沙利度胺对实验性关节炎大鼠滑膜细胞凋亡的影响

目的 研究不同浓度沙利度胺对体外培养成纤维细胞样滑膜细胞(FLS)凋亡的影响.方法 体外培养及纯化FLS,分为6组:对照组,脂多糖(LPS)组,LPS+高剂量沙利度胺(50 mg/L)组,LPS+中剂量沙利度胺(5 mg/L)组,LPS+低剂量沙利度胺(0.5 mg/L)组,LPS+甲氨蝶呤(MTX)组.48小时后显微镜及电镜观察不同处理组FLS形态,膜黏连蛋白-5/碘化丙啶(Annexin- V/PI)联合标记法测定FLS凋亡率及坏死率.结果 ①原代滑膜细胞培养3代后以梭形且呈极向排列FLS为主;②沙利度胺及MTX处理后FLS渐呈不规则形,细胞变圆缩小,透射电镜下可见典型细胞凋亡.③中、高剂量沙利度胺及MTX组凋亡率和坏死率均高于对照组、LPS组及低剂量沙利度胺组,凋亡率分别为(5.72±1.42)％、(8.07±1.45)％、(11.28±2.48)％vs (0.30±0.09)％、(0.24±0.05)％、(0.40±0.04)％(P＜0.05),坏死率分别为(12.96±4.32)％、(20.34±4.43)％、(18.11±1.17)％ vs (0.22±0.08)％、(0.13±o.09)％、(1.94±1.16)％(P＜o.05);低、中、高剂量沙利度胺及MTX组凋亡率均依次升高(P＜0.05);低、中、高剂量沙利度胺组及MTX组坏死率均依次升高(P＜0.05);高剂量沙利度胺组与MTX组坏死率之间比较差异无统计学意义(P＞0.05).结论 沙利度胺能够诱导FLS凋亡,在一定浓度范围内该作用呈剂量依赖性.沙利度胺对FLS早期凋亡作用弱于MTX,对晚期凋亡作用与MTX相同.     

类风湿关节炎滑膜成纤维样细胞的蛋白质组学研究

目的研究类风湿关节炎(RA)和骨性关节炎(OA)关节滑膜成纤维样细胞(FLS)蛋白质组的表达差异,分析差异蛋白在RA关节滑膜增生中的作用。方法原代培养关节滑膜FLS,抽提细胞总蛋白并定量,通过高分辨双向电泳(2-DE)对RA和OA的滑膜FLS进行蛋白分离,找出其中表达差异的蛋白质点并进行质谱(MS)分析鉴定。结果 OA及RA滑膜FLS的2-DE图谱上分别显示有1324和1147个蛋白质点。其中47个蛋白质点在OA或RA滑膜FLS中有3倍以上的量变;12个蛋白质点只存在于OA滑膜FLS;15个蛋白质点只存在于RA滑膜FLS。选择54个蛋白质点进行MS,共成功鉴定34个蛋白质点。其中包括在OA中表达上升的人异天冬氨酸甲基转移酶(PIMT)和PIR(PIR),仅在RA中表达的硫氧还蛋白1(TRX-1)这三个与关节滑膜增生相关的蛋白质。结论 PIMT和PIR在RA中表达降低及TRX-1在RA中表达升高可能是RA关节滑膜增生,引起关节软骨破坏的原因之一。     

