小鼠巨噬细胞α-肿瘤坏死因子基因表达的调节

The expression of tumor necrosis factor-alpha (TNF-alpha) mRNA in murine inflammatory peritoneal macrophages (M phi) was studied with a sensitive liquid hybridization method. Upon exposure to 10-1000 ng/ml of lipopolysaccharide (LPS), M phi were induced to express TNF-alpha mRNA in a dose-dependent manner. mRNA was detectable within 1 h after stimulation, peaked at about 2 h and then gradually declined. A 10 min treatment with LPS was enough to stimulate the maximal level of TNF-alpha mRNA, as determined in a 2 h period. Although calcium ionophore A23187 and macrophage activating factor (MAF) (both can activate M phi to mediate tumoricidal activity) did not induce TNF-alpha mRNA expression by themselves, they did act synergistically with LPS. Treatment of M phi with retinoic acid strongly inhibited LPS-induced TNF-alpha mRNA expression, whereas trifluoperazine had an opposite effect. Cycloheximide not only synergized with LPS but also induced TNF-alpha mRNA expression by itself. In contrast, actinomycin D completely blocked LPS-induced TNF-alpha gene activation. These findings indicate that LPS-induced TNF-alpha mRNA expression is not solely due to an increase in intracellular free calcium ion and is independent of the protein kinase C pathway of signal transduction. In addition, TNF gene activity may be regulated by short-lived protein repressor(s).     

Relationship between the increase of secretion of sTNFα induced by lipopolysaccharides and the enhanced expression of TACE mRNA in HL-60 cells and adhesive cells from human spleen

Summary In order to find out the potential modulators which influence the secretion of TNFα, the relationship between the amount of secreted TNFα(sTNFα) and the level of TNFα converting enzyme (TACE) gene expression was studied before and after the stimulation by lipopolysaccharides (LPS) to HL-60 cells and adhered cells isolated from human spleen using cytological and molecular biology techniques and methods (RT-PCR, Dot-blot hybridization, etc.). The experimental results showed that: (1) LPS could induce the increase of expression of TNFα mRNA and TACE mRNA, reaching the peak value at 6 h and 10 h respectively after addition of LPS into cell culture medium; (2) The anti-sense oligodeoxyribonucleotide (A-ODN) complementary to TACE mRNA sequence really inhibited the secretion of TNFα as a result of blocked translation of TACE gene; (3) Furthermore, it was also observed that RDQ, a kind of injection derived from Chinese traditional herb, had strongly inhibitory effects on the expression of TACE mRNA and secretion of sTNFα stimulated by LPS. The above results suggested that the TACE indeed involved in delivering and processing of pro-TNFα during the period of LPS stimulation and the study about the regulator/inhibitors of TACE gene expression would be very important to develop new types of therapy agents against toxic and side effects of sTNFα during the peroid of infection/inflammation to human body. This project was supported by a grant from Chinese National Natural Sciences Foundation of China (No. 39630320).     

