TNF-α对人糖皮质激素受体表达的影响

目的: 研究TNF-α对人糖皮质激素受体(GR)表达的影响,探讨炎性细胞因子与糖皮质激素抵抗的相关性. 方法: 分离人外周血单个核细胞(PBMCs),用TNF-α刺激培养后,采用RT-PCR方法分析其与未用TNF-α刺激培养的PBMCs在GRα和GRβ mRNA表达方面的变化. 结果: 经TNF-α刺激培养24 h的PBMCs,与未加TNF-α刺激培养的PBMCs相比,GRα mRNA表达无显著变化(P>0.05),GRα/GAPDH mRNA分别为1.16 ±0.73,1.20±0.51,但GRβ mRNA表达明显增加(P<0.05),GRβ/GAPDH mRNA分别为1.62±0.59,0.81±0.66. 结论: TNF-α可以增加GRβ的表达,这可能是炎性细胞因子诱发糖皮质激素抵抗的内在机制之一.     

尼古丁对体外培养人外周血单个核细胞分泌TNF的影响

OBJECTIVE: To observe the effect of nicotine on the secretion of TNF of human peripheral blood mononuclear cells (PBMC) in vitro, and explore the possible mechanism of the high level of TNF caused by smoking. METHODS: Twenty-one non-smokers and 19 smokers were studied. The PBMCs isolated from each volunteer's blood sample were distributed to three groups, and cultured for 72 hours in supplemented RMPI 1640 alone or in supplemented RPMI 1640 containing 50 ng.ml-1 nicotine or 500 ng.ml-1 nicotine respectively. The level of TNF in supernate was quantified with the immuno-radioassay, and PBMC proliferation was observed. RESULTS: The level of TNF spontaneously secreted by PBMCs in smokers was significantly higher than that in non-smokers (P < 0.05). When the concentrations of nicotine were 50 ng.ml-1 and 500 ng.ml-1, the level of TNF increased significantly in non-smokers (P < 0.05, P < 0.01). In smokers, the level of TNF significantly increased when the concentration of nicotine was 50 ng.ml-1; however, the level of TNF significantly decreased (P < 0.05), and the proliferation of PBMCs was inhibited when the concentration of nicotine was 500 ng.ml-1 (P < 0.05, P < 0.05). The level of TNF was positively correlated to the multiple of PBMC proliferation (r = 0.93, P < 0.05). The linear regression equation was Y = 2.913X - 2.955. CONCLUSION: Nicotine can induce PBMCs to secrete more TNF, and the magnitude of the effect is strongly related to the dosage of nicotine, but a great dose of nicotine will inhibit the production of TNF.     

关节内注射不同浓度臭氧对类风湿关节炎大鼠TNF-α、TNFRⅠ和TNFRⅡ的影响

摘要：目的观察不同浓度臭氧(O3)对类风湿关节炎(RA)大鼠体内TNF-α、TNFRⅠ和TNFRⅡ的影响,阐述O3治疗类风湿
关节炎的机理。方法健康雄性Wistar大鼠48只,采用随机数字表法分为CON组、RA组、O2组、O3-10组、O3-20组、O3-30
组、O3-40组、O3-50组,每组6只。除空白对照组外,采用注射弗氏完全佐剂乳化的牛Ⅱ型胶原蛋白的方法建立大鼠RA模
型。造模后21 d,根据分组关节内注射纯氧和浓度分别为10、20、30、40、50 μg/ml的O3各1 ml,每周1次,共注射3周。治
疗前后测量双后肢足爪厚度。治疗结束后1周,检测大鼠血清和滑膜TNF-α、TNFRⅠ和TNFRⅡ的含量。结果治疗结束
后,O3-40 组大鼠足爪厚度减小,与RA组相比差异有统计学意义（P<0.01）。RA组、O2组、及O3治疗各组血清中TNF-α、
TNFRⅠ、TNFRⅡ含量差异无统计学意义（P>0.05）。与RA组相比,O3-40组、O3-50组大鼠滑膜TNF-α、TNFⅡ含量降低,
差异有统计学意义（P<0.01）。与RA组相比,O3-40组大鼠滑膜TNFRⅠ含量升高,差异有统计学意义（P<0.05）。结论关
节内注射O3可减轻RA大鼠的关节肿胀,浓度为40 μg/ml的O3最为明显,作用机制和O3可以降低滑膜内TNF-α、TNFRⅡ
的活性,上调TNFRⅠ的表达有关。     