Smoothened表达对类风湿关节炎成纤维样滑膜细胞RhoA/ROCK通路的影响

目的初步探讨Sonic Hedgehog(Shh)信号通路分子Smoothened(Smo)对类风湿关节炎(RA)成纤维滑膜细胞(FLS)RhoA/ROCK通路相关分子的影响。方法收集病情活动(DAS28≥3.2)RA患者关节镜手术或关节置换术切除的滑膜组织,组织块培养法培养RA-FLS作为细胞模型,流式细胞术检测CD55阳性率鉴定细胞,然后分别予Smo分子激动剂Purmorphamine或抑制剂KAAD-Cyclopamine处理,应用GST-pull down法检测RhoA活性,Western blot检测ROCK活性与Smo蛋白表达。结果与对照组相比,RA-FLS经Purmorphamine刺激后,活性RhoA蛋白、磷酸化MYPT1蛋白及Smo蛋白均上调(P<0.05);经KAAD-Cyclopamine处理后,活性RhoA蛋白、磷酸化MYPT1蛋白及Smo蛋白均下调(P<0.05)。结论 RA-FLS Smo表达可影响RhoA/ROCK信号的传导。Smo可能参与了RA-FLS中Shh信号通路非经典途径的调控。     

TNF-α和CRT双信号对RA患者滑膜组织NLRP3炎症小体的活化作用

目的研究肿瘤坏死因子-α(TNF-α)和钙网织蛋白(CRT)双信号对类风湿性关节炎(RA)患者成纤维样滑膜细胞(FLS)中核苷酸结合寡聚化结构域受体3(NLRP3)炎症小体活化的影响。方法采用间接免疫荧光染色法检测12例RA和10例骨性关节炎(OA)患者滑膜组织NLRP3、衔接蛋白凋亡相关斑点样蛋白(ASC)的表达,并与滑膜衬里层和衬里下层FLS标志物进行共定位研究。采用胶原酶消化法分离RA患者滑膜组织中FLS并进行体外培养。分别用不同浓度的TNF-α或脂多糖(LPS)(刺激剂)处理细胞,采用免疫印迹法和实时荧光定量聚合酶链反应(qRT-PCR)检测细胞NLRP3、白细胞介素1β前体(pro-IL-1β)和白细胞介素18前体(pro-IL-18)的蛋白和mRNA表达。加入尼日利亚菌素(Nigericin)或CRT后采用免疫印迹法检测FLS中半胱氨酸天冬氨酸特异性蛋白酶1(Caspase-1)活化片段p20的表达;收集细胞培养上清液,采用酶联免疫吸附试验(ELISA)检测分泌型白细胞介素(IL)-1β和IL-18水平。结果 RA患者滑膜组织中NLRP3炎症小体的主要成分NLRP3和ASC的平均荧光强度高于OA患者(P<0.01)。NLRP3、ASC和IL-1β裂解片段(cleaved IL-1β)与RA患者滑膜衬里层FLS表面标志物PDPN和衬里下层FLS表面标志物CD248均有共定位。体外细胞实验结果显示TNF-α可以促进FLS中NLRP3和pro-IL-1β的蛋白表达;与对照组(无刺激剂)相比,二者的mRNA表达显著增加(P<0.05、P<0.001),而LPS对RA患者FLS中NLRP3炎症小体的预活化无影响。TNF-α/Nigericin或TNF-α/CRT双信号能够促进FLS中Caspase-1的活化,导致Caspase-1活化片段p20呈浓度依赖性升高;与对照组相比,TNF-α/CRT组分泌型IL-1β显著升高(P<0.05)。结论 TNF-α/CRT双信号可促进RA患者FLS中NLRP3炎症小体的活化。     

雷公藤内酯醇抑制血管平滑肌细胞增殖的研究

目的:探讨雷公藤内酯醇对血清诱导的大鼠血管平滑肌细胞(VSMC)增生的影响及其作用机制。方法:体外培养大鼠胸主动脉平滑肌细胞,用细胞计数法观察雷公藤内酯醇对细胞增生的抑制作用,采用流式细胞仪进行细胞周期分析,逆转录-聚合酶链反应(RT-PCR)法检测细胞c-fos的mRNA表达水平。结果:雷公藤内酯醇明显抑制血清诱导的大鼠VSMC增生;阻断细胞周期中细胞由G0/G1期向S期转化;RT-PCR检测显示雷公藤内酯醇能明显抑制原癌基因c-fos的表达,其作用呈剂量依赖性。结论:雷公藤内酯醇可抑制血清诱导的大鼠VSMC增生,下调原癌基因c-fos的表达。     

