小檗碱对人宫颈癌细胞生物学行为及裸鼠成瘤的影响

目的:研究小檗碱对人宫颈癌细胞生物学行为及裸鼠成瘤的影响。方法:将不同浓度小檗碱(5、20、40mg/L)作用于HeLa及CaSki细胞,MTT法检测HeLa及CaSki细胞的增殖能力;光学显微镜观察细胞形态变化;流式细胞仪检测细胞凋亡率;侵袭及迁移实验研究细胞侵袭和迁移能力;Western blot法检测细胞中Bcl-2、Bax及MMP-9表达。显微镜下观察HeLa及CaSki细胞中NF-κB p65蛋白阳性表达情况并计算阳性表达率。建立宫颈癌裸鼠模型,腹腔给予不同浓度小檗碱(10、30、50mg/L),观察裸鼠肿瘤体积变化及肿瘤组织中Bcl-2、Bax、MMP-9、NF-κB p65阳性表达情况。结果:小檗碱对HeLa及CaSki细胞增殖的抑制作用呈剂量和时间依赖性,细胞抑制率随着作用时间及作用剂量的增加而升高。小檗碱促进HeLa及CaSki细胞凋亡、降低细胞侵袭迁移能力。随着小檗碱浓度增加,HeLa及CaSki细胞内Bax蛋白表达量增加,而Bcl-2、MMP-9、NF-κB p65蛋白表达量显著降低。小檗碱能明显降低肿瘤体积,且呈剂量依赖性降低Bcl-2、MMP-9、NF-κB p65蛋白表达,提高Bax蛋白表达。结论:小檗碱能有效抑制宫颈癌增殖及侵袭转移并诱导其发生凋亡,其作用机制可能与降低Bcl-2、MMP-9、NF-κB p65表达及提高Bax表达有关。     

百里醌对人卵巢癌细胞株OVCAR-3增殖和凋亡的影响

目的:探讨百里醌对人卵巢癌细胞株OVCAR-3的作用及相关机制。方法:采用不同浓度(0、10μmol/L、20μmol/L和40μmol/L)百里醌处理OVCAR-3细胞。MTT法检测细胞增殖活性,流式细胞仪检测细胞凋亡。RT-PCR和ELISA检测炎症因子白介素-6(IL-6)、白介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)表达水平。Western blot法检测JAK2、STAT3、p-JAK2、p-STAT3、p-p65和p65表达。结果:百里醌呈浓度依赖性抑制OVCAR-3细胞的增殖和诱导OVCAR-3凋亡,降低IL-6、IL-1β、TNF-α、p-JAK2、p-STAT3和p-p65表达,而对JAK2、STAT3和p65表达无影响。结论:百里醌可抑制OVCAR-3细胞的增殖和诱导凋亡,其作用机制与抑制JAK2/STAT3/p65介导的炎症相关。     

白细胞介素-1β对人早孕期细胞滋养层细胞核因子-κB的激活机制

目的:研究核因子-κB(NF-κB)在离体培养的人早孕期细胞滋养层细胞激活的机制。方法:离体培养人早孕期细胞滋养细胞,以白细胞介素-1β(IL-1β)和NF-κB抑制剂——吡咯烷二硫代氨基甲酸酯(PDTC)作为处理因素,通过免疫荧光检测NF-κB的亚基p65的核转位;凝胶电泳迁移率改变分析(EMSA)法检测NF-κBDNA结合活性。结果:①IL-1β作用30min后细胞滋养细胞出现了p65的核转位;②细胞滋养细胞NF-κBDNA结合活性显著增高;PDTC能显著降低NF-κBDNA结合活性。结论:IL-1β在离体培养的人早孕期细胞滋养细胞可引起NF-κB的短暂激活,PDTC可以抑制IL-1β诱导的NF-κB的激活。这为进一步研究激活或抑制NF-κB是否能调节NF-κB的靶基因、尤其是在胚胎着床和妊娠发挥重要作用的靶基因提供了理论基础。     