关节内注射不同浓度臭氧对类风湿关节炎大鼠TNF-α、TNFRⅠ和TNFRⅡ的影响

目的观察不同浓度臭氧(O3)对类风湿关节炎(RA)大鼠体内TNF-α、TNFRⅠ和TNFRⅡ的影响,阐述O3治疗类风湿关节炎的机理。方法健康雄性Wistar大鼠48只,采用随机数字表法分为CON组、RA组、O2组、O3-10组、O3-20组、O3-30组、O3-40组、O3-50组,每组6只。除空白对照组外,采用注射弗氏完全佐剂乳化的牛Ⅱ型胶原蛋白的方法建立大鼠RA模型。造模后21d,根据分组关节内注射纯氧和浓度分别为10、20、30、40、50μg/ml的O3各1ml,每周1次,共注射3周。治疗前后测量双后肢足爪厚度。治疗结束后1周,检测大鼠血清和滑膜TNF-α、TNFRⅠ和TNFRⅡ的含量。结果治疗结束后,O3-40组大鼠足爪厚度减小,与RA组相比差异有统计学意义(P<0.01)。RA组、O2组、及O3治疗各组血清中TNF-α、TNFRⅠ、TNFRⅡ含量差异无统计学意义(P>0.05)。与RA组相比,O3-40组、O3-50组大鼠滑膜TNF-α、TNFⅡ含量降低,差异有统计学意义(P<0.01)。与RA组相比,O3-40组大鼠滑膜TNFRⅠ含量升高,差异有统计学意义(P<0.05)。结论关节内注射O3可减轻RA大鼠的关节肿胀,浓度为40μg/ml的O3最为明显,作用机制和O3可以降低滑膜内TNF-α、TNFRⅡ的活性,上调TNFRⅠ的表达有关。     

黄芪对冠心病患者PBMC凋亡及血浆ET、IL-6的影响

黄芪是传统中药，对心血管疾病具有很高应用价值。我们应用黄芪注射液与西药相结合治疗冠心病，观察冠心病患者外周血单个核细胞（PBMC）凋亡及血浆内皮察（ET）、白细胞介素-6（IL-6）变化，研究黄芪对冠心病患者PBMC凋亡及血浆ET、IL-6影响。     

TNF-α信号通路主要受体衔接蛋白在SLE患者外周血单个核细胞中的表达及其意义

目的探讨肿瘤坏死因子（TNF）α信号通路主要受体衔接蛋白-肿瘤坏死因子相关死亡结构域蛋白（TRADD）、Fas相关死亡结构域蛋白（FADD）、受体相互作用蛋白1（RIP-1）和肿瘤坏死因子受体相关因子2（TRAF-2）在系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMC）中的表达变化及意义。方法收集本院确诊的SLE住院患者（51例）和健康志愿者（17例）外周静脉血,分离PBMC,采用实时荧光定量PCR检测上述4种受体衔接蛋白及半胱氨酸蛋白水解酶（Caspase）3和白细胞介素（IL）-1β的表达水平,比较SLE患者与健康志愿者之间以及不同狼疮活动度SLE患者中上述4种受体衔接蛋白的表达差异,以及与狼疮活动指数之间的相关关系。结果正常对照组PBMC中可表达TRADD、FADD、RIP-1和TRAF-2。SLE患者PBMC中TRADD、FADD、RIP-1和TRAF-2表达分别为正常对照组的0．38,0．69,0．59和0.55倍（P〈0．05）;SLE高活动度组4种受体衔接蛋白的表达分别为正常对照组的0．31,0．59,0.52和0．49倍（P〈0．05）,但低活动度组与正常对照组之间的差异无统计学意义（P〉0．05）。SLE患者PBMC中Caspase 3表达明显升高（P〈0．01）,而IL-1β表达与正常对照组之间差异无统计学意义（P〉0．05）。SLE患者PBMC中TRADD、FADD、RIP-1和TRAF-2的表达与狼疮活动指数（SLEDAI）均呈负相关关系,相关系数分别为-0．285,-0．280,-0．307和-0．298（P〈0．05）。结论TNF-α信号通路中主要的4种受体衔接蛋白TRADD、FADD、RIP-1和TRAF-2在SLE患者PBMC中的表达均降低,且与狼疮活动指数呈负相关关系,其异常表达可能参与了TNF—α信号网络紊乱所介导的SLE免疫病理损伤。     