沙利度胺对AnnexinⅡ基因的调节作用

目的 了解沙利度胺在体外对人多发性骨髓瘤(MM)细胞系RPMI8226、人微血管内皮细胞系HMEC-1中Annexin Ⅱ(AnxA2)基因表达的调节作用,探讨沙利度胺诱发血栓的可能机制.方法 实时荧光定量PCR检测在不同浓度下沙利度胺对两种细胞系AnxA2 mRNA表达的影响,采用流式细胞术、激光共聚焦仪检测不同浓度下沙利度胺对细胞膜表面AnxA2蛋白表达的影响.结果 当沙利度胺浓度为12.5、25.0、50.0 μg/ml时,RPMl8226细胞AnxA2 mRNA表达水平为0.60±0.15、0.33±0.14、0.42±0.16,较对照组(1.07±0.16)降低(P<0.05),HMEC-1细胞AnxA2 mRNA表达水平为0.21±0.20、0.08 ±0.08、0.17±0.16,较对照组(1.16 ±0.24)降低(P<0.05).沙利度胺浓度为12.5、25.0、50.0 μg/ml时,RPMI8226细胞膜表面AnxA2蛋白表达水平为3.39±0.32、2.82 ±0.28、3.21 ±0.23,较对照组(5.53 ±0.32)降低(P<0.05),HMEC-1细胞膜表面AnxA2蛋白表达水平为0.72 ±0.11、0.64 ±0.08、0.67±0.08,较对照组(1.40±0.15)降低(P<0.05).结论 沙利度胺能抑制RPMI8226和HMEC-1细胞AnxA2 mRNA和蛋白表达,可能为其诱发骨髓瘤血栓形成的原因之一.     

IPAF和NALP1在RA-FLS中的蛋白表达情况及意义

目的：研究人IL-1β转换酶蛋白激活因子（IPAF）和富含亮氨酸重复结构域蛋白（NALP）1在类风湿关节炎（RA）成纤维样滑膜细胞（FLS）中的表达及其意义。方法：收集来自4例骨关节炎、6例RA患者、3例关节创伤患者的滑膜组织标本,分为骨关节炎组、RA组、正常对照组。分离培养FLS,应用蛋白免疫印迹法检测并比较3组IPAF、NALP1蛋白表达水平,比较3组FLS中IPAF和NALP1蛋白表达。6份RA-FLS分别加入胎牛血清培养液（空白对照组）、脂多糖、脂多糖加ZVAD-FMK、TNF-α、TNF-α加ZVAD—FMK处理24h,比较各样本IPAF和NALP1的蛋白表达水平。结果：IPAF蛋白表达水平由高至低依次为骨关节炎组、RA组、正常对照组,骨关节炎组IPAF蛋白表达水平明显高于RA组及正常对照组（P〈0．05）。NALP1蛋白表达水平由高至低依次为骨关节炎组、RA组、正常对照组,但3组比较差异无统计学意义（P〉0．05）。RA-FLS经不同药物干预后,TNF-α处理组IPAF蛋白表达水平均高于其他4组（P〈0．01或0．05）;各组NALP1蛋白表达水平比较差异无统计学意义（P〉0．05）。结论：RA-FLS中IPAF蛋白表达水平升高,TNF-α可促进RA—FLS中IPAF表达。     

