siRNA阻断NF-κB信号通路抑制胃癌SGC-7901细胞的增殖及侵袭

目的采用RNA干扰(RNA interference,RNAi)技术下调胃癌细胞株SGC-7901中NF-κB p65基因的表达,探讨其对肿瘤细胞增殖活性和侵袭能力的影响。方法利用阳离子脂质体LipofectamineTM2000将化学合成的人NF-κB p65的小干扰RNA(small interference RNA,siRNA)转染入胃癌细胞株SGC-7901中。采用RT-PCR法测定细胞内NF-κB p65mRNA的表达;酶联免疫吸附测定法(ELISA)检测NF-κB亚单位p65的DNA结合活性的改变;采用MTT法测定细胞增殖活性的变化;利用Transwell侵袭实验检测细胞体外侵袭能力的变化。结果与对照组比较,化学合成的人NF-κB p65siRNA能有效地抑制SGC-7901细胞中NF-κB p65mRNA的表达(P<0.05);RelAsiRNA组的p65亚单位与DNA结合活性明显低于对照组(P<0.05),并且RelA siRNA组中SGC-7901细胞的体外侵袭力减弱,增殖活性降低。结论特异的siRNA可以有效阻断NF-κB信号通路,影响人胃癌细胞的增殖活性和体外侵袭能力。     

AP1与NF-κB对PPARδ基因的转录调控作用

目的研究在过氧化物酶体增长因子活化受体δ(PPARδ)表达中的相关调控因子。方法用RT-PCR扩增人类PPARδ启动子序列,通过Deletion及观测各重组体转染活性,通过电泳迁移率变更分析(EMSA)、突变等方法确定核转录因子激活蛋白1(AP1)及NF-κB对PPARδ表达的作用。结果 Deletion及重组转染后发现AP1及NF-κB因子各自结合位点突变后转染活性明显降低,同时突变其转染活性无明显变化。结论 AP1及NF-κB均可以增强PPARδ的表达活性。     

利多卡因对脑缺血再灌注损伤大鼠海马组织细胞间黏附分子-1及核转录因子-κB表达的影响

目的观察利多卡因对大鼠脑缺血再灌注损伤后海马组织细胞间黏附分子-1(ICAM-1)及核转录因子-κB(NF-κB)蛋白和mRNA表达的影响,探讨利多卡因脑保护作用的机制。方法雄性SD大鼠32只,随机分为假手术组(A组)、缺血再灌注组(B组)、利多卡因小剂量组(C组)和利多卡因大剂量组(D组),缺血前10 min腹腔注射。脑缺血10 min再灌注24 h时,断头处死大鼠。用RT-PCR技术检测海马组织ICAM-1及NF-κB mRNA的表达,用免疫组织化学、蛋白印记(Western blot)方法检测ICAM-1及NF-κB蛋白表达情况。结果脑缺血再灌注后海马组织ICAM-1和NF-κB mRNA及蛋白表达水平增高,利多卡因可下调ICAM-1及NF-κB表达;缺血再灌注后,海马区神经元出现明显坏死,利多卡因可减轻海马区神经元损伤。结论利多卡因可能通过抑制ICAM-1与NF-κB基因表达而对脑缺血再灌注损伤起到一定的保护作用。     

CpG寡核苷酸对人外周血单个核细胞的免疫刺激作用

目的　观察CpGODN、GM CSF、CD40 L对正常人外周血单个核细胞 (PBMC)的免疫刺激作用 ,以期筛选出一种高效的疫苗佐剂。方法　用CpGODN 2 0 0 6、GM CSF、CD40 L刺激正常人PBMC ,48h后采用ELISA法检测培养上清液中IFN γ、IL 12及IL 10水平 ;应用免疫荧光法检测PBMC上CD40、CD86、HLA DR的表达。结果　CpGODN 2 0 0 6能诱导正常人PBMC分泌较高水平的IL 12、IFN γ ,抑制IL 10的分泌 ;上调PBMCCD40、CD86、HLA DR表达的作用最强。结论　CpGODN 2 0 0 6是一种免疫刺激作用强大的新型佐剂 ,可用于多种疫苗的制备。     