α肿瘤坏死因子（TNFα）基因的分离鉴定及在人肝癌细胞中的表达

应用不连续密度梯度法分离获得人肝癌浸润性单个核细胞（ＴＩＭ），ＲＮＡ斑点杂交提示ＴＩＭ中有明显的ＴＮＦαｍＲＮＡ转录。经ＲＴ－ＰＣＲ扩增反应分离得到一特异的７００ｂｐ左右的ＤＮＡ片段，ＤＮＡ序列分析证实这一ＤＮＡ片段中包含编码人ＴＮＦα成熟肽所需的的全部ｃＤＮＡ序列。将这一人ＴＮＦαｃＤＮＡ克隆到真核细胞表达质粒ｐＳＶＫ３，获得真核细胞表达重组ｐＳＶＫ３－ｔｎｆ，用磷酸钙沉淀法将ｐＳＶＫ３－ｔｎｆ导入人肝癌细胞ＳＭＭＣ７７２１中的细胞培养上清中可检测到ＴＮＦα的一过性表达。结果提示ＴＩＭ可成为获取ＴＮＦα基因的来源，获得的ｐＳＶＫ３－ｔｎｆ重组质粒为肝癌的基因治疗奠定了基础。     

肿瘤患者外周血细胞端粒酶表达及临床意义

目的探讨肿瘤患者外周血单个核细胞端粒酶活性的表达.方法采用端粒重复序列扩增-微孔板杂交法测定恶性肿瘤患者48例,良性患者34例和38例正常对照者外周血单个核细胞端粒酶表达.结果恶性肿瘤和良性肿瘤患者组外周血单个核细胞端粒酶活性比对照组明显增高(P＜0.01),且对恶性肿瘤和良性肿瘤患者的检出阳性率分别达77.1%和47.1%.结论外周血单个核细胞端粒酶表达与肿瘤的发生、发展有关,因而检测外用血单个核细胞端粒酶活性有助于肿瘤的早期诊断和治疗.     

复方丹参注射液对人外周血单个核细胞体外分泌TNF-α的影响

[摘要] 目的 探讨复方丹参注射液治疗强直性脊柱炎可能的作用机制。方法 将分离的正常人外周血单个核细胞分成5组,分别给予生理盐水、大剂量复方丹参、中剂量复方丹参、小剂量复方丹参和地塞米松;孵育48h后,提取细胞培养液上清夜,用放免法检测肿瘤坏死因子α的含量。 结果 大剂量复方丹参组和地塞米松组细胞培养液的上清液TNF-α含量明显低于生理盐水组(P<0.05),大剂量复方丹参组的TNF-α的含量水平明显低于中、小剂量复方丹参组(P<0.05)。 结论 抑制人PBMC分泌TNF-α可能是复方丹参注射液治疗AS的途径之一。     

Expression of tumor necrosis factor receptor on peripheral blood mononuclear cells from patients with primary hepatocellular carcinoma and effects of treatment on the expression]

OBJECTIVE: To conduct an intensive investigation of the abnormal immune status of patients with primary hepatocellular carcinoma and explore the possible mechanism of biotherapy for these patients. METHODS: The expression of tumor necrosis factor receptor (TNFR)I and TNFR II on peripheral blood mononuclear cells (PBMCs) obtained from 30 normal subjects and 31 patients with primary hepatocellular carcinoma were examined by flow cytometry and S-P staining assay. The effects of biotherapy and chemotherapy administered in the patients were also investigated in terms of the changes in the expressions. RESULTS: The TNFR I and TNFR II expressions in the patients were (28.35+/-9.09) % and (37.45+/-9.51) % respectively, significantly lower than those of normal subjects [(38.54+/-8.51) % and (44.89+/-9.08) %]. The biotherapy for the patients caused an increase of TNFR I expression to (42.86+/-9.02)%. CONCLUSION: There are significant immune abnormalities in patients with primary hepatocellular carcinoma, and this study may offer some theoretical basis for the implementation of biotherapy in such cases.     

