炎性关节病中成纤维样滑膜细胞的生物学特性及致病机制研究
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摘要
成纤维样滑膜细胞(fibroblast-like synoviocytes,FLSs)是炎症关节中重要的效应细胞。过去认为其只在对单核和淋巴细胞的应答中具有修复功能,即具有降解和重塑细胞外基质的作用;目前认为,FLSs能够向周围基质细胞和浸润免疫细胞提供大量的趋化和活化信号,并具有不需要T细胞介导的直接破坏软骨和骨的能力。体外培养的FLSs能够释放多种细胞因子和生长因子,区别于从非滑膜部位分离的成纤维细胞,具有独特的生物学特性。FLSs释放的效应分子能刺激或(在某些情况下)减弱炎症反应,其具有的参与关节炎症的作用正越来越被人们所关注。
本研究旨在观察类风湿关节炎、骨关节炎以及强直性脊柱炎患者体外培养的FLSs形态学特征及其在静息和刺激条件下的生长增殖曲线及细胞迁移比率;通过流式细胞术检测细胞免疫表型在FLSs的表达和比例;通过实时定量PCR检测炎性细胞因子mRNA的表达情况以及通过CBA法分析细胞培养上清中分泌的炎症因子的表达水平,并比较药物(地塞米松、环孢素A)对其的影响,从而探讨FLSs与关节炎相关的发病机制和治疗机理。具体分为以下三个部分:
第一部分不同类型关节炎FLSs的形态学特征及其在静息和刺激条件下的生长增殖曲线及细胞迁移能力比较
目的:了解体外原代及传代培养的源自不同类型关节炎患者的FLSs的形态学特征,比较其在静息和刺激条件下的细胞生长增殖特性及细胞的迁移能力。
方法:选择骨关节炎(OA)13例,类风湿关节炎(RA)6例,强直性脊柱炎(AS)2例,关节置换术取滑膜组织或抽取膝关节滑液分离FLSs滑膜细胞并培养、传代。在相差显微镜下观察细胞形态,WST-1法观察细胞生长曲线以及对脂多糖(LPS)刺激的增殖反应,Transwell小室观察细胞在静息及LPS刺激下的迁移能力变化。
结果:OA、RA以及AS患者FLSs细胞在形态上无明显差异:原代和早期传代的贴壁细胞均表现为梭形、星形、多角形、树突形等形态上的多样性,传代后并密集生长的FLSs细胞形态逐渐单一纯化,多为梭形且大小均一、呈平行样或漩涡样排列,滑液来源的细胞中圆形或椭圆型的贴壁细胞占多数。RA和OA FLSs在传代后第4天进入对数生长期,在第7天达到生长平台期。LPS刺激组于给药后第2天明显增殖,第3天增殖达到最高,但在维持LPS浓度的第5天,细胞数降至比未刺激组更低的水平。培养第3天的RA FLSs与OA FLSs的OD值进行比较有显著性差异(1.48±0.07 vs 0.808±0.12,P＜0.001)。滑液来源的AS FLSs细胞生长缓慢,对LPS刺激反应不明显。RA FLSs与OA FLSs在LPS刺激下迁移的细胞数比对照组有显著差异(55.58±4.09 vs 30.25±1.79,36.10±3.50 vs 22.00±2.22,P值均＜0.01),RA FLSs在刺激后增加的迁移细胞数与OA FLSs比较也有显著的差异(25.33±3.86 vs 14.11±3.26,P＜0.005)。
结论:不同类型炎性关节病患者体外培养的FLSs在光镜下无明显形态学上的差异;体外培养的RAFLSs具有比较OAFLSs更强的增殖和迁移能力。
第二部分免疫细胞化学相关膜分子在FLSs细胞的表达及变化以及胞内因子TNFα、IFNγ的流式检测
目的:通过流式细胞术筛查FLSs膜分子的免疫细胞化学特征及TNF×、IFNγ等细胞因子在FLSs细胞的表达情况,检测在LPS刺激下FLSs细胞亚群的表型和比例变化。
方法:病例选择同第一部分。选择的膜分子抗体染料试剂为:CD3-FITC,CD4-PE,CD8-APC,CD45-Pacific Orange,CD45RO-APC,CD45RA-PE,TCRαβ-PE,TCRγδ-PE;CD19-PE-Cy5,CD14-APC,CD68-FITC,CD11c-PE,HLA-DR-APC;CD2-PE,CD7-FITC,CD16-PE,CD56-PE,CD57-FITC,CD116-Pacific Blue;CD13-PE,CD33-PE,CD34-PE-Cy7,CD38-Percp-Cy5.5,CD44-Pacific Blue,CD69-PE-Cy5,CD90-APC,CD117-PE。胞内染色的细胞因子为TNF-α-PacificBlue、IFNγ-PercpCy5.5、IL-4-FITC、IL-17-PE。