血红素氧合酶-1对肝硬化大鼠肝脏缺血再灌注损伤的保护作用

目的探讨血红素氧合酶-1(HO-1)对肝硬化大鼠肝脏缺血再灌注损伤的保护作用。方法将大鼠随机分为:正常组(N组)、硬化组(LC组)、假手术组(S组)、手术组(I/R组)和给药组(I/R+hemin组)。除正常组外其余各组大鼠皮下注射体积分数为40%的CCl4溶液,每周2次,11周后形成肝硬化模型;停药1周后进行肝脏缺血再灌注;于再灌注后6 h检测肝功能、抗氧化能力,免疫组织化学检测核转录因子(NF-κB)、天冬半胱酶(caspase-3)和HO-1蛋白的表达。结果给予hemin诱导HO-1高表达能减轻肝脏缺血再灌注后肝细胞损伤,提高锰超氧化物歧化酶(MnSOD)水平,降低caspase-3、NF-κB阳性表达。结论HO-1能有效地减轻肝硬化肝脏缺血再灌注损伤,其机制可能与提高MnSOD水平、降低caspase-3及NF-κB表达有关。     

Ⅰ型胶原及NF-κB与动脉粥样硬化关系的实验研究

目的探讨动脉粥样硬化(AS)时血脂成分的改变以及Ⅰ型胶原mRNA与其蛋白、NF-κBmRNA与其蛋白在兔AS发生中的作用及其机制,为临床预防及治疗AS斑块提供新的理论依据。方法采用HE、免疫组化及原位杂交法染色,光镜观察AS病变,并应用CMIAS真彩色医学图像分析系统分析;检测40只食饵性AS新西兰兔血脂成分的改变及血管壁的病理变化、Ⅰ型胶原mRNA与其蛋白、NF-κBmRNA与其蛋白的表达情况。结果实验兔15周时血胆固醇(TC)升高约43倍,血低密度脂蛋白(LDL)升高约37倍。主动脉内壁可见脂质条纹形成。主动脉AS病变Ⅰ型胶原、NF-κB蛋白及Ⅰ型胶原mRNA、NF-κBmRNA表达定量(A值)分别为0.27±0.02、0.19±0.01及0.30±0.03、0.35±0.03,显著高于对照组(0.08±0.01、0.09±0.01及0.11±0.01、0.10±0.09,P<0.05)。结论AS时Ⅰ型胶原增多刺激NF-κB的活性,可能参与了血管AS的形成。     

益肾胶囊对系膜增殖性肾小球肾炎大鼠肾脏NF-κB及PDGF-β表达的影响

目的探讨益肾胶囊对系膜增殖性肾小球肾炎(MsPGN)大鼠肾脏核转录因子(NF-κB)及血小板源生长因子β(PDGF-β)表达的影响。方法观察治疗组大鼠血脂、肾功能及24 h尿蛋白(Pro/24 h)的变化,同时用SABC免疫组化方法检测各组肾小球、肾小管NF-κB及PDGF-β的表达情况。结果经过8周的观察,治疗组大鼠血脂、肾功能及Pro/24 h与模型组比较,甘油三酯(TG)、胆固醇(TC)、低密度脂蛋白(LDL-C)、血肌酐(Scr)、血尿素氮(BUN)及Pro/24 h均明显降低(P<0.05或P<0.01),而高密度脂蛋白(HDL-C)明显升高(P<0.05);且益肾胶囊可明显抑制肾组织NF-κB、PDGF-β的表达(P<0.05或P<0.01)。结论益肾胶囊可明显降低MsPGN大鼠血脂及Pro/24 h,改善肾功能,其作用机制可能与抑制肾组织NF-κB、PDGF-β的表达有关。     

β2受体阻滞剂对胰腺癌细胞侵袭能力的影响及其机制

目的研究β2受体阻滞剂对胰腺癌细胞的增殖及侵袭转移能力的影响及其作用机制。方法β2受体特异性阻滞剂ICI118,551、广谱β受体阻断剂普萘洛尔和β1受体特异性阻滞剂美托洛尔干预胰腺癌细胞MIA PaCa-2和BxPC-3后,通过MTT分析、流式细胞术,细胞侵袭实验,Western blot和EMSA等技术阐明β2受体阻滞剂对胰腺癌侵袭能力的抑制作用,进一步检测核转录因子NF-κB和AP-1的活性及其下游相关分子VEGF、MMP-2、MMP-9和COX-2的表达。结果在优势浓度(100μmol/L)下:普萘洛尔、ICI118,551诱导胰腺癌细胞周期G1/S期阻滞和抑制增殖、侵袭效应强于美托洛尔(P<0.05);三种阻滞剂均可下调NF-κB和AP-1的活性,并降低其相关下游分子VEGF、MMP-2、MMP-9和COX-2的表达(P<0.05),普萘洛尔、ICI118,551对上述分子抑制率强于美托洛尔,对BxPC-3的抑制强于MIA PaCa-2细胞。结论β2受体阻滞剂通过下调核转录因子及其相关下游分子的表达而抑制胰腺癌细胞的增殖及侵袭转移能力。     

