趋化因子CXCL1与脊柱关节病外周关节炎及病因相关性研究

目的 探讨趋化因子在诱导脊柱关节病(SpA)外周关节炎症中的作用.方法 分别在体外应用5例SpA患者的关节滑液诱导5名健康志愿者的外周血单个核细胞(PBMC)1.5h,提取总RNA后,应用基因芯片技术中的Superarray芯片技术筛选分别含有96个目的细胞因子和趋化因子的基因表达谱.应用ELISA技术分别检测54例强直性脊柱炎(AS)患者和30例健康者(正常对照组)外周血CXCL1的蛋白表达水平;比较 33例SpA和14例骨关节炎(OA)患者关节液中CXCL1的表达水平,同时还检测生物制剂英利昔单抗治疗前后CXCL1的蛋白表达水平.结果 在关节液刺激后,基因芯片筛选出PBMC中的CXCL1、CXCL2、CXCL3和IL-8基因表达增高2倍以上,炎性细胞因子(如TNF-α、IL-6、IL-1等)和其他趋化因子的表达并未随关节液的刺激而增加;ELISA结果发现AS患者外周血中CXCL1的蛋白水平显著高于正常对照组(P＜0.01);SpA患者关节液中的CXCL1蛋白表达水平也显著高于OA患者(P＜0.01).结论 CXCL1在诱导脊柱关节病外周关节炎症和在脊柱关节病的发病机制中发挥一定的作用.     

细胞核因子κB受体活化因子配基在强直性脊柱炎的表达及意义

目的 检测细胞核因子κB受体活化因子配基(RANKL)在强直性脊柱炎(AS)患者脊柱外关节滑膜组织中的表达．并以类风湿关节炎(RA)、骨关节炎(OA)患者和健康者外周关节滑膜组织为对照,了解RANKL表达在AS患者关节炎症病理机制中的意义。方法 应用单克隆抗体,通过免疫组织化学方法检测13例AS、16例RA、17例OA及6例健康对照关节滑膜组织中RANKL、CD68蛋白表达及分布情况,通过计算机辅助图像分析系统和半定量分析方法确定RANKL在各滑膜组织中的表达水平,分析RANKL的表达与炎性指标及关节X线分期之间的相关性。结果 AS和RA患者组滑膜组织RANKL表达水平明显升高,阳性细胞主要见于滑膜组织衬里层和滑膜软骨交界区,OA患者组和健康对照组滑膜组织中则未见阳性表达信号。AS和RA患者滑膜组织中RANKL表达水平与关节X线分期呈正相关(相关系数r分别为0．41、0．73,P值分别为0．021、0．003)。结论 RANKL在AS患者骨质破坏的病理机制中具有重要作用,其表达量的多少在一定程度上可反映AS患者骨质破坏程度,AS患者滑膜组织中RANKL表达模式与RA相似,提示AS外周关节骨质破坏的病理机制可能与RA类似。     

强直性脊柱炎患者外周血单个核细胞干扰素诱导蛋白10及Th1/Th2型细胞因子的表达

目的　探讨干扰素诱导蛋白 10 (IP 10 )在强直性脊柱炎 (AS)发病中的作用。方法　收集肝素抗凝的AS患者的外周全血 ,梯度离心得到外周血单个核细胞(PBMC) ,应用半定量RT PCR法测定IP 10、IFN γ和IL 4mRNA表达水平。结果　AS患者PBMC中IP 10、IFN γ和IL 4mRNA表达水平均较健康对照组高(P <0 0 1) ,同时IFN γ/IL 4值也较对照组高 (P <0 0 5 )。IFN γ和IL 4mRNA表达水平与C 反应蛋白呈负相关 ,IP 10mRNA表达水平与IFN γ、IL 4mRNA和IFN γ/IL 4值均呈正相关。结论　AS患者PBMC表达的细胞因子以Th1型为主 ,IP 10表达升高 ,IP 10可能通过促进Th1型细胞向炎症部位的归巢来参与AS的发病     