RNA干扰对活化的小鼠巨噬细胞肿瘤坏死因子α表达的影响

目的：观察靶向小鼠肿瘤坏死因子α（TNF-α）基因的小干扰RNA（siRNA）在体外对小鼠巨噬细胞系RAW264．7表达TNF-α的抑制作用。方法：采用化学法合成针对TNF-α mRNA不同位点设计的3条siRNA序列（siRNA1～3）和1条带有荧光标记的BLOCK—IT^TM荧光Oligo（修饰的荧光标记的dsRNA,siRNA4）通过脂质体包裹后将其分别转染至小鼠巨噬细胞系RAW264．7,同时设立1个无任何靶基因的siRNA作为阴性对照（siRNA4）。荧光显微镜下观察siRNA的转染效率;用实时荧光定量PCR和酶联免疫吸附实验（ELISA）法分别检测siRNA对TNF-α的mRNA和蛋白表达的抑制作用。结果：内毒素刺激后6h,巨噬细胞表达TNF-α mRNA和合成分泌的TNF-α量均增加,于9～12h达高峰。利用荧光标记的Oligo观察到siRNA转染效率达72％～80％。siRNA1～4转染巨噬细胞后,siRNA2、3可见内毒素刺激的TNF-α mRNA（0．158±0．030、0．114±0．028）和TNF-α蛋白表达[（1355±348）pg／ml、（817±138）pg／m1]均明显少于未转染组[TNF-α mRNA0．294±0．147,蛋白（2104±32）pg／ml,均P〈0．05],其中siRNA3的抑制率非常显著,达61．2％（P〈0．01）。阴性对照siRNA4对细胞基因及蛋白表达无影响。结论：内毒素可刺激小鼠巨噬细胞TNF-α的合成。化学合成siRNA转染小鼠巨噬细胞能有效抑制TNF-α mRNA及蛋白的表达。     

Activation of p~(38) mitogen activated protein kinase induced by lipopolysaccharide and its role in TNF a gene expression

Mitogen--activatedproteinkinase(MAPKs)aremajormediatorsineukaryoticcellstotransductextracellularsignalsintocellularresponses['].Thereareatleast4subgroupsofMAPKsidentifiedinmammaliancells:extracellularsignal--regulatedkinase(ERK),c--JunN--terminalkinase(JNK)orstressactivatedproteinkinase(SAPK),ERKSorBMK1andp38['"].MAPKsareactivatedbyvariousstimulitomediatemanycellularprocessesorresponses,includingcellgrowth,death,cellcycle,inflammationandstressresponses,elc.[l.31.ActivationofMAPKs…     

丝裂原活化蛋白激酶通路对脂多糖诱导RAW264.7细胞肿瘤坏死因子α基因表达的协同调节作用

目的 探讨脂多糖（LPS）诱导RAW264.7细胞肿瘤坏死因子α(TNF-α)基因表达过程中丝裂原活化蛋白激酶（MAPK）通路的协同调节作用及其分子机制。方法 用蛋白激酶活性测定分析LPS刺激RAW264．7细胞引起的激酶活性变化;用报告基因技术和反转录聚合酶链反应（RT－PCR）方法研究LPS诱导的TNF－α基因转录的分子机制。结果 LPS刺激RAW264．7细胞可引起细胞外信号调节激酶1（ERK1）、c-Jun氨基末端激酶1（JNK1）和p38MAPK的一过性激活,用MAPK上游激酶的活性突变体分别转染RAW264．7均可不同程度地诱导TNF－α启动子转录活性;而且,这些MAPK通路激活诱导的TNF－α启动子转录活性表现出明显的协同效应;三种MPAPK的无活性突变体均显示出对LPS刺激引起的TNF－α启动子转录激活的抑制效应;RT－PCR的结果证实,ERK、JNK和p38MAPK的特异性抑制剂对TNF－αmRNA表达具有不同程度的抑制作用。结论 LPS刺激引起的TNF－α启动子转录活性增加,可能涉及了ERK、p38和JNK三条通路的激活;这些通路通过协同效应共同发挥对TNF－α基因表达的调控。     

脂多糖激活p38在诱导肿瘤坏死因子α基因表达中的作用

目的探讨防治内毒素休克的新方法,研究脂多糖（ＬＰＳ）诱导肿瘤坏死因子α（ＴＮＦα）表达的分子机制。方法用蛋白激酶活性测定检测ＬＰＳ刺激引起的激酶活性变化;共聚焦激光扫描技术显示ｐ３８激活移位;用反转录聚合酶链反应和报告基因系统研究ＴＮＦα基因转录的分子机制。结果发现ＬＰＳ刺激ＲＡＷ细胞引起ｐ３８激活并由胞浆移位至胞核。ＬＰＳ刺激引起ＴＮＦαｍＲＮＡ表达增加,而且由ＬＰＳ引起的ＴＮＦα的转录活性可被ｐ３８特异性抑制剂所抑制。结论激活的ｐ３８通过磷酸化转录因子增加ＴＮＦα基因转录活性,这是中毒性休克时ＴＮＦα生成增加的一个重要机制。ｐ３８是ＬＰＳ诱导ＴＮＦα基因表达的重要调节物质。     