结果:在筛查的27种表型中,CD13、CD90、CD44呈强阳性表达(分别为98.05±2.18%,96.7±4.8%,88.83±8.2%)。CD34、CD56、HLA-DR呈弱阳性表达,且为独立的亚群(分别为8.36±2.76%,18.75±13.74%,3.28±2.34%),其它21种抗体阳性比例均＜0.5%。RA-FLSs(n=6)中CD34~+、CD56~+、HLA-DR~+细胞亚群的比例与OA FLSs(n=13)比较均有显著差异,其中RA FLSs CD56~+、HLA-DR~+比例高于OA FLSs(分别为33.65±6.12vs16.80±4.36,7.13±1.85 vs 3.75±1.24,P＜0.0001),而CD34~+比例低于OA FLSs(4.34±1.63vs8.89±2.60,P＜0.01)。在LPS刺激2h后,RA FLSs(n=6)CD56~+FLSs、HLA-DR~+FLSs表达比例较未刺激时明显增高(分别为47.61±8.35vs33.65±6.12,P＜0.05;26.68±9.47vs7.13±1.85P＜0.01),而CD34~+FLSs比例则较未刺激时明显下降(1.93±0.92vs4.34±1.63,P＜0.01)。2例OA FLSs与1例RA FLSs在LPS刺激后产生少量的TNF×、IFNγ,IL-4与IL-17在各次检测中均为阴性。
结论:CD90、CD44、CD13是FLSs的主要分子标记,CD56~+FLSs、CD34~+FLSs、HLA-DR~+FLSs亚群可能与滑膜炎症的调控相关,FLSs细胞可在刺激后产生少量TNFα和IFNγ。
第三部分FLSs细胞IL-6、IL-1β、IL-10 mRNA的表达、培养上清中分泌的炎性细胞因子水平以及不同药物对其的影响
目的:在转录水平观察FLSs细胞IL-6、IL-1β、IL-10mRNA的表达,并观察地塞米松(Dex)及环孢素A(CsA)在LPS刺激2h后对其表达的影响;在蛋白水平检测细胞培养上清中IL-6、IL-8、IL-1B、IL-10、IL-12p70以及TNFα等主要炎性因子的表达以及Dex、CsA在LPS刺激24h后对其分泌的细胞因子的影响。
方法:提取FLSs总RNA,合成cDNA第一链,以β-actin为看家基因,实时荧光定量PCR分析FLSs细胞中IL-6、IL-1β、IL-10mRNA的相对含量,比较LPS刺激前后以及分别加入药物Dex、CsA后的影响;CBA法检测FLSs细胞培养上清中分泌的IL-6、IL-8、IL-1β、IL-10、IL-12p70以及TNFa的水平,并比较LPS刺激前后以及分别加入药物Dex、CsA后的影响。
结果:OA和RA FLSs细胞IL-6、IL-1β、IL-10 mRNA在LPS刺激后的表达明显增加,其中IL-1βmRNA表达最高,IL-6 mRNA次之,加入Dex后,各细胞因子mRNA水平均明显降低,而加入CsA后降低的程度不如Dex明显。RA和OA FLSs在静息状态下可分泌低水平的IL-6(分别为1.12±1.05,3.53±3.99 ng/ml)及IL-8(分别为0.15±0.08,0.15±0.07 ng/ml)。在LPS刺激下,IL-6水平增高(分别为42.78±12.49,10.18±3.32 ng/ml),IL-8水平也增高(分别为23.13±7.25,19.07±9.34 ng/ml),其中以RA FLSs增高程度更为明显(P＜0.001)。Dex能够明显抑制IL-6的分泌,对IL-8的抑制作用相对较弱,而CsA均仅起到部分的抑制作用。而其它IL-1β、IL-10、IL-12p70及TNFα的含量均为微量或测不出。
结论:IL-6与IL-8是FLSs分泌的主要炎症相关细胞因子,IL-1β、TNFα等主要由巨噬细胞、T细胞表达的细胞因子在FLSs很少表达,Dex具有比CsA更强的阻断FLSs致病作用的能力。
Fibroblast-like synoviocytes(FLSs) have emerged as a pivotal effector cell in the inflamed joint,based on its ability to degrade the extracellular matrix and to provide chemotactic and activation signals to resident parenchymal cells and infiltrating immunocytes,and have the ability of non T cell-mediated damage to cartilage and bone. In vitro studies have demonstrated that cultured FLSs display unique biological characteristics that set them apart from fibroblasts isolated from non-synovial anatomic sites.These cells,most importantly,release an impressive array of cytokines and growth factors,which have the capacity to stimulate and,in some cases,dampen the inflammatory response.Moreover,the role of FLSs is increasingly concerned about its effect in pathogenesis of inflammatory arthritis.