类风湿关节炎患者蛋白聚糖aggrecan G1区特异性T细胞诱导SCID小鼠产生膝关节炎的实验研究

目的探讨针对蛋白聚糖的自身免疫反应在类风湿关节炎（RA）发病中的作用。方法从人软骨细胞中克隆获得aggrecan G1区的cDNA片段,应用杆状病毒表达系统获取重组人aggrecan G1区蛋白（rAG1）。用rAG1刺激来自RA患者关节液的单个核细胞,采用β-液闪计数仪对培养细胞进行放射性检测并计算刺激增长指数（SI）,取SI超过3者为rAG1特异性T细胞。将取自3个人的rAG1特异性T细胞克隆腹腔注射至8只SCID Beige小鼠,同时在小鼠左膝注射rAG1。于注射后第8天处死小鼠,取双膝关节行组织病理学检查,观察炎细胞浸润及软骨和骨糜烂情况。结果从RA患者关节液单个核细胞分离得到rAG1特异性T细胞,主要为CD4^＋CD8^-T细胞,分泌Th1型细胞因子（γ-干扰素）。部分腹腔注射人rAG1特异性T细胞的小鼠左膝出现了关节滑膜单个核细胞浸润和早期软骨侵蚀改变。结论人特异性rAG1反应性T细胞在rAG1刺激下可向关节局部归巢而导致关节炎症反应,提示针对rAG1的自身免疫反应可能参与了RA的发病。     

哮喘患者血清Rac1表达水平及其与气道炎症相关性分析

目的探讨哮喘患者血清Rac1的表达水平及其与气道炎症的相关性。方法选取自2020年4月至2021年4月上海交通大学附属新华医院崇明分院收治的57例哮喘患者作为哮喘组,另选取同期的57例健康体检者作为健康组。检测两组研究对象的外周血单个核细胞(PBMC)中Rac1 mRNA表达水平及血清白细胞介素(IL)-5、IL-13、IL-33水平。比较哮喘组与健康组,以及不同严重程度哮喘患者PBMC中Rac1 mRNA及血清IL-5、IL-13、IL-33水平。采用Pearson相关法分析哮喘患者的PBMC中Rac1 mRNA表达与血清IL-5、IL-13、IL-33的相关性。结果哮喘组Rac1 mRNA表达低于健康组,血清IL-5、IL-13、IL-33水平高于健康组,差异有统计学意义(P<0.05)。中度组与重度组Rac1 mRNA表达低于轻度组,且重度组低于中度组,差异有统计学意义(P<0.05)。中度组与重度组血清IL-5、IL-13、IL-33水平高于轻度组,且重度组高于中度组,差异有统计学意义(P<0.05)。哮喘患者Rac1 mRNA表达与血清IL-5、IL-13、...     

破骨细胞在强直性脊柱炎滑膜组织中的分化及其在外周关节骨质破坏病理机制中的作用

目的检测破骨细胞在强直性脊柱炎（AS）外周关节滑膜组织中的分化情况,了解破骨细胞的分化与AS患者外周关节骨质破坏病理改变的相关性。方法应用单克隆抗体,通过免疫组织化学及酶组织化学染色的方法检测13例AS、16例类风湿关节炎（RA）、17例骨关节炎（OA）及6例健康对照关节滑膜组织中CD68蛋白表达及TRAP染色阳性滑膜细胞的分布状况,并通过计算机辅助图像分析系统和半定量分析方法确定CD68分子表达水平及TRAP染色阳性细胞在各研究组滑膜组织中的差异性。结果滑膜组织中均有CD68阳性细胞,AS和RA组CD68表达水平较OA组和健康对照组显著增高（P〈0．05）,其阳性细胞主要分布于滑膜衬里层,衬里下层也见少量表达;OA和健康者滑膜中仅有少量CD68阳性细胞分布。TRAP染色阳性细胞位于滑膜组织衬里层、淋巴细胞聚集区及滑膜软骨交界区,其中AS组阳性细胞百分数显著低于RA组,OA及正常对照组阳性细胞少见。RA组阳性细胞百分数与RANKL表达水平呈正相关（r=0．442,P=0．043）。结论炎症关节滑膜组织中表达CD68分子及TRAP染色阳性滑膜细胞数量的增加为破骨细胞的分化、成熟提供了充足的细胞数量基础,破骨细胞数量和活性上调是造成强直性脊柱炎外周关节骨质破坏的重要前提。     

