【作者】 朱鹭冰；

【导师】 李明； 冯林音；

【作者基本信息】 复旦大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 第一部分SSc患者和正常人皮肤Fb克隆分离及胶原合成异质性克隆的筛选目的分离系统性硬皮病(Systemic scleroderma,SSc)患者和正常人皮肤成纤维细胞(Fibroblast,Fb)克隆,筛选出胶原合成异质性克隆。方法以组织块培养法进行SSc患者和正常人皮肤Fb原代培养,采用免疫组化方法进行细胞鉴定,以改良的有限稀释法分离、培养Fb克隆,通过原位杂交方法检测Fb克隆Ⅰ型前胶原α1链(COL1A1)mRNA的表达。结果建立5个SSc患者皮肤Fb细胞系和4个正常人皮肤Fb细胞系,经免疫组化法鉴定培养的细胞为Fb。经分离、培养共获得68个Fb克隆(克隆第四代),包括SSc患者36个Fb克隆和正常人32个Fb克隆。原位杂交法显示SSc组Fb克隆COL1A1mRNA的平均OD值(6.636±2.480)高于正常对照组(3.693±2.007),t=3.690,P=0.001。通过聚类分析将所有克隆的平均OD值分为高、中、低三组。SSc组和正常对照组Fb克隆高、中、低三组的总体构成比不同(P=0.005)。SSc组COL1A1mRNA高表达组克隆的比例高于正常对照组。在此基础上,本课题组另一成员对Fb克隆Ⅰ型前胶原蛋白(免疫印迹和ELISA法)和COL1A1、COL3A1mRNA(实时定量RT-PCR,Taqman探针法)的测定结果表明:(1)SSc组Fb克隆COL1A1mRNA与COL3A1mRNA水平的异质性较正常对照组大;(2)Fb克隆Ⅰ型前胶原蛋白表达与其mRNA水平呈正相关;(3)Fb克隆的COL1A1mRNA水平与COL3A1mRNA水平呈正相关。并将Fb克隆分为高、中、低胶原合成组。SSc组高胶原合成克隆的比例及其平均胶原合成量高于正常对照组高胶原合成克隆。结论SSc患者皮肤Fb高胶原合成克隆的比例及其胶原合成增高的幅度大于正常人。第二部分SSc患者和正常人胶原合成异质性Fb克隆的胶原基因转录特性研究目的了解SSc患者和正常人胶原合成异质性Fb克隆的胶原基因转录特性。方法构建含人COL1A1和COL3A1基因启动子片段的重组质粒,采用瞬时转染法、双荧光素酶报告基因检测技术测定COL1A1和COL3A1基因启动子在胶原合成异质性Fb克隆中的活性。通过染色质免疫沉淀(ChIP)分析法结合定量PCR法检测转录因子Sp1与不同Fb克隆COL1A1基因近端启动区的结合活性。结果经琼脂糖凝胶电泳和测序鉴定,所有重组质粒构建正确。经瞬时转染、报告基因测活发现COL1A1-174bp～+42bp、COL3A1-96bp～+16bp在不同Fb克隆中具有相对最高或较高的活性,并且其活性分别与Fb克隆Ⅰ型/Ⅲ型前胶原表达呈正相关。ChIP、定量PCR结果显示,Sp1与高胶原合成克隆COL1A1近端启动区-174bp～+42bp的结合活性高于低胶原合成克隆(P＜0.05),这种差异在SSc组较正常对照组更为明显(F=24.569,p=0.000)。结论SSc患者高胶原合成Fb克隆的Ⅰ型、Ⅲ型前胶原基因在转录水平即被激活;转录因子Sp1可能参与上调SSc患者高胶原合成Fb克隆COL1A1-174bp～+42bp启动片段的活性。第三部分细胞因子和丹参对SSc患者胶原合成异质性Fb克隆胶原基因转录的调控目的研究细胞因子、丹参及其单体对SSc患者胶原合成异质性Fb克隆Ⅰ型、Ⅲ型前胶原基因转录的影响。方法以瞬时转染方法将含有人COL1A1-174bp～+42bp、COL3A1-96bp～+16bp启动片段的重组质粒转染SSc患者和正常人胶原合成异质性Fb克隆,施加细胞因子、丹参及其单体干预48h后,通过双荧光素酶报告基因检测技术测定COL1A1和COL3A1近端启动区的活性。结果TGF-β1(5ng/mL)、CTGF(20ng/mL)上调COL1A1、COL3A1近端启动区在SSc患者和正常人Fb克隆中的活性,其中CTGF在SSc患者优先上调COL3A1近端启动区在低胶原合成Fb克隆中的活性。丹参注射液(1mg/mL)、丹参酮ⅡA(5μg/mL)抑制COL1A1、COL3A1近端启动区在SSc患者和正常人Fb克隆中的活性,并且优先下调两者在SSc患者高胶原合成Fb克隆中的活性;原儿茶醛(5μg/mL)和丹酚酸B(5μg/mL)分别选择性抑制COL1A1和COL3A1近端启动区在Fb克隆中的活性。未发现IFN-γ(100ng/mL)、丹参素钠(20μg/mL)抑制COL1A1和COL3A1近端启动区在Fb克隆中的活性。结论细胞因子之间可能构成复杂的转录调控网络,参与激活SSc患者Fb胶原基因转录。丹参及其部分单体可抑制SSc患者胶原合成异质性Fb克隆Ⅰ型、Ⅲ型前胶原基因转录,但作用不尽相同。更多还原