The aim of this study was to investigate the morphological characteristics of cultured FLSs in vitro from the patients with rheumatoid arthritis(RA),osteoarthritis(OA) or ankylosing spondylitis(AS),and to evaluate the cell proliferative curve and migration rate in resting or lipopolysaccharide(LPS) stimulating conditions;Further more cellular immune phenotype expression and proportion on FLSs were analyzed by flow cytometry; Finally,mRNA expression of inflammatory cytokines was determined by real-time polymerase chain reaction(PCR),and the levels of secreted inflammatory cytokines in culture supernatant were detected by Cytometric Bead Array(CBA),in comparion with the treatment by dexamethasone(Dex) or cyclosporin A(CsA).The results might provide us a clue to understand the mechanism between the pathogenesis of arthritis and FLSs.The thesis was divided into the following three parts:
PART ONE The morphological characteristics of FLSs from different type of arthritis and the comparison with the proliferative curve and migration rate in resting or stimulated condition
Objective:To investigate the morphological characteristics of FLSs primary and subcultured in vitro from the patients with different type of arthritis and compare with the proliferation of cell growth and the cell migration capacity both in their resting and stimulated conditions.
Methods:FLSs were separated from tissues in joint replacement surgery or extracted synovial fluid FLSs from 13 osteoarthritis,6 rheumatoid arthritis and 2 ankylosing spondylitis,then FLSs were cultured and subcultured.The morphological characterization of the cells were observed under the phase contrast microscopy;The proliferation to LPS was measured by WST-1 assay;The cell migration capacity was evaluated in resting and LPS stimulated by transwell chambers.
Results:OA,RA and AS FLSs were no significantly different in morphology:The adherent cells in primary and early passage were shown for the spindle,stellate, polygonal and dendritic-shaped morphological diversity;After the passage following the intensive growth,the FLS cells gradually formed a single purification,and uniform size for the spindle,with kinds of parallel or whirlpool-like arrangement;The cells in synovial fluid were major round or oval-shaped adherent cells.RA and OA FLSs entered the logarithmic phase on the fourth day after passage,then they reached the growth plateau phase on the seventh day.The LPS stimulated group obviously proliferated on the second day,and researched the maximum on the third day;But on the fifth day maintaining the LPS concentration,the cell count dropped lower than the level of the unstimulated group.The OD values were significantly different between cultured RA and OA FLSs on the third day(1.48±0.07 vs 0.808±0.12,P＜0.001).AS FLSs from synovial fluid grew slowly and showed non obvious response to LPS stimulation.RA and OA FLSs cell migration in stimulated were significantly different with the control group(55.58±4.09 vs 30.25±1.79;36.10±3.50 vs 22.00±2.22,P value＜0.01);The number of RA migrated FLS cells in stimulated was also increased significantly compared with OA(25.33±3.86 vs 14.11±3.26,P＜0.005).
Conclusion:There are no obvious morphological differences among the cultured FLSs in vitro from different type of inflammatory artirtis by the light microscope;FLSs in vitro have more highly capacity in proliferation and migration in RA comepared with OA.
PART TWO Immunocytochemical-associated membrane molecule expression on FLSs and intracellular factors TNFαand IFNγdetected by flow cytometry
Objective:To screen immunocytochemical characterization of membrane molecules on FLSs and detect intracellular factors TNFα,IFNγor other cytokines by flow cytometry;To determined the changes of phenotype and frequency of the cell subsets when stimulated by LPS.