白花蛇舌草黄酮和多糖对与肿瘤相关巨噬细胞共培养的人子宫内膜癌Ishikawa细胞的影响

目的研究白花蛇舌草黄酮和多糖对与肿瘤相关巨噬细胞(tumor associated macro-phages,TAMs)共培养的人子宫内膜癌Ishikawa细胞的作用。方法噻唑蓝比色法(MTS)检测白花蛇舌草黄酮及多糖对人子宫内膜癌Ishikawa细胞株的作用;酶联免疫吸附法(ELISA)测定最佳量效时间药物作用后细胞上清液中白介素-10(IL-10)水平;流式细胞仪检测TAMs特殊表型标志物的表达;蛋白印迹法(Western blot)检测共培养体系核转录分子-κB p65(NF-κB p65)的表达。结果白花蛇舌草多糖无抑制Ishikawa细胞增殖的作用(P0.05)。白花蛇舌草黄酮可显著抑制Ishikawa细胞的增殖,抑制作用呈剂量依赖性,以800μg/mL抑制作用最佳(P0.05),而24、48h比较,差异无统计学意义(P0.05);并能降低细胞培养上清液中IL-10的水平(P0.01)及下调TAMs细胞中NF-κB p65的表达(P0.01)。结论白花蛇舌草抗人子宫内膜癌Ishikawa细胞作用主要依赖于白花蛇舌草黄酮,它能减少细胞产生IL-10和阻断细胞NF-κB p65信号通路的表达,从而抑制与TAMs共培养的Ishikawa细胞的增殖。     

TLR3、NF-κB和IL-1β在宫颈上皮内瘤变及宫颈癌中的表达及意义

目的:检测TLR3、NF-κB及IL-1β在宫颈上皮内瘤变(CIN)与宫颈癌中的表达,探讨3者的相关性及其与临床病理参数的关系。方法:收集中国医科大学附属盛京医院2014年1月至2014年12月妇科手术标本共120例,其中宫颈鳞癌60例,CIN 40例,正常宫颈20例。采用免疫组化法检测TLR3、NF-κB及IL-1β表达。结果:正常宫颈、CIN、宫颈癌中TLR3阳性表达率分别为100%、75%、23.23%,NF-κB阳性表达率为0%、60%、90%,IL-1β阳性表达率为0%、70%、90%,各组间差异均有统计学意义(P0.005)。TLR3表达与宫颈癌临床分期相关(P0.05);NF-κB及IL-1β与临床分期、病理分级及淋巴结转移相关(P0.05)。TLR3与NF-κB、IL-1β表达均呈显著负相关(rs=-0.478,P0.01;rs=-0.45,P0.01);NF-κB与IL-1β表达呈显著正相关(rs=0.589,P0.01)。结论:宫颈癌中TLR3呈低表达,NF-κB与IL-1β均呈高表达,可为TLR激动剂治疗宫颈癌提供一定的理论依据。     

脱氧寡核苷酸对卵巢癌细胞核转录因子-κB活性和下游细胞分子表达的影响

目的 探讨核转录因子(NF)-κB诱捕物脱氧寡核苷酸(ODN)对人卵巢癌细胞株SKOV3细胞NF-κB活性和下游细胞分子如细胞间黏附分子1(ICAM-1)、血管内皮生长因子(VEGF)、尿激酶型纤溶酶原激活剂(uPA)表达的影响。方法 SKOV3细胞转染NF-κB诱捕物ODN后,用白细胞介素113(IL-1β)刺激6、12、24、48 h,采用凝胶电泳滞后实验测定NF-κB DNA结合活性,采用RT-PCR技术检测ICAM-1、VEGF、uPA mRNA表达水平,采用酶联免疫吸附实验(ELISA)检测VEGF蛋白表达水平。结果 (1)SKOV3细胞表达NF—κB DNA结合活性,IL-1β刺激后其活性上升,在转染NF—κB诱捕物ODN后SKOV3细胞NF-κB DNA结合活性被显著抑制,刺激6、12、24、48 h的抑制率分别为99.6％、86.4％、80.1％、21.6％。各时间点间比较,差异均有显著性(P<0.05～0.01)。(2)SKOV3细胞表达ICAM-1、VEGF、uPA mRNA和VEGF蛋白,IL-1β刺激后其表达率上升,转染NF-κB诱捕物ODN后其表达率下降。结论NF—κB诱捕物ODN转染SKOV3细胞后可能通过抑制NF-κB活性,从而抑制ICAM-1、VEGF、uPA的表达。NF—κB诱捕物ODN有望应用于卵巢癌的基因治疗。     