外周血中BAFF、BAFF-R及Bcl-xL的表达与B细胞非霍奇金淋巴瘤疾病进展的相关性研究

目的研究B细胞性非霍奇金淋巴瘤(B-NHL)患者外周血B cell-activating factor(BAFF)、其受体BAFF-R及Bcl-xL基因的表达,探讨其与B-NHL疾病进展的相关性。方法应用实时荧光定量PCR法检测36例B-NHL患者外周血单个核细胞(PBMC)BAFF、BAFF-R、Bcl-xL mRNAs的相对表达水平,以7例健康献血员作为对照,并据性别、淋巴瘤国际预后指数(IPI)、年龄、临床分期、是否耐药进行分组比较。结果 B-NHL患者PBMC BAFF、BAFF-R及Bcl-xL的mRNAs表达较正常对照组显著升高(P<0.05),Ⅲ/Ⅳ期高于Ⅰ/Ⅱ期,耐药B-NHL组高于非耐药组(P<0.05)。结论外周血单个核细胞BAFF、BAFF-R、Bcl-xL mRNAs的表达水平与B-NHL临床分期有关,在耐药B-NHL外周血中明显升高,提示BAFF/BAFF-R介导的信号通路可能参与B-NHL的临床进展及耐药机制。     

维持性血液透析患者外周血单个核细胞早期凋亡

目的　研究维持性血液透析 (MHD)患者外周血单个核细胞 (PBMC)早期凋亡 (Annexin V)。　方法　应用流式细胞仪 (FCM)定量检测终末期肾衰竭未透析患者 (ND)、醋酸纤维膜透析患者 (CA)、聚砜膜透析患者 (PS)和健康自愿者 (C)各10例的 PBMC体外无刺激培养 2 4 h后 Annexin V的表达。　结果　 ND组 Annexin V表达显著高于 C组和 PS组 ,CA组显著高于 C组和 PS组 (P<0 .0 5 ) ,而 PS组与 C组、CA组与 ND组差异不显著。 Annexin V与 Ccr间呈显著负相关 (P<0 .0 5 )。　结论　透析和尿毒症本身可诱导外周血单个核细胞凋亡增加 ,生物相容性较好的透析膜可减轻细胞凋亡     

Correlation between expressions of BAFF, BAFF-R and Bd-xL in peripheral blood and progression in B cell non-Hodgkin's lymphoma patients

目的 研究B细胞性非霍奇金淋巴瘤(B-NHL)患者外周血B ceil-activating factor(BAFF)、其受体BAFF-R及Bcl-xL基因的表达,探讨其与B-NHL疾病进展的相关性.方法 应用实时荧光定量PCR法检测36例B-NHL患者外周血单个核细胞(PBMC)BAFF、BAFF-R、Bcl-xL mRNAs的相对表达水平,以7例健康献血员作为对照,并据性别、淋巴瘤国际预后指数(IPI)、年龄、临床分期、是否耐药进行分组比较.结果 B-NHL患者PBMC BAFF、BAFF-R及Bcl-xL的mRNAs表达较正常对照组显著升高(P<0.05),Ⅲ/Ⅳ期高于I/Ⅱ期,耐药B-NHL组高于非耐药组(P<0.05).结论 外周血单个核细胞BAFF、BAFF-R、Bcl-xL mRNAs的表达水平与B-NHL临床分期有关,在耐药B-NHL外周血中明显升高,提示BAFF/BAFF-R介导的信号通路可能参与B-NHL的临床进展及耐药机制.     

