蛋白酪氨酸激酶在IL-1β和TNF-α诱导类风湿关节炎成纤维样滑膜细胞MAPKs活化中的作用

蛋白酪氨酸激酶在IL-1β和TNF-α诱导类风湿关节炎成纤维样滑膜细胞MAPKs活化中的作用
2011-02-27 孙铁铮　吕厚山　药立波

【摘　要】　目的　比较类风湿关节炎(rheumatoid arthritis，RA)与骨关节炎(osteoarthritis，OA)成纤维样滑膜细胞(fibroblast-like synoviocytes，FLS)蛋白酪氨酸磷酸化状态的差异；探讨RA FLS中，蛋白酪氨酸激酶(protein tyrosine kinase，PTK)在IL-1β和TNF-α作用下有丝分裂原活化蛋白激酶(mitogen activated protein kinase,MAPKs)活化中的作用。方法　滑膜细胞原代培养，流式细胞仪鉴定滑膜细胞型别，提取蛋白进行SDS-PAGE单相电泳和ICE-PAGE双相电泳分离后，用Western blots 检测FLS蛋白磷酸化的状态。结果　滑膜细胞培养至第三代时，CD3、CD14、CD20、CD11b、von Willebrand factor阳性细胞比例小于1%；RA FLS蛋白酪氨酸磷酸化程度是OA FLS的4～6倍；IL-1β和TNF-α可以瞬时引起RA FLS蛋白酪氨酸磷酸化程度增加，并在短时间内激活MAPKs(ERK2，JNK2和p38为主)：genistein对ERK2活化抑制作用显著，而对于JNK2、p38，活化的抑制则较弱。结论　原代培养的滑膜细胞当传代至第三代时即可以获得型别均一的成纤维样滑膜细胞；RA FLS蛋白酪氨酸磷酸化状态较OA FLS明显增高；IL-1β和TNF-α可以瞬时引起RA FLS蛋白酪氨酸磷酸化程度增加，激活MAPKs通路(EPK2、JNK2、p38)，两种因子诱导MAPKs活化过程中PTK依赖途径和PTK非依赖途径并存。
【关键词】　关节炎；　滑膜；　成纤维细胞；　蛋白质酪氨酸激酶；　有丝分裂原活化蛋白激酶；　白细胞介素1；　肿瘤坏死因子

Role of protein tyrosine kinase in activation of MAPKs induced by IL-1β and TNF-α in fibroblast-like synoviocytes of rheumatoid arthritis

SUN Tiezheng,L Houshan,YAO Libo
（Arthritis Clinical and Research Center,People's Hospital,Beijing Medical University,Beijing 100044,China）

【Abstract】　Objective　To evaluate the change of tyrosine phosphorylation status in rheumatoid arthritis fibroblast-like synoviocytes (RA FLS),compared with osteoarthritis (OA) FLS;and to explore the role of protein tyrosine kinase (PTK) in signaling of IL-1β and TNF-α,especially in activation of MAPKs in rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) under the stimulation of IL-1β and TNF-α.Methods　RA and OA FLS were primarily cultured; FLS type was defined by flow cytometry.After proteins were isolated from RA FLS and OA FLS,SDS-PAGE single-dimensional electrophoresis and ICE-PAGE two-dimensional electrophoresis were used to fractionate them,followed by Western blots to determine tyrosine phosphorylation status using specific anti-protein tyrosine phosphorylated antibodies.Results　In passage 3,the positive cells′ proportion of CD3,CD14,CD11b,CD20,and von Willebrand

factor less than 1% respectively.The extent of protein
tyrosine phosphorylation in RA FLS is 4～6 times higher than that of OA FLS.IL-1β and TNF-α transiently increased protein tyrosine phosphorylation,and activated MAPKs cascades (mainly ERK2,JNK2,and p38) in RA FLS.The activation of ERK2 induced by the two cytokines was inhibited obviously by genistein,but the inhibitory role on that of JNK2 and p38 was relatively weak.Conclusion　Synoviocytes of passage 3 are homogenous population of fibroblast-like synoviocytes;tyrosine phosphorylation status in RA FLS increases much higher than that of OA FLS.During signal transductionf IL-1β and TNF-α in RA FLS,tyrosine phosphorylation is increased transiently;MAPKs cascades are activated in a few minutes (mainly ERK2,JNK2 and p38),PTK has a role in activation of ERK,but has weak effect on that of JNK and p38.
【Key words】　Arthritis;　Synovial membrane;　Fibroblasts;　Protein tyrosine kinase;　Mitogen activated protein kinase;　Interleukin-1;　Tumor necrosis factor

类风湿关节炎(rheumatoid arthritis，RA)是一种慢性系统性疾病，它的病情进展中综合体现了炎症、自身免疫、滑膜组织增生三种病理生理过程。异常增生的滑膜组织类似于局限性侵袭生长的肿瘤，对关节软骨、骨造成进行性破坏，最终导致关节畸形和不同程度的功能障碍。最近研究发现，RA成纤维样滑膜细胞(fibroblast-like synoviocyte，FLS)具有转化细胞特征，而在骨关节炎中未发现此转化现象的存在。这种转化特性可能是RA疾病发生的早期特征［1］。蛋白酪氨酸激酶在细胞转化和生物信号转导中发挥关键作用［2］。本研究通过比较RA和OA两种不同发病机制的关节病变中成纤维样滑膜细胞酪氨酸磷酸化状态的差异，探讨RA成纤维样滑膜细胞中酪氨酸激酶活性的改变；应用IL-1β、TNF-α作用于RA FLS， 观察两种细胞因子对有丝分裂原活化蛋白激酶(mitogen activated protein kinase,MAPKs)的激活情况，并应用genistein(特异性酪氨酸激酶抑制剂)，探讨酪氨酸激酶在上述因子诱导RA FLS MAPKs亚家族成员活化的信号转导过程中发挥的作用。

1　资料与方法

1.1　滑膜细胞的培养：滑膜均取材于北京医科大学人民医院骨关节病诊疗研究中心行膝关节置换术或滑膜切除术的病人，其中RA 20例，OA 20例，所有病人均符合国际诊断标准［3］。术中取出滑膜组织，无菌条件下剪碎，1.0 mg/ml Ⅰ型胶原酶(GIBCO-BRL) 37 ℃消化4 h，离心收集细胞，加入20% FBS-DMEM (GIBCO-BRL)培养液中，置于37 ℃、5% CO2孵箱中令其贴壁生长，24 h后PBS冲洗除去悬浮细胞，待细胞贴壁生长至85%汇满时，胰蛋白酶-EDTA消化进行1∶3传代。本实验采用均为3～5代滑膜细胞。
1.2　滑膜细胞类型鉴定：分别

取1～3代滑膜细胞，用0.125 mmol EDTA缓慢消化下来，PBS洗涤，台盼蓝染色计算活细胞数
目，分别加入FITC标记的小鼠抗人CD3、CD14、CD20、von Willebrand factor (Serotech UK)和PE标记的小鼠抗人CD11b；4 ℃作用45 min；以含有1%马血清的PBS溶液充分混匀作用1 h，80%酒精固定。实验均按三个平行管设计，重复6次。每次均以FITC和PE标记的鼠源性抗体作为阴性对照。样品集中进行流式细胞仪测定相对荧光强度及各种表面标记阳性细胞比例。