Expression of toll-like receptor 9 in peripheral blood mononuclear cells from patients with different hepatitis B and C viral loads

The aim of the present study was to investigate the expression of toll-like receptors （TLR） 9 in peripheral blood mononuclear cells （PBMC） of patients with chronic hepatitis B and C with different virus copies. The study group included 90 patients （60 with chronic hepatitis B, and 30 with chronic hepatitis C）, and 20 healthy people served as control group. The protein and mRNA levels of TLR9 were detected by using flow cytometry and real-time PCR. The serum viral copies of HBV and HCV were measured in all patients, and the correlation between HBV-DNA copies or HCV-RNA copies and the TLR9 expression was analyzed. Our results demonstrated that HBV or HCV infection led to a decreased expression of TLR9 mRNA and protein compared to the control group （P〈0.05）. The TLR9 protein and mRNA levels were negatively correlated with serum viral copies of HBV and HCV （r=-0.632, r=-0.909, P〈0.01）. It was concluded that TLR9 mRNA and protein are down-regulated in PBMC of HBV-infected or HCV-infected patients, and they are negatively correlated with serum viral copies and play an important role in detecting viral replication of HBV and HCV.     

肝细胞癌患者外周血循环肿瘤细胞阳性提示预后不良

目的评价外周血循环肿瘤细胞（CTCs）检测在肝细胞癌临床诊治中的应用价值。方法随机选取2013年7月~2015年1 月期间南方医院肝胆外科收治的肝细胞癌患者296例为肝细胞癌组,并同时收集39例非肿瘤或肝脏良性肿瘤患者为对照组,采 用广州益善生物技术股份有限公司开发的CanPatrolTM CTCs检测技术检测肝细胞癌组及对照组患者外周血中CTCs的分布情 况,并分析外周血CTCs阳性与肝细胞癌患者临床病理参数之间的关系,以及CTCs与肝细胞癌患者术后无瘤生存时间的关 系。结果在对照组患者外周血中未检测到CTCs,在肝细胞癌组中,191例患者外周血中检测到CTCs,阳性率为64.5%,显著高 于对照组（P<0.05）;肝细胞癌患者外周血中CTCs阳性与患者血清甲胎蛋白水平、肿瘤数量、TNM分期、BCLC分期、门静脉癌栓 及转移等显著相关（P<0.05）;在127例接受根治性手术治疗的肝细胞癌患者中,CTCs阳性的肝细胞癌患者术后无瘤生存时间 显著缩短（P<0.05）。结论肝细胞癌患者外周血CTCs阳性提示预后不良,CTCs可以成为监测肝细胞癌患者预后一个较好的 辅助指标。     

Expression of Toll-like receptor 4 in neonatal cord blood mononuclear cells in patients with preeclampsia

The expression of Toll-like receptor 4 (TLR4) in neonatal cord blood mononuclear cells (MNCs) and serum TNF-α were investigated in order to explore the roles of TLR4 in the pathogenesis of preeclampsia.The study enrolled 27 patients suffering from preeclampsia (experimental group) and 21 normal pregnancy patients (control group).After MNCs were separated, the expression of TLR4 mRNA and protein was detected by using real-time quantitative PCR and Western blotting respectively, and the expression of TNF-α by using ELISA.The results showed the TLR4 mRNA level in cord blood MNCs (2-CT:0.07±0.17), TLR4 protein expression level (absorbance ratio:0.81%±0.15%) and TNF-α level (9.5±1.73 pg/mL) were all increased in experimental group as compared with control group with the differences being statistically significant (P<0.05).There was a positive correlation between the expression of TLR4 mRNA and TNF-α in both experimental group and control group (r=0.54 and 0.53, respectively, P<0.05).It was concluded that TLR4 expression in the experimental group of cord blood MNCs was increased and there was a positive correlation between the expression of TLR4 mRNA and TNF-α in both groups.TLR4-mediated release of inflammatory cytokines may be one of the important reasons leading to preeclampsia.     