核转录因子-κB与妊娠期糖尿病发病机制的研究

目的:通过调控核转录因子-κB(NF-κB)在细胞信号转导过程中的状态,研究NF-κB与妊娠期糖尿病(GDM)炎症反应发生发展以及终末炎症因子之间的关系,探讨NF-κB在GDM的炎症反应发生中的作用。方法:选择正常孕妇(W组)和GDM孕妇(G组)各30例,检测血清中脂多糖(LPS)的量。将每个样本各分为4个处理组,分别进行未处理、加入LPS(1μg/ml)刺激、加入NF-κB抑制剂毗咯烷二硫代氨基甲酸(PDTC)(50 mmol/L)干预、加入PDTC预处理1小时后再给予LPS刺激,并分别标记为W(G)、W(G)+L、W(G)+P、W(G)+LP组。采用Western blot法检测各组NF-κB蛋白的活化量,采用ELISA法检测各组肿瘤坏死因子(TNF-α)、白介素-1(IL-1)、白介素-10(IL-10)的水平,并行Pearson相关分析。结果:1GDM孕妇组与正常孕妇组比较,血清中LPS的量明显增加(P0.05)。2两组组内的比较:加入LPS刺激后,W(G)+L组、W(G)+LP组中细胞内NF-κB蛋白活化量和炎症因子TNF-α、IL-1、IL-10水平均明显高于W(G)组(P0.05);使用PDTC后,W(G)+LP组中细胞内NF-κB蛋白活化量和炎症因子TNF-α、IL-1、IL-10水平均明显低于W(G)+L组(P0.05)。3GDM组各处理组中NF-κB蛋白活化量和炎症因子TNF-α、IL-1、IL-10的水平均较正常孕妇组中相同处理组明显升高(P0.05)。4Pearson相关分析示:两组细胞中NF-κB的活化量与终末炎症因子TNF-α、IL-1、IL-10水平呈正相关关系(P0.05)。结论:NF-κB作为细胞内多条传导通路的汇合点,介导信号向胞核内转导,通过调控终末炎症因子水平,参与了GDM炎症反应的发生,并可能在其中起着类似"开关点"的作用。     

RAGE/NF-κB通路对妊娠期糖尿病大鼠胎盘滋养细胞凋亡及胎盘屏障功能改变的影响

目的:探讨晚期糖基化终末产物受体(RAGE)/核转录因子-κB(NF-κB)通路对妊娠期糖尿病(GDM)大鼠胎盘滋养细胞凋亡及胎盘屏障功能改变的影响。方法:将受孕雌性SD大鼠随机分为正常妊娠组、模型组(GDM组)、RAGE抑制剂组(FPS-ZM1组)、RAGE抑制剂阴性对照组(FPS-ZM1 NC组)、RAGE激活剂组(RAGE组)、RAGE激活剂阴性对照组(RAGE NC组),每组20只。除正常妊娠组外,其余各组均建立GDM模型,并在妊娠第5天开始给药,连续给药2周后,比较各组空腹血糖(FBG)、晚期糖基化终末产物(AGE)、胎盘重量、胎鼠重量、胎盘通透性、胎盘滋养细胞凋亡率;胎盘组织RAGE、NF-κB、紧密连接蛋白(TJ)、囊膜蛋白-2(Syncytin-2)、调解超家族蛋白2 A(MFSD2 A)、抗凋亡蛋白B淋巴细胞瘤-2(Bcl-2)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)表达水平的差异。结果:与正常妊娠组相比,GDM组大鼠FBG及AGE水平、胎盘重量及胎鼠重量、胎盘组织伊文思蓝(EB)含量、RAGE和NF-κB阳性表达水平、IL-6、TNF-α、Bcl...     