Methods:The cases chosen was the same as the first part;The membrane dye reagents for antibody were CD3-FITC,CD4-PE,CD8-APC,CD45-Pacific Orange, CD45RO-APC,CD45RA-PE,TCRαβ-PE,TCRγδ-PE;CD19-PE-Cy5,CD14-APC, CD68-FITC,CD11c-PE,HLA-DR-APC;CD2-PE,CD7-FITC,CD16-PE,CD56-PE, CD57-FITC,CD116-Pacific Blue;CD 13-PE,CD33-PE,CD34 -PE-Cy7, CD38-Percp-Cy5.5,CD44-Pacific Blue,CD69-PE-Cy5,CD90-APC and CD 117-PE;The intracellular cytokine staining for antibody were TNF-α-PacificBlue,IFNγ-PercpCy5.5, IL-4-FITC and IL- 17-PE.
Results:FLSs were highly expressing CD44,CD90,CD13(98.05±2.18%,96.7±4.8%,88.83±8.2%,respectively),and CD56,CD34,HLA-DR expression were in low levels,but formed there independent subsets(8.36±2.76%;18.75±13.74%;3.28±2.34%,respectively) in the screening of 27 kinds of phenotype,and other 21 antibodies' positive ratio was＜0.5%.The frequency of CD34,CD56 and HLA-DR of FLSs subsets in RA(n = 6) were significant differences in comparison with OA(n = 13),in which the positive proportion for CD56 and HLA-DR of RA FLSs were higher than OA (respectively 33.65±6.12vs16.80±4.36;7.13±1.85 vs 3.75±1.24,P＜0.0001),and the frequency for CD34 of RA was lower than OA(4.34±1.63vs8.89±2.60,P＜0.01).After stimulated by LPS two hours,RA FLSs(n = 6) were more highly expressing CD56 and HLA-DR frequency significantly than unstimulated(47.61±8.35vs33.65±6.12,P＜0.05; 26.68±9.47vs7.13±1.85,P＜0.01,respectively),while the proportion of the CD34 positive FLSs subset was decreased significantly than unstimulated(1.93±0.92vs4.34±1.63,P＜0.01).A little TNFαand IFNγexpression was shown in two cases of OA FLSs and one case of RA FLSs when stimulated by LPS,and IL-4 and IL-17 were negative expression in each test.
Conclusion:CD90,CD44 and CD13 are the main molecular markers on FLSs; CD56~+/CD34~+/HLA-DR~+ FLSs subsets may be associated with the controls of synovitis; A little TNFαand IFNγcan be produced by FLSs after stimulated.
PART THREE The expression of IL-6,IL-1βand IL-10 mRNA produced by FLSs and the level of inflammatory cytokines secreted in culture supernatant in different experimental condition
Objective:To determined the expression of IL-6,IL- 1βand IL- 10 mRNA produced by FLSs and evaluate the changes of their expression when treated with dexamethasone or cyclosporin A at 2h after LPS stimulating in transcriptional levels;To detect IL-6, IL-8,IL-1β,IL-10,IL-12p70,TNFαand other major inflammatory factor expression in cell culture supernatant in protein levels when treated with Dex or CsA at 24h after LPS stimulating.
Methods:The total RNA were extracted from FLSs and synthesized first strand cDNA,then madeβ-actin as a housekeeping gene to analysize IL-6,IL-1βand IL-10mRNA relative concentration by real-time PCR with or without the effect on Dex and CsA before and after LPS stimulating;Finally,the levels of secreted IL-6,IL-8, IL-1βand other inflammatory cytokines in culture supernatant were detected by CBA assay with or without Dex and CsA.
Results:IL-6,IL-10 and IL-1βmRNA expression were significantly increased both in OA and RA FLSs after stimulated,in which IL-1βmRNA was the highest,and IL-6 mRNA the next;After addition of Dex,these cytokine mRNA expression was in significant low levels.However,addition of CsA had no obvious impact on these cytokines;RA and OA FLSs in resting can secret a low level of IL-6(1.12±1.05;3.53±3.99 ng/ml,respectively) and IL-8(0.15±0.08;0.15±0.07 ng/ml,respectively).When stimulated by LPS,IL-6 levels(42.78±12.49;10.18±3.32 ng/ml,respectively)and IL-8 levels(23.13±7.25;19.07±9.34 ng/ml,respectively) in RA and OA are increased,and RA FLSs were more obviously increased(P＜0.001).Dex could inhibit the secretion of IL-6 significantly,and the inhibition to IL-8 was relatively weak,and CsA were only played part of the inhibitory effect.And the levels of other cytokines such as IL-1β, IL-10,IL-12p70 and TNFαwere measured trace or not.
