.
Conclusion
The expression of caveolin-1 differ in thy-1+ and thy-1- subsets,and the decrease level of cavolin-1 may be responsible for the high responsibility of thy-1- fibroblast to profibroblastic factors.The different positive rate of thy-1 in different cell types and distinct response to profibroblastic factors may be another proof of the fibroblast heterogeneity.Caveolin-2 was involved in the progress of pulmonary fibrosis, Treatment approaches that augment caveolin-1 bioavailability may help store normal alveolar epithelial repair and regeneration and slow the progression of fibrosis.
小窝蛋白1,即caveolin-1,是细胞表面的穴样内陷(caveolae)家族的最重要的一员,具有多种生物学功能。最近研究提示caveolin-1与肺纤维化关系密切。成纤维细胞是肺纤维化主要的生物合成细胞,近年来的研究表明,成纤维细胞是一个异质性的群体,根据细胞表面蛋白thy-1可以将成纤维细胞分为thy-1+和thy-1-两个亚组,两亚组在诸多方面具有不同的特性,但是两个亚组caveolin-1蛋白的表达是否相同目前国内外尚无报导。本研究目的是从体外方法研究NIH/3T3成纤维细胞在致纤维化因子的刺激下caveolin-1表达水平的变化以及不同亚组caveolin-1表达的差异。博来霉素是肺纤维化模型是经典的模型,我们同时检测了博来霉素致纤维化不同时期caveolin-1表达的变化,初步探讨了caveolin-1在肺纤维化动物模型中的变化意义。
材料与方法
一、材料
1、NIH3T3成纤维细胞株
2、转化生长因子β1
3、caveolin-1抗体
4、thy-1抗体
5、DMEM培养基
6、胎牛血清。
7、wistar大鼠
二、方法
1、流式细胞术
2、荧光激活细胞分选技术
3、免疫印迹技术
4、免疫组化技术
实验结果
1、TGF-betal能够导致成纤维细胞caveolin-1蛋白表达水平下降,并呈时间和剂量依赖性。
2、TGF-beta 1并不能导致NIH3T3成纤维细胞thy-1表型改变。
3、Thy-1+亚组较thy-1-亚组caveolin-1表达水平明显升高。
4、随着纤维化进展,caveolin-1表达水平呈下降趋势。
讨论
肺纤维化是一种进展性、致死性的疾病。成纤维细胞是主要的生物合成细胞,具有异质性。根据表面蛋白thy-1利用荧光激活细胞分选技术将成纤维细胞分为thy-1+和thy-1-亚组,可以更深入的了解成纤维细胞的功能和特性。Cav-1是细胞表面穴样内陷的重要组成部分,参与了多个系统、多种疾病的发生发展。我们首次探讨了两亚组cav-1表达水平,发现thy-1-亚组高于thy-1+亚组,初步推测thy-1-亚组对于致纤维化因子的高反应性可能与cav-1的低表达有关,进一步的验证可以通过siRNA或者腺病毒转染的方法验证。成纤维细胞的异质性不仅表现在不同亚组对不同刺激反应性不同,而且不同部位、不同疾病的成纤维细胞thy-1表达本身也有所不同。气管内滴注博来霉素是肺纤维化的经典模型,通过免疫组化对肺纤维化模型不同时期cav-1的变化做了初步的探讨,表明肺纤维化的进展中同样伴随了cav-1的表达变化,进一步的研究会对疾病的发病机制和治疗提供新的思路。
结论
基于thy-1蛋白的两亚组成纤维细胞cav-1基础表达水平不同,thy-1-亚组对于致纤维化因子的高反应性可能与cav-1的低表达有关;不同的成纤维细胞thy-1阳性率不同,对于致纤维化因子的反应性亦有差异,进一步说明了成纤维细胞的异质性;Cav-1参与了肺纤维化的发展过程,恢复或增加cav-1生物活性的治疗措施可能有助于恢复上皮细胞的正常再生过程和延缓纤维化的进展。
Preface
Caveolin-1 is one of the most important member of a family of membrane proteins that are the major coating proteins of caveolae which are 50～100nm flask-shaped invaginations that represent a morphologically identifiable subset of lipid raft.Recent findings indicate that there is a close relationship between caveolin-1 and pulmonary fibrosis.Fibroblasts which are major biosynthetic cells in pulmonary fibrosis were shown in recent studies that they are not homogeneous subsets.Based on cell membrane glycoprotein thy-1,fibroblasts are divided into two characteristically subsets ie,thy-1+ subset and thy-1- subset.However,expression of caveolin-1 in the two subsets was not explored currently.This article also sought to determine the expression of caveolin-1 in NIH3T3 fibroblasts under the influence of profibroblastic factor TGF-beta1.Further,we also replicated rat fibrosis model by bleomycin instillation intratracheally.And,the caveolin-1 level was also investigated.
Materials and Methods
一、Materials
1、NIH3T3 cell line
2、TGF-β1
3、Caveolin-1 antibody
4、Thy-1 antibody
5、DMEM culture medium
6、Fetal bovine serum
7、wistar rats
二、Methods
1、Flow cytometry
2、fluorescent activated cell sorting
3、immunoblotting
4、immunohistochemistry
Results
1、TGF-βlean decrease the level of caveolin-1 in fibroblast in a time and dose dependent manner.
2、TGF-β1 has little influence on the expression of thy-1 in NIH3T3 fibroblast.
3、Thy-1+ fibroblast subset has a higher baseline level of caveolin-1 than thy-1-subsets.
4、The level of caveolin-1 decreases as fibrosis progress.
Discussion
Idiopathic pulmonary fibrosis is a progressive and lethal disease,whose main biosynthetic cell is fibroblasts.Based on cell membrane glycoprotein thy-1,fibroblasts can be divided into two characteristically subsets.caveolin-1,the most important component of eaveolae,plays a role in multiple systems and diseases.To our knowledge,we are the first to explore the level of caveolin-1 of the two subsets and find thy-1- subset has a higher baseline level.We presume that the decrease level of cavolin-1 was responsible for the high responsibility of thy-1- fibroblast to profibroblastic factors.So far bleomycin is the standard agent for induction of experimental pulmonary fibrosis in animals.In this experiment we also replicated this model in wistar rats and investigated the expression of caveolin-1.Further research on caveolin-1 may provide a new way for the pathogenesis and therapy of idiopathic pulmonary fibrosis.
Conclusion
The expression of caveolin-1 differ in thy-1+ and thy-1- subsets,and the decrease level of cavolin-1 may be responsible for the high responsibility of thy-1- fibroblast to profibroblastic factors.The different positive rate of thy-1 in different cell types and distinct response to profibroblastic factors may be another proof of the fibroblast heterogeneity.Caveolin-2 was involved in the progress of pulmonary fibrosis, Treatment approaches that augment caveolin-1 bioavailability may help store normal alveolar epithelial repair and regeneration and slow the progression of fibrosis.
引文
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44 Koumas L,Smith TJ,Phipps RP: Fibroblast subsets in the human orbit: Thy-1~+ and Thy-1~-subpopulations exhibit distinct phenotypes.Eur J Immunol 2002,32:477-485.
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49 Matthew E.Felch,Deborah Brown,and Richard P.Phipps.Differential collagen and fibronectin production by Thy 1+ and Thy 1- lung fibroblast subpopulations.Am.J.Physiol.1992;263( Lung Cell.Mol.Physiol.7): L283-L290.
50 Fluck J,Querfeld C,Cremer A,Niland S,Krieg T,Sollberg S.Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels.J.invest Dermatol.1998; 110(2): 153-7.