【Abstract】 PartⅠIsolation of dermal fibroblast clones from systemic scleroderma patients and normal controls and separation of clones into different subpopulations by amount of collagen producedObjective To isolate dermal fibroblast clones from patients of systemic scleroderma(SSc) and normal controls and separate the clones into different subpopulations by the amount of collagen produced.Methods Fibroblast cell lines from sclerotic skin of SSc patients and skin of normal controls were established and identified by immunohistology.By means of modified limiting dilution cloning,single cells were isolated and cultured.In situ hybridization was used to detect mRNA steady-state level ofα1(Ⅰ) procollagen gene(COL1A1) of fibroblast clones.Results Five fibroblast cell lines from SSc and four from normal controls were established,from which 68 fibroblast clones(4th-passage fibroblast clones) were obtained,consisting of 36 clones from SSc group and 32 clones from normal controls. The mean COL1A1 mRNA steady-state level of fibroblast clones from SSc group (6.636±2.480) was much higher than normal controls(3.693±2.007)(t=-3.690, P=0.001).All the clones were divided into 3 subpopulations(high,intermediate and low) by K-means cluster analysis of COL1A1 mRNA levels.The total constituent ratios of the high,intermediate and low COL1A1 mRNA levels subpopulations between SSc group and normal controls were significantly different(P=0.005).A larger proportion of high COL1A1 mRNA level subpopulation was observed in SSc group compared with normal controls.Based on the above observations,another member of our project examined the typeⅠprocollagen production of the fibroblast clones by western blot and ELISA and COL1A1/α1(Ⅲ) procollagen(COL3A 1) mRNA levels by real-time RT-PCR(Taqman). The results showed the heterogeneity of COL1A1/COL3A1 mRNA of the fibroblast clones from SSc patients was more significant than clones from normal controls.The amounts of typeⅠprocollagen produced by the fibroblast clones paralleled their mRNA levels,and the COL1A1 mRNA levels paralleled their COL3A1mRNA levels. All the fibroblast clones were divided into high,intermediate and low collagen-producing subpopulations.A larger proportion and a higher extent of increased collagen production of high collagen-producing subpopulation were observed in SSc group compared with normal controls.Conclusions The proportion and the extent of increased collagen production of high collagen-producing subpopulation were elevated in fibroblast clones from patients with SSc compared with normal controls.PartⅡTranscription characteristics of procollagen gene in subpopulations of SSc fibroblast clones with different amounts of collagen producedObjective To study the transcription characteristics of COL1A1 and COL3A1 gene in subpopulations of SSc fibroblast clones with different amounts of collagen produced.Methods Five recombinant plasmids containing various lengths of sequences of human COL1A1 and COL3A1 promoter were constructed.By means of cationic liposome-mediated transient transfection and dual-luciferase reporter assay system, the activity of different fragments of COL1A1 and COL3A1 promoter in fibroblast clones subpopulations from SSc and normal controls with different amounts of collagen produced was measured.By ChIP and qPCR,the binding activity between Sp1 and COL1A1 proximal promoter was examined.Results All of the recombinant plasmids targeting on COL1A1 and COL3A1 promoter were confirmed by agarose electrophoresis and DNA sequencing analysis of the inserted segments.In most of the examined clones from different subpopulations,maximal activity was involved COL1A1 promoter region encompassing -174bp～+42bp and COL3A1 promoter region encompassing -96bp～+16bp,which paralleled the amounts of their typeⅠ/typeⅢprocollagen production.ChIP and qPCR showed that the binding activity between Sp1 and COL1A1-174bp～+42bp was elevated in high collagen-producing subpopulation, compared with low collagen-producing subpopulation(P＜0.05),and the difference was more significant in SSc group compared with normal controls(F=24.569, P=0.000).Conclusions The expression of COL1A1 and COL3A1 gene in high collagen-producing fibroblast clones from patients with SSc may be upregulated at the transcriptional level,in which Spl may be involved.PartⅢTranscription regulation of cytokines and Radix Salviae Miltiorrhizae on procollagen gene in subpopulations of SSc fibroblast clones with different amounts of collagen producedObjective To study the transcription regulation effect of cytokines and Radix Salviae Miltiorrhizae on COL1A1 and COL3A1 gene in subpopulations of SSc fibroblast clones with different amounts of collagen produced.Methods By transient transfection and dual-luciferase reporter assay system,the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in subpopulations of SSc fibroblast clones with different amounts of collagen produced under cytokines, Radix Salviae Miltiorrhizae and its active monomers were detected.Results TGF-β1(5ng/mL) and CTGF(20ng/mL) upregulated the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in fibroblast clones from patients with SSc and normal controls,and CTGF preferentially upregulated the activity of COL3A1-96bp～+16bp in low collagen-producing subpopulations of SSc fibroblast clones.No inhibiting effects of IFN-γ(100ng/mL) on COL1A1-174bp～+42bp or COL3A1-96b～+16bp activities were observed.The water soluble extracts of Radix Salviae Miltiorrhizae(1mg/mL) and Yanshinone IIA(5μg/mL) inhibited the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in fibroblast clones from SSc patients and normal controls,preferentially downregulating the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in high collagen-producing subpopulations of SSc fibroblast clones.Protocatechuic aldehyde(5μg/mL) and Salvianolic acid B(5μg/mL) respectively inhibited the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in fibroblast clones.There were no significant effects of Danshensu(20μg/mL) on COL1A1-174bp～+42bp and COL3A1-96bp～+16bp activities in fibroblast clones.Conelusions Cytokines may constitute a complex network of transcriptional regulation involved in activation of procollagen gene in SSc fibroblasts.Radix Salviae Miltiorrhizae and some of its active monomers played different roles in inhibiting COL1A1 and COL3A1 gene transcription in SSc fibroblasts.更多还原