健康高龄者外周血NK细胞活性及肿瘤坏死因子分泌的研究

目的 为探讨健康高龄者外周血自然杀伤细胞的活性及经诱导活化后肿瘤坏死因子（tumor necrotic fac-tor,TNF）的分泌。方法 采用乳酸脱氢酶释放法,以K562细胞和SGC-7901细胞为靶细胞测定外周血NK细胞的活性;白介素2（IL-2）与脂多糖诱导活化后的TNF的分泌活性测定以L-929细胞作为靶细胞,用MTT法测定其存活率表示。结果 与健康中青年组比较,高龄组人群的NK细胞对K562和SGC-7901细胞的杀伤活性均降低,但差异无统计学意义（P〉0.05）,经IL-2与LPS诱导的外周血淋巴细胞分泌的TNF活性均增高,差别有显著性（P〈0.01和P〈0.05）。结论 高龄组人群的外周血淋巴细胞经诱导分泌的TNF活力增高可能补偿了部份免疫功能的下降。     

人脾贴壁细胞中TNFα转换酶cDNA的克隆以及LPS对其表达的影响

根据TNFα转换酶（TNFαconvertingenzyme,TACE）cDNA序列和TACE分子结构特征设计并合成引物,采用逆转录PCR（RT-PCR）技术,首次从健康人脾贴壁细胞中扩增出TACEcDNA解整合素结构片段（disintegrindomain）,并用大肠杆菌脂多糖（LPS）刺激人脾贴壁细胞,观察其对TACEmRNA表达的调节作用。结果表明不同健康人脾贴壁细胞中均存在TACEmRNA的自然表达,并且LPS对该表达具有上调作用,其上调程度与LPS刺激时间呈正比关系。     

肿瘤坏死因子-α对少突胶质前体细胞的影响及其机制的研究

目的：探讨肿瘤坏死因子‐α（T N F‐α）对少突胶质前体细胞（O PC ）的作用及机制,为脑室周围白质软化（PV L ）的治疗提供策略。方法将分离纯化的OPC分为空白对照组、TNF‐α组、肿瘤坏死因子受体1（TNFR1）抗体组,将50 ng／mL TNF‐α作用于TNF‐α组、TNFR1抗体组,免疫荧光化学检测OPC的分化情况,四甲基偶氮唑盐（MTT）法检测3组细胞的相对活力, RT‐PCR检测细胞TNFR1的mRNA水平的表达情况。结果与空白对照组和TNFR1抗体组相比,TNF‐α组OPC的细胞活力明显下降（P＜0．05）,并且不能沿着少突胶质谱系细胞进行分化,即分化为原少突胶质细胞。TNF‐α组TNFR1的mRNA表达明显的上调,空白对照组和TNFR1抗体组的mRNA表达无明显的变化。结论 TNF‐α主要通过 TNFR1引起OPC的凋亡,抑制O PC分化。     

杀菌-通透性增强蛋白对分泌性中耳炎大鼠中耳黏膜TNF-α和IL-6表达的影响

目的：探讨杀菌-通透性增强蛋白（BPI）对分泌性中耳炎（OME）大鼠中耳黏膜TNF-α和IL-6 mRNA及蛋白表达的影响和意义。 方法：采用大鼠中耳腔注射脂多糖（Lipopolysaccharide,LPS）制备OME模型,动物分为LPS实验组和BPI治疗组。利用ELISA和RT-PCR方法检测中耳黏膜组织TNF-α和IL-6 mRNA及其蛋白的表达。结果：LPS实验组和BPI治疗组在第1、2天均有IL-6的表达,但LPS组表达量高于BPI组（P<0.05）。在第7天后两组均无表达;而TNF-α的表达贯穿于两组实验的始终,但LPS组表达量高于BPI组（P<0.05）。结论：IL-6和TNF-α在中耳炎病变的形成中起作用;BPI通过抑制细胞因子表达和分泌,减轻炎症刺激;BPI是一个潜在的治疗OME 的有效药物,可能预防慢性OME的发生。     