炎症因子对人颗粒细胞凋亡调控基因P53、Bax、Casepase-3、Bcl-2、Survivin表达的影响

目的:探讨炎症因子对人颗粒细胞凋亡调控基因P53、Bax、Casepase-3、Bcl-2、Survivin表达的影响。方法:选择行体外受精-胚胎移植(IVF-ET)治疗的非多囊卵巢综合征(PCOS)患者50例,提取卵泡液中颗粒细胞进行体外培养,随机分为炎症因子处理组和对照组,处理组分别加入5nmol/L白细胞介素-1(IL-1)、白细胞介素-6(IL-6)、肿瘤坏死因子(TNFα),比较各组颗粒细胞的增殖速率及凋亡调控基因的表达是否存在差异。结果:IL-1、IL-6、TNFα处理组体外培养48小时后颗粒细胞增殖速率低于对照组(P0.05);处理组促凋亡基因P53、Bax、Casepase-3的表达水平高于对照组(P0.05),而抗凋亡基因Bcl-2、Survivin的表达水平低于对照组(P0.05)。结论:炎症因子IL-1、IL-6、TNFα抑制人颗粒细胞增殖,并诱发颗粒细胞凋亡。     

核因子-κB在小鼠胚泡着床前后子宫表达的初步研究

探讨核因子-κB(NF-κB)在小鼠胚泡着床过程中是否存在的短暂激活。方法:采用免疫组织化学检测小鼠子宫内膜NF-κB的亚基p65蛋白的定位和表达,Western印迹法检测子宫内膜NF-κB的抑制性蛋白IκBα的降解。结果:p65在小鼠胚胎着床前后子宫内膜均有表达,其部位主要在腔上皮细胞和腺上皮细胞的胞浆部分,基质细胞和子宫肌层也有微弱表达。妊娠后子宫内膜p65的表达增加,妊娠d5达最高值,d8仍然维持较高水平;而d5IκBα的降解最明显。结论:NF-κB可能通过其短暂激活来参与调节小鼠胚泡着床。     

NF-κB、MIF在子宫内膜异位症的表达及意义

目的:探讨NF-κB、MIF与子宫内膜异位症(EMs)发病的关系,及在子宫内膜异位症表达的相关性。方法:采用免疫组化(SABC法)分别检测NF-κB、MIF在35例EMs在位内膜、60例EMs异位内膜及30例正常子宫内膜(对照组)中的表达;ELISA法检测TNF-α、PDTC+TNF-α干预后子宫内膜基质细胞(ESC)上清液中NF-κB、MIF的浓度,并统计分析检测结果。结果:(1)NF-κB、MIF在3组内膜中均有表达,以异位内膜的表达最强;(2)NF-κB、MIF的表达与月经周期无关,其在3组内膜的表达有相关性,相关系数为0.875、0.882、0.927;(3)NF-κB、MIF在经TNF-α、PDTC+TNF-α处理过的ESC上清液中有表达,TNF-α促进二者表达,而PDTC可抑制此作用,且两者表达有相关性,相关系数为0.947。结论:NF-κB、MIF在EMs内膜高表达,MIF可能通过NF-κB途径在EMs发病机制中起作用。     

