NF-kappa B

摘要： 糖尿病是一组由多病因引起的以慢性高血糖为特征的代谢性疾病,是由于胰岛素分泌和利用缺陷所引起。糖尿病同时也伴随着多种并发症,近年来有研究表明核因子-kB (Nuclear factor-kappa B)在糖尿病并发症中起重要作用。

摘要： In order to investigate the roles of expression of Hedgehog and NF-κB signaling pathway signal molecules and in-flammatory cytokines in the alveolar epithelial typeⅡ cells (AECⅡ) infected with Mycobacterium tuberculosis(Mtb),the hu-man AECⅡ cell line A549 was infected with Bacillus Calmette-Guérin (BCG,attenuated strain) and H37Rv (international standard virulent strain) respectively,and the signal molecules of Hedgehog and NF-κB signaling pathway and inflammatory cytokines in A549 cells of immune response to Mtb was detected by qPCR,and Western blot. The results showed that, the expression of Shh,Ptch and Gli1 in Hedgehog signaling pathway and the phosphorylation of NF-κB in NF-κB signaling path-way was increased in A549 cells when infected with H37Rv and BCG,and at the same time,the expression of IL-8 and TNF-α was also upregulated. And the protein expression of Shh,Ptch,Gli1 and NF-κB in A549 cells infected with H37Rv was higher than that infected with BCG. The Hedgehog and NF-κB signaling pathway could be activated, and the expression of inflammatory cytokines could be promoted in AECⅡ cells when infected with Mtb. The activation level of the both signal-ing pathway infected with Mtb virulent strain was higher than that of infected with BCG strain.%探讨结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染Ⅱ型肺泡上皮细胞(Alveolar epithelial typeⅡcells,AECⅡ)的免疫应答过程中,Hedgehog与NF-κB信号通路及炎症细胞因子的表达变化规律.利用结核分枝杆菌BCG(Bacillus Calmette-Guérin,BCG,卡介苗,弱毒株)和H37Rv(国际标准强毒株)株分别感染人AECⅡ细胞株A549,利用qPCR和Western blot检测Hedgehog信号通路以及NF-κB信号通路相关因子和炎症细胞因子的表达变化.结果表明,H37Rv和BCG感染A549细胞后,Hedgehog信号通路中Shh、Ptch和Gli1与NF-κB信号通路中p-NF-κB mRNA的表达水平升高,同时,伴随着炎症细胞因子IL-8和TNF-α mRNA的表达水平也升高.并且,H37Rv感染A549细胞引起信号分子Shh、Ptch、Gli1和NF-κB蛋白的表达水平高于BCG. Mtb感染AECⅡ细胞都能激活Hedgehog信号通路和NF-κB信号通路并促进细胞的炎症因子分泌,强毒株感染对信号通路的活化强于弱毒株.

摘要： NF－κB转录因子家族广泛存在于真核细胞中，调控着细胞的生存、生长和凋亡过程，参与炎症、免疫性疾病及肿瘤等疾病的发生与发展。在大肠癌的疾病进程中，NF－κB作为炎性因子介导慢性结肠炎症向癌症的转化。同时，它又能通过调控细胞周期而抑制细胞的凋亡，促进大肠癌的发生，并在化学药物治疗的过程中通过抑制凋亡而介导大肠癌细胞的多药耐药。研究发现一些中西药制剂能够直接或间接作用于NF－κB，抑制大肠癌的发生以及治疗过程中出现的多药耐药，这为大肠癌的治疗提供了新的思路与靶点。%In eukaryotic cells ,NF-κB transcription factor family regulates many processes like cell sur-vival,growth,and apoptosis.It participates in the development of a variety of diseases ,including inflammatory,im-mune disease ,and cancer .As an inflammatory factor , NF-κB mediates the transformation of chronic colitis to cancer during the course of colorectal cancer .Furthermore,it could inhibit cell apoptosis through regulating cell cycles,which promotes the development of colorectal cancer and mediates the multidrug resistance of the tumor cells.Therefore,targeting NF-κB,a large number of preparations involved both Chinese and western medicine has been researched .The further research and the use of them may be an effective method to cure the colorectal cancer in clinical work .