肺泡表面活性物质对肺挫伤后肺泡巨噬细胞功能的影响

目的 观察研究外源性猪肺表面活性物质(PPS)对肺挫伤大鼠肺泡巨噬细胞(AM)的吞噬功能和分泌功能的影响,探讨补充PPS对肺挫伤大鼠的治疗作用及其机制.方法 采用贴壁培养的方法,分离肺挫伤大鼠肺泡灌洗液中的AM,将分离的AM于普通培养液、含PPS(100μg/ml或200 μg/ml)的培养液、含LPS(20 μg/ml)的培养液或含LPS(20 μg/ml)+PPS(100 μg/ml或200 μg/ml)的培养液中培养2h后,采用真菌吞噬法测定各组细胞的吞噬功能;采用RTPCR方法测定各组细胞中TNF-α mRNA含量.结果 与对照相比,PPS单独作用对AM的吞噬功能和TNF-α mRNA含量无明显影响.经LPS刺激后AM的吞噬功能增强,TNF-α mRNA含量明显增高.PPS对LPS导致的吞噬功能增强无明显作用,但是能明显降低TNF-α mRNA含量.结论 PPS对AM的吞噬功能影响不大,但可以抑制TNF-α mRNA表达.     

肿瘤坏死因子基因和蛋白及受体在子宫内膜组织的表达

目的探讨子宫内膜癌组织肿瘤坏死因子（ＴＮＦ）基因和蛋白及受体的表达及意义。②方法分别采用非同位素原位杂交方法、免疫组化方法对４８例子宫内膜癌组织ＴＮＦｍＲＮＡ,ＴＮＦ蛋白及ＴＮＦ受体（ＴＮＦＲ）的表达进行检测。③结果子宫内膜癌组织中１６例ＴＮＦｍＲＮＡ呈阳性表达,均为间质的单个核细胞和平滑肌细胞,腺癌细胞无阳性表达,对照组未见阳性表达。１５例病人ＴＮＦ蛋白呈阳性表达,２５例ＴＮＦＲ呈阳性表达,对照组子宫内膜腺体也有ＴＮＦＲ阳性表达,两组比较差异无显著性（χ２＝２．４５,Ｐ＞０．０５）。ＴＮＦｍＲＮＡ与ＴＮＦ蛋白表达存在不一致性,ＴＮＦ与ＴＮＦＲ表达与组织学分化及肌层浸润深度差异均无显著性（χ２＝２．１７,０．８１,Ｐ＞０．０５）;ＴＮＦＲ蛋白表达明显高于ＴＮＦ,两者比较差异有极显著性（χ２＝４．２９,Ｐ＜０．０５）;ＴＮＦＲ在正常内膜表达与子宫内膜癌表达差异无显著性（χ２＝２．４５,Ｐ＞０．０５）。④结论子宫内膜癌间质细胞能够表达ＴＮＦｍＲＮＡ,并产生ＴＮＦ蛋白。     

Influence of Radix Isatidis on the Endotoxin-induced Release of TNF-α and IL-8 from HL-60 Cells

The effects of active antiendotoxin chemical fraction isolated from Radix Isatidis (fraction D) on TNF-α and IL-8 secretion in HL-60 cells induced by lipopolysaccharide (LPS) were studied. The appropriate densities of cell suspension and fraction D solution were determined by MTT colorimetric method. Fraction D and LPS were added to HL-60 cell suspension with three different methods respectively. The contents of TNF-α and IL-8 in the cultured supernatant induced by LPS were detected by using ELISA method. The results showed that the absorbance (A) was directly proportional to the number of cells and the linearity was good in the range from 0.25 × 105 to 2 ×105 cell/mL cell suspension. The fraction D significantly inhibited the oversecretion of TNF-α and IL-8 in HL-60 cells induced by LPS at the concentration of 7. 812 mg/mL which had no cytotoxicity. It was indicated that the antiendotoxin mechanism of the active fraction from Radix Isatidis was contributed to the inhibition of the oversecretion of cytokines induced by LPS.     