BMP-2和bFGF在强直性脊柱炎活动期病理性表达及意义

目的：探讨骨形态发生蛋白2（BMP-2）和碱性成纤维细胞生长因子（bFGF）在强直性脊柱炎（AS）活动期的病理性表达及意义。方法：通过原位杂交技术检测活动期AS患者与对照组（骨盆骨折患者）骶髂关节滑膜组织中BMP-2、bFGF的表达,用图像分析系统检测阳性细胞与阴性细胞灰度值。结果：在活动期AS患者骶髂关节滑膜组织中BMP-2和bFGF阳性表达,而对照组骶髂关节滑膜组织中BMP-2和bFGF阴性表达,图像灰度值比较有统计学差异（P（0.01）。结论：BMP-2和bFGF均为强直性脊柱炎病理性成骨过程中重要的成骨因子,它们与强直性脊柱炎骶髂关节成骨硬化过程密切相关。控制BMP-2、bFGF的表达可能阻断AS的病理性成骨硬化,为治疗强直性脊柱炎提供新的思路。     

超声检测类风湿关节炎膝关节滑膜改变及其血流的临床应用价值

目的：通过观察类风湿关节炎（rheumatoid arthritis ,RA ）膝关节滑膜病变的超声声像图表现,分析滑膜病变超声检查结果与RA活动度的相关性,探讨超声检查RA膝关节滑膜病变的临床应用价值。方法对38例RA患者患侧膝关节和20例正常对照者双侧膝关节行超声检查,根据DAS28评分结果将RA患者分组,分别对比各组与对照组的滑膜厚度,髌上囊积液;分析RA组超声检测结果与DAS28评分之间的相关性。结果 RA组滑膜厚度较对照组明显增厚,髌上囊积液明显增多（ P ＜0．05）。RA组膝关节滑膜厚度、关节积液及滑膜内血流信号分级与RA患者DAS28评分之间存在一定的相关性（ P ＜0．05）。结论超声检查对RA膝关节滑膜病变具有一定的的诊断价值,与DAS28评分的相关性较好,可以作为评估RA膝关节滑膜病变常规检查方法之一。     

UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotease-mediated shedding

Abstract. Purpose : L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. Materials and methods : Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITClabelled annexin-V in the presence of propidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. Results : PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. Conclusion : UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte tra Ýcking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.     

123I-interleukin-2 scintigraphy for in vivo assessment of intestinal mononuclear cell infiltration in Crohn's disease.

Activated mononuclear cells expressing interleukin-2 (IL2) receptors (IL2-Rs) heavily infiltrate the Crohn's disease (CD) gut wall. A new technique for the in vivo detection of tissue infiltrating IL2-R positive (IL2R+ve) cells was developed based on 123I-IL2 scintigraphy. The aim of this study was to investigate whether 123I-IL2 accumulates in the CD gut wall in different phases of the disease and to evaluate the specificity of 123I-IL2 binding to activated IL2R+ve cells infiltrating the gut wall. METHODS: Fifteen patients with ileal CD (10 active and 5 inactive) and 10 healthy volunteers were studied by 123I-IL2 scintigraphy. Six patients with active CD were studied before and after 12 wk of steroid treatment. After scintigraphy, patients were followed up for 29-54 mo. Ex vivo autoradiography was performed to determine specificity of 125I-IL2 binding to IL2R+ve cells. For bowel scintigraphy, 123I-IL2 (75 MBq) was injected intravenously and gamma camera images were acquired after 1 h. Bowel radioactivity was quantified in 64 regions of interest (ROIs). RESULTS: Autoradiography showed specific binding of 125I-IL2 to IL2R+ve mononuclear cells infiltrating the CD gut wall. Intestinal 123I-IL2 uptake assessed by the number of positive ROIs was higher in patients with active or inactive CD than in healthy volunteers (P < 0.0001 and P = 0.03, respectively) and positively correlated with the CD activity index (P = 0.01). 123I-IL2 intestinal uptake significantly decreased in patients with CD in steroid-induced remission (P = 0.03). A significant correlation was observed between the number of positive ROIs and time to disease relapse. CONCLUSION: 123I-IL2 accumulates in the diseased CD gut wall by specific binding to IL2R+ve cells, infiltrating the involved tissues. 123I-IL2 scintigraphy may be an objective tool for the in vivo assessment of intestinal activated mononuclear cell infiltration.     

UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotese-mediated shedding

PURPOSE: L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITC-labelled annexin-V in the presence ofpropidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. RESULTS: PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. CONCLUSION: UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte trafficking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.     

原代白血病细胞IL-6受体α表达的实验研究

目的：分析不同类型原代白血病细胞IL-6R基因及IL-6R蛋白的表达情况。方法：采用RT-PCR方法对取自29例白血病患者的原代白血病细胞和8例健康人外周血单个核细胞的IL-6R基因表达情况进行了分析，并应用原位杂交技术和免疫组织化学方法对细胞表面的IL-6受体蛋白进行了检测。结果：20例急性髓细胞白血病均明显表达IL-6R mRNA；9例急性淋巴细胞白血病中有2例为阳性，其余7例为阴性；8例正常人PBMC中6例阴性。2例有弱表达。免疫组化和原位杂交结果与RT-PCR实验结果相一致。结论：IL-6R可作为急性髓系白血病免疫治疗的候选靶位点。     

Evaluation of the degree of clinical rheumatoid arthritis activity based on the concentrations of cytokines TNF-alpha, IL-12, IL-15, and IL-18 in serum and synovial fluid

BACKGROUND/AIM: Experimental in vitro and in vivo investigations in a mouse model have proved that TNF-alpha, IL12, IL-15 and IL18 participate in the pathogenesis of erosive inflammatory arthritis. The aim of this research was to determine the clinical significance of cytokines in the evaluation of the activity of rheumatoid arthritis (RA). METHODS: Inside a 4-year period we followed-up 64 patients with RA as newly ocurred or in the phase of worsening. We observed the clinical manifestation of the disease upon which we divided the patients in to 3 groups: the patients with low active RA, patients with moderate active RA, and the patients with wild active RA. The control group (n=25 patients) included the patients with osteoarthrosis (OA), and arthritis of the knee. In the samples of serum of all of the patients the concentratin of cytokines TNF-alpha, IL-12, IL-15, and IL-18 were determined using the immunoenzymatic methods in mice for human interleukines. By comparing the concentrations in 30 patients with the high, 14 patiens with moderate, and 20 patiens with the mild activity of RA it was determined that the patients with the high degree of the disease activity, had significantly high (p < 0.01; p < 0.05) concentrations of the examined cytokines in blood and synovial fluid as compared to the patients with the moderate and mild active disease. There was a relationship (p < 0.01) between the concentrations of cytokines in blood and synovial fluid with the quantity of the Disease Activity Score in 28 joints. CONCLUSIONS: Cytokines concentrations could be good indicators of the degree of the general activity of RA. This research could contribute to the interpretation of insufficiently well known views of the pathogenesis role and significance of citokines in an active disease.     

淋巴瘤患者外周血单个核细胞FOXp3与HLA-B基因表达研究

 目的 探讨淋巴瘤患者外周血单个核细胞(peripheral blood mononuclear cells, PBMC)FOXp3与人白细胞抗原B (human leukocyte antigen, HLA-B),的基因表达水平及对淋巴瘤的临床应用价值.方法 设计FOXp3和HLA-B基因引物和实时荧光PCR相对定量法反应体系,在实时荧光定量PCR仪上检测69例淋巴瘤患者和30例健康人外周血单个核细胞内基因表达水平并分析其差异.结果 淋巴瘤患者FOXp3和HLA-B的mRNA相对表达量分别为1.46±0.45和0.74±0.22,健康组分别为1.15±0.30和1.05±0.28,两组差异均有统计学意义.在淋巴瘤患者组中,Ⅰ期+Ⅱ期的HLA-B相对表达量高于Ⅲ期+Ⅳ期,差异有统计学意义,而FOXp3未见区别.结论 淋巴瘤患者外周血单个核细胞的FOXp3基因表达水平上调,HLA-B的基因表达水平下调且HLA-B不同分期间存在差异. 检测PBMC的FOXp3、HLA-B基因表达水平有助于淋巴瘤诊断和研究.