摘要： 目的：针对人S100A10基因设计siRNA序列并构建shRNA表达载体；重组载体转染人关节软骨（HCs）细胞，观察细胞内S100A10基因表达及其对细胞内核转录因子κB（NF-κB）活性的影响。方法登陆美国国家生物技术信息中心（NCBI）数据库，根据人钙结合蛋白A10（S100A10）基因信息，设计3条针对其编码区（ CDS）区的小干扰RNA（ siRNA）序列。根据设计的siRNA序列，设计两条互补的shRNA序列，构建shRNA重组表达载体；阳离子脂质体法转染shRNA重组表达载体到HCs细胞，转染后48 h，收集细胞，提取细胞总 RNA及总蛋白，使用实时PCR（ RT-PCR）及蛋白质印迹法（ Western blot）的方法检测转染后细胞中S100A10 mRNA及蛋白相对含量；Western blot检测细胞内NF-κB的P65及P50亚基磷酸化。数据统计使用单因素方差分析。结果成功设计siRNA序列并成功构建了shRNA表达载体；mRNA及蛋白含量检测结果显示，三条siRNA序列对目的基因均有沉默效果，其中1号siRNA序列最为-有效，其转染后48 h，细胞内mRNA及蛋白含量相比较未转染组细胞，分别下降了74.2％和76.4％，差异均有统计学意义（t ＝5.21，P＜0.01）；HCs 细胞内S100A10基因沉默，能够明显抑制核转录因子NF-κB活性，与对照组比较，P65及P50亚基磷酸化明显减弱（ t＝3.02，P＜0.01）。结论 S100A10基因沉默可以有效抑制HCs细胞内NF-κB活性。%Objective To design siRNA sequences targeting human S100A10 gene, construct shRNA expression vectors with a plasmid expression vector and observe the effects on inhibition of NF-kappa B activity in human chondrocytes by transfecting with the recombinant vectors.Methods The human S100A10 gene sequence was obtained from National Center for Biotechnology Information ( NCBI) database.According to human S100A10 (NM_002966) gene information, three siRNA sequences were designed for the ( Sequence coding for amino acids in protein, CDS) of S100A10 gene.According to the designed siRNA sequences, two complementary DNA sequences were designed to construct recombinant shRNA expression vectors.Human articular chondrocytes ( HCs) were transfected with the recombinant shRNA expression vectors by cationic liposomes, and the transfection;48 h after the transfection, the cells were collected, the total RNA and total protein contents were extracted respectively.Relative S100A10 mRNA and protein levels in the transfected cells were detected by real-time PCR and Western blot, and NF-κB activity was detected by analysis of P65 and P50 subunit phosphorylation.One-way analysis of variance were used for data analysis.Results S100A10 gene information was found, siRNA sequences were designed and the shRNA expression vectors were constructed successfully.The results of the mRNA and protein showed that all three siRNA sequences had inhibitory effects on the target gene, and the 1st siRNA sequence was the most effective, which decreased mRNA and protein levels in the transfected group by 74.2%and 76.4%, respectively;the difference compared with the untransfected cells was statistically significant (t=5.21, P<0.01).Compared with the control group, P65 and P50 subunit phosphorylation decreased obviously (t =3.02, P <0.01) in the gene silencing HCs, which meant S100A10 gene silence could obviously inhibit the transcription factor nuclear factor kappa B activity in HCs.Conclusion S100A10 gene silence can effectively reduce the NF-kappa B activity in HCs.

摘要： 目的 探讨JSH-23阻断NF-κB信号通路后人舌鳞癌细胞Tca8113增殖和凋亡的情况及NF-κB信号通路相关凋亡抑制基因Bcl-2的表达变化,进而为临床舌鳞癌的干预和治疗寻找新的药物提供实验依据.方法 采用体外培养人舌癌Tca8113细胞的方法,经不同浓度JSH-23作用不同时间后,用MTT法、RT-PCR及Western blot等方法检测JSH-23对Tca8113细胞增殖、凋亡的影响.采用SPSS13.0统计软件对数据进行方差分析及t检验.结果 Tca8113细胞经JSH-23作用后,增殖受到明显抑制,20 μmol/L JSH-23作用48 h时,抑制作用最为显著,细胞生长抑制率达到60.45％.同时,Bcl-2凋亡抑制基因表达减少,与对照组相比,JSH-23作用48 h后,Bcl-2表达显著减少47.85％,差异有统计学意义(P＜0.05);Bcl-2蛋白含量也明显减少,作用48 h后,Bcl-2蛋白表达相对光密度值分别下降50.93％ (P ＜0.01).结论 JSH-23作为NF-κB信号通路抑制剂,可以明显抑制人舌鳞癌细胞的增殖,同时显著下调Bcl-2基因及蛋白的表达.