生存素基因在子宫内膜异位症中的表达及促性腺激素释放激素激动剂、环氧合酶-2抑制剂对其表达的影响

目的 探讨生存素(survivin)基因在子宫内膜异位症(FMs)发病机制中的作用.方法 检测生存素在正常与异位子宫内膜组织中的表达;观察促性腺激素释放激素激动剂(GnRHa)、环氧合酶-2(COX-2)抑制剂对体外培养的EMs异位内膜细胞及正常子宫内膜细胞中survivin基因mRNA表达的调节作用及对体外培养的异位内膜凋亡率的影响.结果 ①EMs异位组、在位组survivin mRNA的表达明显强于对照组(均P<0.05),且无周期性变化.②GnRHa可呈浓度依赖性下调体外培养的异位内膜细胞及正常子宫内膜细胞中survivin mRNA的表达,COX-2抑制剂亦呈浓度依赖性下调体外培养的异位内膜细胞及正常子宫内膜细胞中survivln mRNA的表达,GnRHa100μL加COX-2抑制荆40 μmol/L可以促进体外培养的异位内膜细胞凋亡(P<0.05),二者无明显协同作用(P>0.05)结论 ①异位内膜细胞高表达survivin,对凋亡的敏感性低,使异位灶存活并发展;②GnRHa、COX-2抑制剂可通过抑制survivin的表达来促进体外培养的EMs异位内膜细胞的凋亡.     

液态芯片技术分析RA患者血清免疫复合物对滑膜细胞分泌细胞因子的影响

目的 用液态芯片技术研究类风湿关节炎(RA)患者血清免疫复合物(IC)对关节成纤维样滑膜细胞(FLS)分泌细胞因子的影响.方法 用聚乙二醇(PEG)沉淀法分别提取10份抗环瓜氨酸肽抗体(ACCP)阳性的RA患者及健康人对照血清IC,间接ELISA法测定IC中ACCP特异性IC水平.体外培养RA患者关节FLS,分别用RA...     

沙利度胺及氨甲蝶呤抗CIA大鼠滑膜血管新生及其作用机制的研究

目的研究沙利度胺、氨甲蝶呤对大鼠Ⅱ型胶原诱导型关节炎血管新生的影响及相关的机制。方法建立类风湿性关节炎大鼠模型.自造模次日治疗组分别给予沙利度胺、甲氨蝶呤和沙利度胺联合甲氨蝶呤治疗,在第6周取膝关节应用免疫组化检测其滑膜的微血管密度(MVD),取血清进行Western Blot检测大鼠体内血管内皮细胞生长因子(VEGF)、基质金属蛋白酶-1、2、3、9的表达并计算相对含量。结果①免疫组织化学染色显示沙利度胺、氨甲蝶呤和沙利度胺联合氨甲蝶呤治疗模型大鼠可使关节滑膜中新生血管数量明显减少.微血管密度(MVD)明显降低(P<0.05).以沙利度胺联合氨甲蝶呤组作用最强。②沙利度胺、氨甲蝶呤和沙利度胺联合氨甲蝶呤可抑制VEGF、MMP-1、2、3、9表达(P均<0.05)。结论沙利度胺和氨甲蝶呤可能通过抑制VEGF,MMP-1、2、3、9的表达而发挥抗滑膜血管新生的作用,且两者有协同作用。     

敲减Akt表达对类风湿关节炎成纤维样滑膜细胞增殖的影响

目的:研究慢病毒介导Akt靶向的RNA干扰对类风湿关节炎成纤维样滑膜细胞(RAFLS)增殖的影响。方法:慢病毒表达载体pL/Akt转染RAFLS并表达靶向Akt的siRNA序列,采用RT-PCR和Western Blot检测Akt表达,MTT法检测RAFLS增殖能力的变化。结果:转染pL/Akt后,RAFLS中Akt1、Akt2的mRNA分别下降了65%和68%以上,Akt蛋白的表达量在48h后下降76.19%(P<0.05),RAFLS细胞增殖在48、72、96h分别下降了27.50%、44.69%和56.80%(P<0.05)。结论:慢病毒表达载体pL/Akt能够降低Akt蛋白表达水平,并显著抑制RAFLS增殖。     

Inhibition of miR-16 enhances the sensitivity of fibroblast-like synovial cells to methotrexate by restraining MDR1/P-gp expression via NF-κB pathway