来曲唑对子宫内膜异位症大鼠TNF-α、IL-6、NF-κB表达的影响

目的:研究不同剂量来曲唑对大鼠子宫内膜异位症(EMs)模型的治疗作用及其对异位病灶中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、核转录因子-κB(NF-κB)表达的影响。方法:将建模成功的大鼠随机分为来曲唑组(低、中、高剂量组)、米非司酮组及生理盐水空白对照组。灌胃4周后采用自定义疼痛评分及阴道痛觉过敏实验评价大鼠疼痛情况;观察异位内膜生长情况;放射免疫法测定血清E2、P、T水平;应用免疫组化法检测TNF-α、IL-6、NF-κB蛋白表达;用逆转录聚合酶链反应(RT-PCR)技术检测TNF-α mRNA、IL-6 mRNA、NF-κB mRNA的含量。结果:来曲唑各组异位病灶体积明显缩小或萎缩,与空白对照组比较差异均有统计学意义(P<0.05);来曲唑中、高剂量组与低剂量组、米非司酮组相比较,异位病灶体积缩小更明显(P<0.05)。治疗后各组大鼠血清E2、P水平均无显著差异(P>0.05),但来曲唑组大鼠血清T水平升高,且呈剂量依赖性(P<0.05)。来曲唑治疗后模型鼠疼痛评分降低,与对照组比较差异有统计学意义(P<0.05),且高剂量来曲唑组较米非司酮组疼痛减轻更明显(P<0.05)。在20、30、40mmHg阴道压力的刺激下,米非司酮组和来曲唑组大鼠的逃避率较空白组显著降低(P<0.05),较少发生痛觉过敏,而在压力为5、10mmHg时,各组间逃避率的差异无统计学意义(P>0.05)。各治疗组异位内膜中TNF-α、IL-6、NF-κB蛋白和mRNA的表达,均较空白组显著降低(P<0.05),尤以高剂量来曲唑组蛋白表达最低。NF-κB与TNF-α、IL-6的表达均呈正相关(P<0.05)。结论:来曲唑对大鼠异位病灶体积影响明显,可缓解EMs引起的疼痛。这可能是通过降低异位病灶局部雌激素水平,降调TNF-α、IL-6、NF-κB在异位内膜组织中的表达而发挥治疗作用的。     

STOX1在子痫前期滋养细胞炎症反应中的作用研究#br#

Objective?To investigate the STOX1 (Storkhead box1) in the inflammatory response of trophoblast cells simulated by human choriocarcinoma cell line JEG3 cells. Methods?The human choriocarcinoma cell line JEG3 cell line was used to mimic trophoblast cells, and a stable transitional cell line model with overexpression/knockdown of STOX1 gene was constructed. 0.05 mmol/L aspirin was added to the experimental and control groups, respectively. Cell migration ability was measured by Transwell, and apoptosis was detected in each group by flow cytometry, and real-time quantitative PCR and protein immunoblotting were used to detect the expression of cell-associated inflammatory, hypoxic and apoptotic factors, biological behavior, and the relative expression of mRNA and protein of molecular biology. Results?Hypoxia-inducible factor α(HIF-1α), endothelial-type nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), and apoptosis B lymphocytoma-2 (Bcl-2) were significantly reduced in the overexpression STOX1 trophoblast group (P<0.01); meanwhile, inflammatory response-related factor nuclear factor κB (NF-κB), tumor necrosis factor α (TNF-α), interleukin 8 (IL-8) and cysteine protease-3 (Caspase-3) were significantly increased at both gene and protein levels (P<0.05). The expression of hypoxia-inducible genes HIF-1α (P<0.01), eNOS (P<0.05), and iNOS (P<0.001) and anti-apoptotic genes Bcl-2 (P<0.01) was elevated after knockdown of STOX1, while inflammatory responses NF-κB (P<0.05), TNF-α (P<0.01), IL-8 (P<0.01), and Caspase-3 (P>0.05) levels were reduced. Conclusion?STOX1 induces inflammatory response, promotes hypoxia and apoptosis-related gene expression in JEG3 cell line, aspirin may inhibit the effect of STOX1, reduce apoptosis in JEG3 cells, decrease inflammatory response and improve hypoxia in trophoblast cells, and knockdown of STOX1 has synergistic effect with the use of aspirin.     

17β-Estradiol inhibits TNF-α-induced proliferation and migration of vascular smooth muscle cells via suppression of TRAIL