Conclusion:IL-6 and IL-8 are the major inflammatory cytokines secreted by FLSs; IL-1βand TNFαmainly secreted by macrophages and T cells are rarely expressed on FLSs;Dex play stronger role than CsA on preventing the pathogenesis of inflammatory joints by FLSs.
本研究旨在观察类风湿关节炎、骨关节炎以及强直性脊柱炎患者体外培养的FLSs形态学特征及其在静息和刺激条件下的生长增殖曲线及细胞迁移比率;通过流式细胞术检测细胞免疫表型在FLSs的表达和比例;通过实时定量PCR检测炎性细胞因子mRNA的表达情况以及通过CBA法分析细胞培养上清中分泌的炎症因子的表达水平,并比较药物(地塞米松、环孢素A)对其的影响,从而探讨FLSs与关节炎相关的发病机制和治疗机理。具体分为以下三个部分:
第一部分不同类型关节炎FLSs的形态学特征及其在静息和刺激条件下的生长增殖曲线及细胞迁移能力比较
目的:了解体外原代及传代培养的源自不同类型关节炎患者的FLSs的形态学特征,比较其在静息和刺激条件下的细胞生长增殖特性及细胞的迁移能力。
方法:选择骨关节炎(OA)13例,类风湿关节炎(RA)6例,强直性脊柱炎(AS)2例,关节置换术取滑膜组织或抽取膝关节滑液分离FLSs滑膜细胞并培养、传代。在相差显微镜下观察细胞形态,WST-1法观察细胞生长曲线以及对脂多糖(LPS)刺激的增殖反应,Transwell小室观察细胞在静息及LPS刺激下的迁移能力变化。
结果:OA、RA以及AS患者FLSs细胞在形态上无明显差异:原代和早期传代的贴壁细胞均表现为梭形、星形、多角形、树突形等形态上的多样性,传代后并密集生长的FLSs细胞形态逐渐单一纯化,多为梭形且大小均一、呈平行样或漩涡样排列,滑液来源的细胞中圆形或椭圆型的贴壁细胞占多数。RA和OA FLSs在传代后第4天进入对数生长期,在第7天达到生长平台期。LPS刺激组于给药后第2天明显增殖,第3天增殖达到最高,但在维持LPS浓度的第5天,细胞数降至比未刺激组更低的水平。培养第3天的RA FLSs与OA FLSs的OD值进行比较有显著性差异(1.48±0.07 vs 0.808±0.12,P＜0.001)。滑液来源的AS FLSs细胞生长缓慢,对LPS刺激反应不明显。RA FLSs与OA FLSs在LPS刺激下迁移的细胞数比对照组有显著差异(55.58±4.09 vs 30.25±1.79,36.10±3.50 vs 22.00±2.22,P值均＜0.01),RA FLSs在刺激后增加的迁移细胞数与OA FLSs比较也有显著的差异(25.33±3.86 vs 14.11±3.26,P＜0.005)。
结论:不同类型炎性关节病患者体外培养的FLSs在光镜下无明显形态学上的差异;体外培养的RAFLSs具有比较OAFLSs更强的增殖和迁移能力。
第二部分免疫细胞化学相关膜分子在FLSs细胞的表达及变化以及胞内因子TNFα、IFNγ的流式检测
目的:通过流式细胞术筛查FLSs膜分子的免疫细胞化学特征及TNF×、IFNγ等细胞因子在FLSs细胞的表达情况,检测在LPS刺激下FLSs细胞亚群的表型和比例变化。
方法:病例选择同第一部分。选择的膜分子抗体染料试剂为:CD3-FITC,CD4-PE,CD8-APC,CD45-Pacific Orange,CD45RO-APC,CD45RA-PE,TCRαβ-PE,TCRγδ-PE;CD19-PE-Cy5,CD14-APC,CD68-FITC,CD11c-PE,HLA-DR-APC;CD2-PE,CD7-FITC,CD16-PE,CD56-PE,CD57-FITC,CD116-Pacific Blue;CD13-PE,CD33-PE,CD34-PE-Cy7,CD38-Percp-Cy5.5,CD44-Pacific Blue,CD69-PE-Cy5,CD90-APC,CD117-PE。胞内染色的细胞因子为TNF-α-PacificBlue、IFNγ-PercpCy5.5、IL-4-FITC、IL-17-PE。
结果:在筛查的27种表型中,CD13、CD90、CD44呈强阳性表达(分别为98.05±2.18%,96.7±4.8%,88.83±8.2%)。CD34、CD56、HLA-DR呈弱阳性表达,且为独立的亚群(分别为8.36±2.76%,18.75±13.74%,3.28±2.34%),其它21种抗体阳性比例均＜0.5%。RA-FLSs(n=6)中CD34~+、CD56~+、HLA-DR~+细胞亚群的比例与OA FLSs(n=13)比较均有显著差异,其中RA FLSs CD56~+、HLA-DR~+比例高于OA FLSs(分别为33.65±6.12vs16.80±4.36,7.13±1.85 vs 3.75±1.24,P＜0.0001),而CD34~+比例低于OA FLSs(4.34±1.63vs8.89±2.60,P＜0.01)。在LPS刺激2h后,RA FLSs(n=6)CD56~+FLSs、HLA-DR~+FLSs表达比例较未刺激时明显增高(分别为47.61±8.35vs33.65±6.12,P＜0.05;26.68±9.47vs7.13±1.85P＜0.01),而CD34~+FLSs比例则较未刺激时明显下降(1.93±0.92vs4.34±1.63,P＜0.01)。2例OA FLSs与1例RA FLSs在LPS刺激后产生少量的TNF×、IFNγ,IL-4与IL-17在各次检测中均为阴性。