MicroRNAs (miRNAs) are demonstrated to contribute to the regulation of drug resistance in a number of diseases. Nevertheless, little is known about the role and the underlying mechanism of miR-16 in rheumatoid arthritis (RA) methotrexate resistance. In this study, we firstly examined the miR-16 expression in the serum and synovial fluid from RA patients who were unresponsive to methotrexate monotherapy (UR-MTX patients) and responsive RA patients (R-MTX patients). Secondly, the miR-16 expression was measured in both fibroblast-like synovial cells (FLS) and methotrexate resistance RA-FLS cells (FLS-MTX). FLS cells used in this study were isolated from synovial tissue specimens obtained from patients with RA who underwent total joint replacement. FLS-MTX cells were conducted by gradually increasing the concentration of methotrexate in the medium. The construction of FLS-MTX cells was confirmed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. Thirdly, in order to further investigate the role of miR-16 in FLS-MTX cells, we introduced miR-16 inhibitor into FLS-MTX cells to knockdown the expression of miR-16, used fluorescence quantitative PCR to detect the inhibition efficiency. The effects of miR-16 inhibition on cell viability, cell cycle arrest and apoptosis in FLS-MTX cells were monitored with MTT and flow cytometry analysis, respectively. And the regulation of miR-16 on P-glycoprotein (P-gp) was performed using qRT-PCR, western blotting, and immunofluorescence staining. Fourthly, ammonium pyrrolidinedithiocarbamate (PDTC), a NF-κB pathway inhibitor, was applied to verify the mechanism by which miR-16 involved in to regulate the P-gp expression, and thus contributing to the methotrexate resistance in FLS-MTX cells. MiR-16 was upregulated in the in serum and synovial fluid from UR-MTX patients as well as in FLS-MTX cells. Inhibition of miR-16 re-sensitized the FLS-MTX cells to methotrexate by suppressing the cell viability, cell promoting cycle arrest at G0/G1 phase and enhancing apoptosis. Knockdown of miR-16 significantly reduced MDR1 mRNA expression and P-gp protein expression in FLS-MTX cells. Furthermore, inhibition of NF-κB pathway by PDTC reinforced the effect of miR-16 knockdown on P-gp expression, cell viability, cell cycle arrest and apoptosis. In conclusion, our study illustrated that inhibition of miR-16 in FLS-MTX cells alleviated methotrexate resistance by inhibiting MDR1/P-gp expression through inactivation of the NF-κB pathway.

MicroRNAs (miRNAs) are demonstrated to contribute to the regulation of drug resistance in a number of diseases.     

Selective inhibition of cyclooxygenase-2 suppresses the growth of pancreatic cancer cells in vitro and in vivo

Cyclooxygenase-2 (COX-2), a prostaglandin synthetase, is involved in development of certain tumors. We therefore analyzed COX-2 expression in pancreatic cancer tissues (53 samples) and Panc-1 human pancreatic cancer cells by immunohistochemistry, RT-PCR and western-blotting analyses. Also, immunohistochemistry of proliferating cell nuclear antigen (PCNA) was performed. We found expression of COX-2 was dramatically upregulated in 36 of 53 cases (67.9%) and the expression of COX-2 was associated with the diameter (> 3 cm) of the tumors (p < 0.05), but not with the age, gender, tumor location, differentiation, lymph-node metastases and TNM stage. The positivity rate of PCNA expression in the pancreatic cancer cells of the COX-2 positive group (32.88 +/- 13.26%) was significantly higher than that in the COX-2 negative group (24.56 +/- 11.51%) (p < 0.05). Then we investigated the effect of selective inhibitors of COX-2 (NS398 and celecoxib) on proliferation of Panc-1 cells by 3-(4,5 dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) assay. Either NS398 or celecoxib suppressed proliferation of Panc-1 cells dose-dependently in vitro. Furthermore, Panc-1 cells were implanted into nude mice, and celecoxib was administrated orally with feed. The volume of the tumor xenografted into nude mice was decreased by 51.6% in the celecoxib group (p < 0.01). In conclusion, the increased expression of COX-2 may be responsible for rapid proliferation of pancreatic cancer, and specific inhibition of COX-2 suppresses proliferation of Panc-1 cells in vitro and in nude mice. The selective inhibitor of COX-2 may be an effectual agent for pancreatic cancer chemoprevention.     