SpA 刺激单核巨噬细胞表达早期致炎因子的实验研究

目的：探讨金黄色葡萄球菌蛋白A（SpA）对单核巨噬细胞表达早期致炎因子 TNF‐α、IL‐1和IL‐6的影响。方法用一定浓度SpA与THP‐1细胞培养不同时间,用MTT法测定SpA对THP‐1细胞增殖的影响;用ELISA测定培养液中TNF‐α、IL‐1和IL‐6的水平;用RT‐PCR检测细胞TNF‐α、IL‐1和IL‐6相应mRNA的表达,并对结果进行统计学分析。结果 SpA对THP‐1细胞增殖的影响与其作用剂量有关。SpA可促进巨噬细胞分泌 TNF‐α、IL‐1和IL‐6及表达相应mRNA ,且呈一定剂量‐效应和时间‐效应关系。SpA刺激巨噬细胞分泌TNF‐α、IL‐1、IL‐6和表达相应mRNA均在12 h达高峰。SpA刺激组TNF‐α、IL‐1和IL‐6的表达和释放均显著升高（P＜0．01）。结论 SpA可明显促进单核巨噬细胞表达和分泌早期致炎细胞因子 TNF‐α、IL‐1和IL‐6。SpA在启动金葡菌性脓毒症及促进其发展中,具有不可忽视的作用。     

足叶乙甙诱导HL-60细胞凋亡及其bcl-2基因表达

研究HL-60细胞在足叶乙甙（Vp16）诱导的细胞凋亡中bcl－2基因表达，以说明药物致调亡的分子机制。方法：采用细胞存活率，形态改变，DNA梯形片段分析和原位缺口标记法，分析药物引起的细胞凋亡，以细胞原位杂交和免疫组化分析bcl－2mRNA及其蛋白的表达水平。结果：Vp16有效地诱导HL60细胞凋亡，12h形成DNA缺口，16h出现DNA梯形片段，24h形成调亡小体，48hDNA完全降解；细胞凋亡时其bcl－2mRNA和蛋白水平都较处理前明显降低。结论:bcl－2基因表达的下调在Vp16诱导的HL-60细胞调亡中起一定作用。     

解毒维康片对HL-60细胞体外增殖影响的研究

目的探讨中药复方制剂解毒维康片(JDWKP)对白血病细胞HL-60体外增殖的影响。方法采用血清药理学方法,以大鼠为实验动物,制备出JDWKP的含药血清,再采用噻唑蓝(MTT)比色法观察JDWKP对HL-60细胞增殖的影响。通过逆转录-聚合酶链式反应(RT-PCR)检测分析JDWKP对细胞凋亡相关基因的影响。通过酶联免疫吸附法(ELISA)法,从抗肿瘤新生血管形成的角度,检测JDWKP对HL-60细胞培养上清中血管内皮生长因子(VEGF)表达的影响。结果1)解毒维康片能明显抑制HL-60细胞的增殖并呈时间与剂量相关效应。2)解毒维康片在诱导细胞凋亡过程中随药物浓度的增加,凋亡相关基因Bcl-2 mRNA表达逐渐下调,C-myc mRNA表达逐渐增加。3)解毒维康片能明显抑制HL-60细胞VEGF的表达。结论解毒维康片可有效地抑制HL-60细胞的体外增殖,其机制与解毒维康片能诱导细胞凋亡,并下调VEGF的表达水平有关。     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.