结论:CD90、CD44、CD13是FLSs的主要分子标记,CD56~+FLSs、CD34~+FLSs、HLA-DR~+FLSs亚群可能与滑膜炎症的调控相关,FLSs细胞可在刺激后产生少量TNFα和IFNγ。
第三部分FLSs细胞IL-6、IL-1β、IL-10 mRNA的表达、培养上清中分泌的炎性细胞因子水平以及不同药物对其的影响
目的:在转录水平观察FLSs细胞IL-6、IL-1β、IL-10mRNA的表达,并观察地塞米松(Dex)及环孢素A(CsA)在LPS刺激2h后对其表达的影响;在蛋白水平检测细胞培养上清中IL-6、IL-8、IL-1B、IL-10、IL-12p70以及TNFα等主要炎性因子的表达以及Dex、CsA在LPS刺激24h后对其分泌的细胞因子的影响。
方法:提取FLSs总RNA,合成cDNA第一链,以β-actin为看家基因,实时荧光定量PCR分析FLSs细胞中IL-6、IL-1β、IL-10mRNA的相对含量,比较LPS刺激前后以及分别加入药物Dex、CsA后的影响;CBA法检测FLSs细胞培养上清中分泌的IL-6、IL-8、IL-1β、IL-10、IL-12p70以及TNFa的水平,并比较LPS刺激前后以及分别加入药物Dex、CsA后的影响。
结果:OA和RA FLSs细胞IL-6、IL-1β、IL-10 mRNA在LPS刺激后的表达明显增加,其中IL-1βmRNA表达最高,IL-6 mRNA次之,加入Dex后,各细胞因子mRNA水平均明显降低,而加入CsA后降低的程度不如Dex明显。RA和OA FLSs在静息状态下可分泌低水平的IL-6(分别为1.12±1.05,3.53±3.99 ng/ml)及IL-8(分别为0.15±0.08,0.15±0.07 ng/ml)。在LPS刺激下,IL-6水平增高(分别为42.78±12.49,10.18±3.32 ng/ml),IL-8水平也增高(分别为23.13±7.25,19.07±9.34 ng/ml),其中以RA FLSs增高程度更为明显(P＜0.001)。Dex能够明显抑制IL-6的分泌,对IL-8的抑制作用相对较弱,而CsA均仅起到部分的抑制作用。而其它IL-1β、IL-10、IL-12p70及TNFα的含量均为微量或测不出。
结论:IL-6与IL-8是FLSs分泌的主要炎症相关细胞因子,IL-1β、TNFα等主要由巨噬细胞、T细胞表达的细胞因子在FLSs很少表达,Dex具有比CsA更强的阻断FLSs致病作用的能力。
Fibroblast-like synoviocytes(FLSs) have emerged as a pivotal effector cell in the inflamed joint,based on its ability to degrade the extracellular matrix and to provide chemotactic and activation signals to resident parenchymal cells and infiltrating immunocytes,and have the ability of non T cell-mediated damage to cartilage and bone. In vitro studies have demonstrated that cultured FLSs display unique biological characteristics that set them apart from fibroblasts isolated from non-synovial anatomic sites.These cells,most importantly,release an impressive array of cytokines and growth factors,which have the capacity to stimulate and,in some cases,dampen the inflammatory response.Moreover,the role of FLSs is increasingly concerned about its effect in pathogenesis of inflammatory arthritis.