关节液中抗环瓜氨酸肽抗体在类风湿关节炎诊断和评估中的意义

目的:分析关节液中抗环瓜氨酸肽(CCP)抗体对类风湿关节炎(RA)的诊断和评估意义。方法:用ELISA法检测37例RA患者血清和关节液中的抗CCP抗体和13例对照组关节炎患者关节液中的抗CCP抗体,并测定类风湿因子(RF),记录RA患者的一些临床参数。对检测结果及临床参数进行统计分析。结果:关节液中抗CCP抗体对RA诊断的敏感度为72.9%,特异度为92.3%。RA组和对照关节炎组患者的关节液中抗CCP抗体的差异有显著性。RA组血清与关节液中抗CCP抗体的浓度高度相关(P<0.001),且关节液中抗CCP抗体与关节液中RF浓度有较高相关性(P<0.01)。RA组关节液中抗CCP抗体与病程相关,病程越长,抗CCP抗体滴度越高;而与肿胀关节数、压痛关节数、疾病活动评分(DAS28)、血清RF、红细胞沉降率(ESR)、C反应蛋白(CRP)和Steinbrocker放射线分级不相关。结论:关节液中抗CCP抗体对RA的诊断有高度特异性和较好敏感性,对其他关节炎有显著性差异,独立于一些临床参数,同时又与血清中抗CCP抗体及关节液中的RF相关。     

Cytokines in chronic inflammatory arthritis. I. Failure to detect T cell lymphokines (interleukin 2 and interleukin 3) and presence of macrophage colony-stimulating factor (CSF-1) and a novel mast cell growth factor in rheumatoid synovitis

Because previous studies showed low levels of IFN-gamma in rheumatoid arthritis (RA) synovial fluid (SF) and synovial tissue (ST) explant supernatants, we assayed RA SF and ST for IL-2 and IL-3-like activity. Using an IL-2 dependent murine CTLL line, 6 of 14 RA SF caused increased thymidine uptake (greater than three times control). The activity was distinct from IL-2 because it was not blocked by antibody to IL-2-R. In addition, IL-2 was not detected (less than 50 pg/ml) in 16 joint samples using an ELISA. Multi-colony-stimulating factor (CSF) activity was measured using two assays that can detect murine IL-3 (mast cell proliferation, and bone marrow CSF). In the mast cell assay, [3H]TdR uptake was 493 +/- 67 cpm for medium, 2,910 +/- 329 cpm in the presence of RA SF (p less than 0.001), 1,246 +/- 156 cpm in the presence of SF from patients with seronegative spondyloarthropathies (p less than 0.001), and 736 +/- 100 cpm in the presence of osteoarthritis SF (p greater than 0.1). In the CSF assay, four of five RA SF and five of five RA ST induced colony formation from bone marrow nonadherent cells. Macrophage colonies were most common, although mixed colonies and granulocytes were occasionally observed. The multi-CSF activity in RA is not due to IL-3 since human rIL-3 was not active in either murine assay, and IL-3 mRNA was not detected in RA synovium. Sephadex column chromatography of RA SF revealed that the mast cell growth factor (approximately 6 x 10(3) mol wt) and the CSF (approximately 40 and 100 x 10(3) mol wt) are distinct. The colony-stimulating aspect of the "IL-3-like" activity in RA SF is likely due to CSF-1 because it is the appropriate mol wt and because the activity was neutralized by specific anti-CSF-1 antibody. Finally, an RIA detected 1.6-25 ng/ml of CSF-1 in RA SF and ST and CSF-1 mRNA was detected in four of five RA synovial tissue samples tested.     

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.