摘要： 表观遗传学使人们可以从生物学机制层面对环境影响表型的途径进行研究。通过定量分析的方法，研究了基因MLL1对可诱导转录因子NF-kappaB下游基因的调控作用，探索组蛋白H3K4甲基化酶参与基因表达的机制。结果表明，MLL1的调控主要是通过对NF-kappaB靶基因启动子进行H3K4甲基化来实现的，表观遗传学已经成为参与调节基因转录和细胞身份的关键因素。

摘要： 目的:探讨NF-KappaB(NF-κB)SiRNA修饰供体树突状细胞(DC)对异基因大鼠间皮肤移植术后免疫耐受诱导的效果,从而为该基因治疗方法应用于临床皮肤移植提供依据.方法 1.PGPU6/GFP/Neo-siRNA–NF-κB 表达载体的构建.2.体外SD大鼠骨髓干细胞体外定向诱导扩增培养DC后,将 NF-κB SiRNA及阴性对照SiRNA以LipofectamineTM2000 转染入大鼠DC,行 RT- PCR 和Western blot 检测.3.NF-κB SiRNA转染的DC 2×106个/只经尾静脉注入受鼠体内,分为对照组,未修饰DC组,NF-κB SiRNA基因修饰DC组,5天后行皮肤移植,移植5天后部分受鼠移植皮肤送病理检测,其余观察皮肤存活时间,TUNEL法检测移植皮肤淋巴细胞调亡情况.结果 构建的PGPU6/GFP/N-eo-siRNA-NF-κB通过脂质体转染到DC后,能明显抑制NF-κB基因和蛋白的表达.目的基因修饰组皮肤移植存活情况明显优于其它两组.结论 PGPU6/GFP/Neo-siRNA–NF-κB表达载体成功转染DC.NF-κB SiRNA修饰的供体DC预处理受体(经尾静脉注入),可明显延长移植皮肤的存活时间.

摘要： Objective To investigate the crosstalk among ROS, JNK and NFkappaB of C2C12 myoblast under cyclic strentch. Methods By using Flexercell strain system, cultured C2C12 myoblasts could be stretched by different stretch magnitudes. Cell counting and MTT was used to estimate the viability of the cells. Rugrescent dye was introduced to test ROS level. Fluorescent reporter and Western Blot were applied to teat NFkappaB activity and pJNK1 level and total JNK1 and the crosstalk of them.Results With the stretch magnitudes increased, consistent with the increased apoptosis, JNK1 were gradually activated, with no obvious changes of JNK1 expression. In contrast, NFkappaB activity increased with the extension rale increasing within the magnitudes no more than 10%, and it began to decline when the extension rate is over 15%. Conclusion JNK activation occurred only when ROS production accumulated sufficiently while NFkappaB was activated at a much lower threshold of ROS abundance.%目的 探讨不同应力作用下成肌细胞内ROS产生的量、JNK和NFkappaB的活化程度以及它们间的交互对话.方法 利用Flexercell细胞加力仪对体外培养C2C12成肌细胞施加不同频率、不同幅度的牵张应力.细胞计数、MTT实验检测应力条件下细胞的活性,通过荧光染料检测ROS的生成,荧光报告系统检洲NFkappaB的活性以及Western Blot检测磷酸化和总体JNK1的水平,分析三者之间的交互对话关系.结果 随着周期性应力的增加,细胞内ROS生成逐渐增加.随着应力的增大,细胞的凋亡逐渐增加,与此同时,JNK1活性逐渐增高,而JNK1表达水平并没有明显改变.与ROS生成量和JNK1活化逐渐增加不同,应力在10％以下时NFkappaB的活性随着应力的增大而增大,而当应力大于15％时,NFkappaB活性开始下降.结论 当ROS的量积累到一定的时候,JNK开始激活,而ROS的量较低的时候,NFkappaB被激活.