The aim of this study was to investigate the morphological characteristics of cultured FLSs in vitro from the patients with rheumatoid arthritis(RA),osteoarthritis(OA) or ankylosing spondylitis(AS),and to evaluate the cell proliferative curve and migration rate in resting or lipopolysaccharide(LPS) stimulating conditions;Further more cellular immune phenotype expression and proportion on FLSs were analyzed by flow cytometry; Finally,mRNA expression of inflammatory cytokines was determined by real-time polymerase chain reaction(PCR),and the levels of secreted inflammatory cytokines in culture supernatant were detected by Cytometric Bead Array(CBA),in comparion with the treatment by dexamethasone(Dex) or cyclosporin A(CsA).The results might provide us a clue to understand the mechanism between the pathogenesis of arthritis and FLSs.The thesis was divided into the following three parts:
PART ONE The morphological characteristics of FLSs from different type of arthritis and the comparison with the proliferative curve and migration rate in resting or stimulated condition
Objective:To investigate the morphological characteristics of FLSs primary and subcultured in vitro from the patients with different type of arthritis and compare with the proliferation of cell growth and the cell migration capacity both in their resting and stimulated conditions.
Methods:FLSs were separated from tissues in joint replacement surgery or extracted synovial fluid FLSs from 13 osteoarthritis,6 rheumatoid arthritis and 2 ankylosing spondylitis,then FLSs were cultured and subcultured.The morphological characterization of the cells were observed under the phase contrast microscopy;The proliferation to LPS was measured by WST-1 assay;The cell migration capacity was evaluated in resting and LPS stimulated by transwell chambers.
Results:OA,RA and AS FLSs were no significantly different in morphology:The adherent cells in primary and early passage were shown for the spindle,stellate, polygonal and dendritic-shaped morphological diversity;After the passage following the intensive growth,the FLS cells gradually formed a single purification,and uniform size for the spindle,with kinds of parallel or whirlpool-like arrangement;The cells in synovial fluid were major round or oval-shaped adherent cells.RA and OA FLSs entered the logarithmic phase on the fourth day after passage,then they reached the growth plateau phase on the seventh day.The LPS stimulated group obviously proliferated on the second day,and researched the maximum on the third day;But on the fifth day maintaining the LPS concentration,the cell count dropped lower than the level of the unstimulated group.The OD values were significantly different between cultured RA and OA FLSs on the third day(1.48±0.07 vs 0.808±0.12,P＜0.001).AS FLSs from synovial fluid grew slowly and showed non obvious response to LPS stimulation.RA and OA FLSs cell migration in stimulated were significantly different with the control group(55.58±4.09 vs 30.25±1.79;36.10±3.50 vs 22.00±2.22,P value＜0.01);The number of RA migrated FLS cells in stimulated was also increased significantly compared with OA(25.33±3.86 vs 14.11±3.26,P＜0.005).
Conclusion:There are no obvious morphological differences among the cultured FLSs in vitro from different type of inflammatory artirtis by the light microscope;FLSs in vitro have more highly capacity in proliferation and migration in RA comepared with OA.
PART TWO Immunocytochemical-associated membrane molecule expression on FLSs and intracellular factors TNFαand IFNγdetected by flow cytometry
Objective:To screen immunocytochemical characterization of membrane molecules on FLSs and detect intracellular factors TNFα,IFNγor other cytokines by flow cytometry;To determined the changes of phenotype and frequency of the cell subsets when stimulated by LPS.
Methods:The cases chosen was the same as the first part;The membrane dye reagents for antibody were CD3-FITC,CD4-PE,CD8-APC,CD45-Pacific Orange, CD45RO-APC,CD45RA-PE,TCRαβ-PE,TCRγδ-PE;CD19-PE-Cy5,CD14-APC, CD68-FITC,CD11c-PE,HLA-DR-APC;CD2-PE,CD7-FITC,CD16-PE,CD56-PE, CD57-FITC,CD116-Pacific Blue;CD 13-PE,CD33-PE,CD34 -PE-Cy7, CD38-Percp-Cy5.5,CD44-Pacific Blue,CD69-PE-Cy5,CD90-APC and CD 117-PE;The intracellular cytokine staining for antibody were TNF-α-PacificBlue,IFNγ-PercpCy5.5, IL-4-FITC and IL- 17-PE.