Atherosclerosis is an inflammatory disease and involves migration of vascular smooth muscle cells (VSMCs). Estrogen inhibits VSMCs migration, while the underlying mechanism remains to be revealed. Recent years, there is emerging evidence showing that TNF-related apoptosis-inducing ligand (TRAIL) increases proliferation and migration of VSMCs. In this study, we investigated the regulatory effect of estrogen on TRAIL expression in VSMCs. TNF-α greatly enhanced TRAIL protein expression and stimulated VSMCs proliferation and migration. This effect was partially inhibited by the addition of TRAIL neutralizing antibody, suggesting that TRAIL is important in TNF-α-induced migration. 17β-estradiol (E2) inhibited TRAIL expression under TNF-α stimulation in a time- and concentration-dependent manner. This effect was was mimicked by ERα agonist 4′,4″,4?-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), but not ERβ agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN), indicating that ERα is involved in this action. TNF-α led to nuclear factor kappa B (NF-κB) p65 phosphorylation and the inhibitor pyrrolidine dithiocarbama (PDTC) inhibited TRAIL expression, suggesting that NF-κB signaling is crucial for TARIL production. E2 suppressed p65 phosphorylation in VSMCs and the overexpression of p65 subunit reversed the inhibitory effect of E2 on TRAIL expression and cell proliferation and migration. Taken together, our results indicate that E2 inhibits VSMCs proliferation and migration by downregulation of TRAIL expression via suppression of NF-κB pathway.     

Tumor necrosis factor-alpha activates nuclear factor-kappaB but does not regulate progesterone production in cultured human granulosa luteal cells

Background. The role of tumor necrosis factor-α (TNF-α) in granulosa luteal cell function and steroidogenesis is still controversial. Our aim was to examine the steroidogenic response, together with the simultaneous expression and activation of nuclear factor-κB (NF-κB), in cultured human granulosa luteal cells (GLCs) following administration of TNF-α.

Materials and methods. This prospective controlled study was conducted in the Human Reproduction Division at the Institute of Maternal and Child Research, Faculty of Medicine, University of Chile and the San Borja Arriarán Hospital, National Health Service, Santiago, Chile. GLCs were obtained from aspirates of follicles from women undergoing in vitro fertilization (IVF). Thirty-two women undergoing IVF for tubal-factor and/or male-factor infertility participated in this study. Protein levels of NF-κB, the NF-κB inhibitor IκBα and steroidogenic acute regulatory protein (StAR) were determined by Western blot and localization of NF-κB was studied by indirect immunofluorescence. Progesterone production was determined by radioimmunoassay.

Results. TNF-α did not affect the expression of StAR protein or the synthesis of progesterone. NF-κB was expressed in the GLCs and activated by TNF-α, resulting in degradation of IκBα and mobilization of the p65 NF-κB subunit into the nucleus.

Conclusions. These results indicate that TNF-α did not modulate steroidogenesis in cultured human GLCs. However, NF-κB was activated by TNF-α. Therefore the activation of NF-κB via the TNF-α pathway is likely associated with other preovulatory granulosa cell processes important for human ovarian function.     

Different levels of IL-1α, IL-6, TNF-α, NF-κB and PPAR-γ in monocyte cultures exposed by plasma preeclampsia and normotensive pregnancy

ObjectiveTo determine different levels of proinflammatory cytokines IL-1α, IL-6, TNF-α, nuclear NF-κB p50 and PPAR-γ in monocyte cultures exposed to normotensive pregnancy plasma compared with those exposed to preeclamptic plasma.Study designThe study involved primigravidae with preeclampsia (12) and normotensive pregnancy (12) in which their blood plasma was given to monocyte cultures from isolated PBMC of healthy and non-pregnant women. They were divided into 2 groups, the first group was incubated for 24 h and the second one was incubated for 48 h (step 1). The levels of IL-1α, IL-6, TNF-α, and nuclear NF-κB p50 as well as PPAR-γ of both groups were subsequently measured and compared (step 2). Data were analyzed to determine the differences and interaction between both treatment groups using one-way ANOVA.ResultsThere was a significantly different level (p-value <0.05) of IL-1α in monocyte cultures incubated for 24 h compared with those incubated for 48 h, as shown in step 1 of the study. Meanwhile, step 2 of the study found significantly different levels of IL-1α, IL-6, TNF-α, and NF-κB p50 in monocyte cultures exposed to preeclamptic plasma compared with those exposed to normotensive pregnancy, in which the latter showed higher levels. Both groups also showed decreased levels of PPARγ, in which monocyte culture exposed to severe preeclamptic plasma (p value <0.05).ConclusionPreeclamptic plasma significantly increased levels of proinflammatory cytokines IL-1a, IL-6, and TNF-a in monocyte cultures. This condition was consistent with the increasing of NF-κB p50 and decreasing of PPARγ.