摘要： Objective To study the effects of 131 I on the expression and function of nuclear factorkappa B (NF-κB) in a DTC cell line and to investigate the possibility of combined therapy of 131 I and a NF-κB inhibitor ( Bay 11-7082).Methods By cell survival assay,effects of monotherapy and the combination of 131I and Bay 11-7082 were assessed.The absorbent values (As) were read at 450 nm.Cell survival rate was calculated using the following formula:(Atherpy -Ablank )/( Acontrol -Ablank ) × 100％.By DNA binding assay,nuclear proteins extracted at 6,24 and 48 h after treatment were added to the NF-κB DNA binding probes.The A was read at 450 nm.DNA binding rate was obtained according to the formula:(Atherapy -Ablank )/( A 1 -A blank ) × 100％.The changes of NF-κB protein expression after different therapies were detected by Western blot,and β-actin was used as control for semi-quantitative analysis.Paired t test,F test and q test were performed for statistical analysis.Results Significant differences of cancer cell survival rates were shown after monotherapy of different 131 I activities and different concentrations of Bay 11-7082 (F =281.07 and 173.84,both P ＜ 0.01 ).The cancer survival rates after 131 I treatment alone,Bay 11-7082treatment alone and combined therapy were (67.33 ± 5.65 ) ％,(61.83 ± 6.68 ) ％ and ( 36.67 ± 5.35 ) ％respectively (F =45.79,P ＜0.01 ).Also,significant reduction of cell survival rates were shown between the combined therapy and monotherapy of 131 I or Bay 11-7082 (q =8.20 and 8.35,both P ＜ 0.01 ).DNA binding assay showed 131I could greatly induce NF-κB binding rate,which reached a peak of (255.33 ±29.86 ) ％ at 24 h.Combined therapy suppressed NF-κB binding rates to 22.10％,39.75％ and 43.18％at 6,24 and 48 h respectively,which were significantly different compared with those of 131I monotherapy ( t:24.58,26.29,8.20,all P ＜ 0.01 ).The most suppressed NF-κB p65 binding rate ( 35.33 ± 8.21 ) ％was observed at 6 h,which was significantly different from that of control group ( t =28.98,P ＜ 0.01 ).The relative protein expression levels of p65 and p50 increased 6 h after 131I therapy.However,the combined treatment could effectively decrease the expression levels of p65 and p50.Both p65 and p50 were significantly different after different treatments ( F =100.93 and 193.55,both P ＜ 0.01 ).Statistical difference was found with p65 and p50 expression levels when compared 131 I and combined therapy with the control group ( q =4.75 and 8.22,both P ＜ 0.05 for 131 I ; q =30.80 and 22.83,both P ＜ 0.01 for combined therapy).Conclusions 131I could induce the expression and function activation of NF-κB in DTC cells.However,NF-κB inhibitor could effectively suppress the changes and enhance therapeutic effects synergistically.%目的 探讨131I对DTC细胞核因子κB(NF-κB)表达和功能的影响,以及1311和NF-κB抑制剂Bay 11-7082联合治疗的可行性.方法 用不同放射性浓度131I和不同浓度Bay 11-7082处理DTC细胞,加入四氮唑盐显色,测定450 nm的吸光度(A)值,用公式(A药物处理组- A空白)/(A对照组-A空白)×100％计算癌细胞存活率(A空白为空白孔的吸光度,A对照组为单纯细胞孔的吸光度);选择癌细胞存活率约60％左右的131I和Bay 11-7082浓度进行联合实验.将131I和Bay 11-7082作用于DTC细胞6、24和48 h后,提取核蛋白,分别与NF-κB结合序列探针相结合,测定450 nm的A值,NF-κB的DNA结合率=(A药物处理组-A空白)/(A对照组- A空白)×100％.用Western blot鉴定单用131I、Bay 11-7082和联合用药6h后NF-κB核蛋白相对表达水平的变化,并以β-actin作为内参对照进行半定量分析.用配对t检验、F检验和q检验进行统计分析.结果 细胞存活分析发现不同放射性浓度131 I和不同浓度Bay 11-7082处理后癌细胞的存活率不同(F=281.07和173.84,P均＜0.01);单用131I组、单用Bay 11-7082组与联合用药组癌细胞的存活率分别为(67.33 ±5.65)％、(61.83±6.68)％和(36.67±5.35)％,差异有统计学意义(F =45.79,P＜0.01),其中前2组分别与联合处理组相比q=8.20和8.35,P均＜0.01.DNA结合实验证实131I可以诱导癌细胞内NF-κB结合率增高,24h为(255.33±29.86)％(最高);而联合使用Bay 11-7082后,在6、24和48 h时间点能够抑制到相应刺激状态下NF-κB功能的22.10％、39.75％和43.18％,联合处理组与单用131I组比较,3个时间点差异均有统计学意义(t:24.58、26.29和8.20,P均＜0.01);6h时NF-κB p65功能受抑程度最明显,DNA结合率仅为(35.33±8.21)％,与对照组相比差异有统计学意义(t=28.98,P＜0.01).半定量分析:131I作用6h后p65和p50相对表达水平均升高,Bay 11-7082联合131I作用6h后两者的表达水平均受到明显抑制;不同给药方式作用后p65和p50的相对表达水平差异均有统计学意义(F=100.93和193.55,P均＜0.01),131I组和对照组两两比较,q=4.75和8.22,P均＜0.05,联合处理组与对照组两两比较,q=30.80和22.83,P均＜0.01.结论 131I会导致DTC细胞NF-κB表达增加、功能增强,联合使用NF-κB抑制剂可以抑制这种改变,获得协同疗效.

摘要： 目的 利用NF-kappaBp50基因敲除小鼠建立子宫内膜异位症模型探索NF-KappaBp50亚单位在子宫内膜异位癌发生发展中的作用.方法 利用子宫内膜移植法建立P50基因敲除小鼠及野生型对照小鼠的子宫内膜异位症动物模型,检测建模后异位病灶大小、免疫组化检测异位病灶、在位内膜以及阴道磷酸化p65 (p-p65)、PKCepsilon和TRPV1的表达.结果 p50基因敲除小鼠异位病灶明显减小,并且,p50基因敲除小鼠异位病灶,在位内膜,以及阴道的p-p65 and PKCepsilon的表达也下降.结论 NF-kappaB p50亚单位在子宫内膜异位症的发生发展中起重要作用,可能是潜在的治疗靶标.