Results:FLSs were highly expressing CD44,CD90,CD13(98.05±2.18%,96.7±4.8%,88.83±8.2%,respectively),and CD56,CD34,HLA-DR expression were in low levels,but formed there independent subsets(8.36±2.76%;18.75±13.74%;3.28±2.34%,respectively) in the screening of 27 kinds of phenotype,and other 21 antibodies' positive ratio was＜0.5%.The frequency of CD34,CD56 and HLA-DR of FLSs subsets in RA(n = 6) were significant differences in comparison with OA(n = 13),in which the positive proportion for CD56 and HLA-DR of RA FLSs were higher than OA (respectively 33.65±6.12vs16.80±4.36;7.13±1.85 vs 3.75±1.24,P＜0.0001),and the frequency for CD34 of RA was lower than OA(4.34±1.63vs8.89±2.60,P＜0.01).After stimulated by LPS two hours,RA FLSs(n = 6) were more highly expressing CD56 and HLA-DR frequency significantly than unstimulated(47.61±8.35vs33.65±6.12,P＜0.05; 26.68±9.47vs7.13±1.85,P＜0.01,respectively),while the proportion of the CD34 positive FLSs subset was decreased significantly than unstimulated(1.93±0.92vs4.34±1.63,P＜0.01).A little TNFαand IFNγexpression was shown in two cases of OA FLSs and one case of RA FLSs when stimulated by LPS,and IL-4 and IL-17 were negative expression in each test.
Conclusion:CD90,CD44 and CD13 are the main molecular markers on FLSs; CD56~+/CD34~+/HLA-DR~+ FLSs subsets may be associated with the controls of synovitis; A little TNFαand IFNγcan be produced by FLSs after stimulated.
PART THREE The expression of IL-6,IL-1βand IL-10 mRNA produced by FLSs and the level of inflammatory cytokines secreted in culture supernatant in different experimental condition
Objective:To determined the expression of IL-6,IL- 1βand IL- 10 mRNA produced by FLSs and evaluate the changes of their expression when treated with dexamethasone or cyclosporin A at 2h after LPS stimulating in transcriptional levels;To detect IL-6, IL-8,IL-1β,IL-10,IL-12p70,TNFαand other major inflammatory factor expression in cell culture supernatant in protein levels when treated with Dex or CsA at 24h after LPS stimulating.
Methods:The total RNA were extracted from FLSs and synthesized first strand cDNA,then madeβ-actin as a housekeeping gene to analysize IL-6,IL-1βand IL-10mRNA relative concentration by real-time PCR with or without the effect on Dex and CsA before and after LPS stimulating;Finally,the levels of secreted IL-6,IL-8, IL-1βand other inflammatory cytokines in culture supernatant were detected by CBA assay with or without Dex and CsA.
Results:IL-6,IL-10 and IL-1βmRNA expression were significantly increased both in OA and RA FLSs after stimulated,in which IL-1βmRNA was the highest,and IL-6 mRNA the next;After addition of Dex,these cytokine mRNA expression was in significant low levels.However,addition of CsA had no obvious impact on these cytokines;RA and OA FLSs in resting can secret a low level of IL-6(1.12±1.05;3.53±3.99 ng/ml,respectively) and IL-8(0.15±0.08;0.15±0.07 ng/ml,respectively).When stimulated by LPS,IL-6 levels(42.78±12.49;10.18±3.32 ng/ml,respectively)and IL-8 levels(23.13±7.25;19.07±9.34 ng/ml,respectively) in RA and OA are increased,and RA FLSs were more obviously increased(P＜0.001).Dex could inhibit the secretion of IL-6 significantly,and the inhibition to IL-8 was relatively weak,and CsA were only played part of the inhibitory effect.And the levels of other cytokines such as IL-1β, IL-10,IL-12p70 and TNFαwere measured trace or not.
Conclusion:IL-6 and IL-8 are the major inflammatory cytokines secreted by FLSs; IL-1βand TNFαmainly secreted by macrophages and T cells are rarely expressed on FLSs;Dex play stronger role than CsA on preventing the pathogenesis of inflammatory joints by FLSs.
引文
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