原发性胆汁性肝硬化患者外周血单个核细胞趋化因子受体的表达及临床意义

目的:初步探讨趋化因子受体(chemokine receptor)与原发性胆汁性肝硬化(PBC,primary biliary cirrhosis) 发生发展的关系。方法: 采用实时荧光定量PCR和流式细胞术,分别从基因转录和蛋白表达水平检测60例PBC、60例乙型肝炎肝硬化患者（疾病对照）和60例健康对照者的外周血单个核细胞(PBMCs)中CCR1、CCR3和CCR5的表达;生化常规测定PBC 患者和健康对照者的总胆红素(TBIL)、γ-谷氨酰基转移酶(γ-GT)水平,分析它们与趋化因子之间的相关性。结果:PBC患者PBMCs中的CCR1、CCR3、CCR5的mRNA和蛋白表达均明显低于健康对照组和疾病对照组(P＜0.05),而疾病对照组和健康对照组PBMCs中CCR1、CCR3、CCR5 mRNA和蛋白表达无统计学差异(P>0.05);PBC患者Ⅲ、Ⅳ期PBMCs中的CCR1、CCR3、CCR5的mRNA 和蛋白表达较Ⅰ、Ⅱ期明显降低(P＜0.05);PBC患者CCR1蛋白表达水平与TBIL水平显著负相关(r=- 0.445,P<0.01),与γ-GT无线性相关关系(r=-0.230,P>0.05);CCR3蛋白表达水平与TBIL水平无线性相关关系(r=-0.173,P>0.05),与γ-GT水平负相关(r=-0.295,P<0.05);CCR5蛋白表达水平与TBIL、γ-GT水平均负相关(r=-0.531,P<0.01;r=-0.665,P<0.01)。结论:CCR1、CCR3、CCR5的表达与PBC的发生发展存在一定的相关性,可能参与了PBC的调控机制,为PBC的诊断和预防提供了新线索。     

IL-10和TNF-α在人外周血单个核细胞体外感染卡介苗中的表达及相关性研究

目的探讨白细胞介素-10(IL-10)、肿瘤坏死因子(TNF-α)在人外周血单个核细胞(PBMCs)体外感染卡介苗(BCG)中的表达及相互作用。方法 PBMCs体外感染BCG后,分别于3、6、8、24h收集细胞应用荧光定量PCR法检测细胞内IL-10和TNF-α的mRNA含量;此外,分别于8、20、32、48h加入anti-IL-10的抗体孵育,收集细胞培养上清,用ELISA法检测上清中IL-10和TNF-α的蛋白含量。结果 PBMCs体外感染BCG后,相对于空白对照组,IL-10和TNF-α在mRNA和蛋白水平的表达量均增高,差异具有统计学意义(P<0.05),两者表达量呈现负相关趋势;当加入anti-IL-10的抗体后,TNF-α的蛋白表达量要高于未加抗体组,差异具有统计学意义(P<0.05)。结论 IL-10和TNF-α在人PBMCs体外感染卡介苗中的表达量增加,IL-10对TNF-α的表达可能具有抑制作用。     

急性胰腺炎患者外周血单核细胞 TLR9 mRNA 的表达及意义

目的：观察急性胰腺炎（AP）患者外周血单核细胞（PBMCs）Toll样受体9（TLR9）mRNA的表达情况。方法收集52例发病24 h内的AP患者,采集第1、3、5天外周乙二胺四乙酸二钾（EDTAK2）抗凝静脉血,提取血浆低温冻存,用于成批检测胰弹性蛋白酶、促炎细胞因子、抗炎细胞因子等。同法采集其治愈后2～3个月的外周EDTAK2抗凝静脉血行上述检测,作为相应指标的基准水平值。分离外周血单核细胞用于随后成批作逆转录聚合酶链反应（RT‐PCR）检测 TLR9 mRNA的表达变化规律。同法采集其治愈后3个月的外周EDTAK2抗凝静脉血行上述检测,作为相应指标的基准水平值。最终将成功采得治愈后3个月血液标本的前36例患者纳入最终研究。结果 AP患者的外周血PBMCs TLR9 mRNA相对含量高于其痊愈后3个月的基线水平（P＜0．05）。AP患者的外周血PBMCs TLR9 mRNA相对含量表达改变与胰弹性蛋白酶、促炎细胞因子水平呈正相关,与抗炎细胞因子无相关性。结论 AP患者的外周血PBMCs TLR9 mRNA表达显著升高并与促炎细胞因子表达同步上调,提示AP的发生、发展可能通过TLR9介导。