ROLE OF ANTISENSE AND DECOY OLIGONUCLEOTIDE OF NF-κB IN VESSEL STENOSIS AND NEOINTIMA FORMATION IN BALLOON-INJURED RAT ARTERY

Objective To examine antisense and decoy oligonucleotides of nuclear factor kappa B in vivo effects on intima proliferation and balloon-injured monocytes chemotactic protein-1 ( MCP-1 ) and extracellular signal regulated kinase-2 ( ERK2 ) expression in the carotid artery of rats. Methods Sprague-Dawley rats underwent balloon-dilation injury of the left carotid artery. Rats are divided into 7 groups ( n = 18 ) and each group includes 6 time points (6 h, 1, 3, 5, 7, 14 d) ( n =3). Uninjured right carotid artery of the same rat was used as controls. Results In model group, sense group and scramble group, vessel intima area , media area and intima/media ratio increased after 5 d and reached the maximum after 7 d. The effect of antisense plus decoy group on intimal hyperplasia was more obvious than that of antisense group and decoy group alone. MCP-1 mRNA expression was increased expression continuously at 3, 5 and 7 d and decreased at 14 d. Compared with model group, sense group and scramble group, antisense group, decoy group and antisense plus decoy group had lowered MCP-1 mRNA expression in each time point ( P ＜ 0. 05 ). NF-κB p65 was dispersed positive stain 6 h after injury and increased after 1 d and peaked at 7 d, but the protein expression was weak at 14 d. ERK2 protein synthesis increased at 1 d and reached the peak at 7 d, while protein expression after 14 d was similar to that at 7 d. Treatment of antisense group,decoy group and antisense plus decoy group inhibited protein synthesis more significantly than those of model group,sense group and scramble group ( P ＜ 0. 05 ). Conclusion NF-κB modulates genes expression and protein synthesis of MCP-1 and ERK2. Celluar proliferation in vessel wall was dynamically changed after balloon angioplasty injury. Antisense and decoy oligonucleotide of NF-κB by local lipofectamine transfer inhibit NF-κB activating gene modulation and neointimal hyperplasia.     

ICI118551联合吉西他滨对胰腺癌细胞BxPC-3增殖和凋亡的影响及其机制

目的探讨β2肾上腺素能受体阻滞剂ICI 118551与吉西他滨联合应用对胰腺癌细胞BxPC-3增殖和凋亡的影响及可能的机制。方法利用ICI 118551、吉西他滨及ICI 118551+吉西他滨干预胰腺癌细胞BxPC-3,MTT法检测细胞增殖能力,Annexin V-FITC/PI和TUNEL检测细胞凋亡情况,RT-PCR检测Bax、Bcl-2表达,Western blot检测NF-κB亚基P65、Bax、Bcl-2表达变化。结果 ICI 118551明显增强吉西他滨的抗增殖和促凋亡效应,成功阻断由吉西他滨诱导的NF-κB的活化,并且提升了Bax/Bcl-2mRNA和蛋白的表达。结论 ICI 118551能增强吉西他滨对胰腺癌细胞的抑制作用,提示吉西他滨联合β2肾上腺素能受体阻滞剂可作为胰腺癌治疗的一种有效方法。     

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in cultured human periodontal ligament (HPDL) cells. Methods Small interfering RNA (siRNA) eukaryotic expression vector targeted transforming growth factor βⅡ receptor (TGF-β RⅡ) was constructed and transfected into T cells. HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide (LPS) and baicalin. The obtained solution was divided into six groups according to the components (group Ⅰ: HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ: HPDL cells+LPS+T cells transfected with siRNA1; group Ⅲ: HPDL cells+LPS+T cells+baicalin; group Ⅳ: HPDL cells+LPS+T cells; group Ⅴ: HPDL cells+baicalin; group Ⅵ: HPDL cells) and was cultured for 48 hours. RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells. Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ (P<0.01) and higher than that in group Ⅲ (P<0.01); The ratio of RANKL/OPG in gronp Ⅲ was lower than that in group Ⅳ (P<0.01); the ratio of RANKL/ OPG in group Ⅳ was higher than that in group Ⅵ (P<0.01); the ratio of RANKL/OPG in group Ⅴ was lower than TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.     

Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-7) gene promoter and its effect on IFN-7 expression in chronic hepatitis B. Method The authors recruited 30 patients with UBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequeneing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytoldne in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P<0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P<0.01) ; the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P<0.01). Oonclusion IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.     

电针联合扶正理气合剂对大鼠肝癌生长及转移的影响及相关机制

目的观察电针联合扶正理气合剂对大鼠肝癌生长及转移的影响并探讨其作用机制。方法雄性Wistar大鼠200只,随机分为5组。以二乙基亚硝胺(DEN)灌胃诱导大鼠肝癌模型,电针组造模同时给予电针足三里、关元、内关、三阴交、肝俞穴治疗,扶正理气合剂组给予扶正理气合剂灌胃,针药联合组则予上述电针和扶正理气合剂治疗,共16周。造模16周后处死大鼠取肝脏组织标本,肉眼及光镜下观察肿瘤生长和转移情况;免疫组化法测定肝组织NF-κB活性及微血管密度(MVD),RT-PCR法检测肝组织中转化生长因子β1(TGF-β1)mRNA及血管内皮生长因子(VEGF)mRNA表达,放射免疫法测定肝组织活性氧(ROS)、抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性、脂质过氧化产物(LPO)含量。结果与正常对照组比较,模型组大鼠的肿瘤生长和转移参数、ROS、LPO含量、NF-κB活性、TGF-β1 mRNA、VEGF mRNA、MVD表达显著增加(P<0.01),而T-AOC、SOD、GSH-Px、CAT活性明显下降(P<0.01);与模型组比较,各治疗组大鼠的肿瘤生长和转移参数、ROS、LPO含量、NF-κB活性、TGF-β1mRNA、VEGF mRNA及MVD表达显著下降,而T-AOC、SOD、GSH-Px、CAT活性明显上升(P<0.01),联合治疗组上述指标优于其他治疗组(P<0.05)。NF-κB活性与TGF-β1mRNA及VEGF mRNA呈正相关关系(r1=0.554,P<0.05;r2=0.572,P<0.05)。结论电针和扶正理气合剂均能显著降低实验性肝癌大鼠的NF-κB活性、TGF-β1mRNA及VEGF mRNA及MVD表达,从而降低肿瘤生长和转移参数,这与其清除自由基有密切关系。     

乌司他丁对大鼠蛛网膜下腔出血后迟发性脑血管痉挛的影响及其脑保护作用

目的通过乌司他丁(UTI)对雄性SD大鼠枕大池二次注血建造的蛛网膜下腔出血(SAH)模型的干预,观察其对迟发性脑血管痉挛(delayed cerebral vasospasm,DCVS)的防治及脑保护作用。方法将30只雄性SD大鼠随机分为空白对照组(Normal组)、假注血组(sham组)、注血组(SAH组)和乌司他丁治疗组(SAH+UTI组)。通过枕大池二次注血法建立SAH模型,光镜下观察基底动脉组织学变化和海马CA1区神经元的形态变化,计算血管舒张度(D/T)及海马CA1区神经元密度;应用免疫组化方法检测基底动脉NF-κB的表达。结果神经功能缺损评分:SAH+UTI组评分较SAH组明显改善(P<0.05);基底动脉测量发现,SAH组较Sham组血管周径及D/T值明显减低(P<0.05),SAH+UTI组较SAH组血管周径及D/T值增大(P<0.05);海马CA1区HE染色:与Normal及Sham组比较,SAH组神经元密度明显减少(P<0.05);SAH+UTI组与SAH组比较神经元密度增大(P<0.05);基底动脉NF-κB的表达:SAH组及SAH+UTI组基底动脉NF-κB的表达均较Sham组及Normal组增多(P<0.05);应用乌司他丁可以减轻SAH后NF-κB的表达(P<0.05)。结论乌司他丁可以减轻SAH后DCVS,并减轻神经细胞损伤,改善大鼠SAH后神经功能缺损症状。     

Microfluidics-based assay on the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors

Abstract A new microfluidic system with four different microchambers （a circle and three equilateral concave polygons） was designed and fabricated using poly（dimethylsiloxane） （PDMS） and the soft lithography method. Using this microfluidic device at six flow rates （5, 10, 20, 30, 40, and 50 μL/h）, the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors were investigated. Escherichia coli HB101 pGLO, which could produce a green fluorescent protein induced by L-arabinose, was utilized as the model bacteria. The results demonstrated that bacterial adhesion was significantly related to culture time, microenvironment geometry, and aqueous flow rates. Adhered bacterial density increased with the culture time. Initially, the adhesion occurred at the microchamber sides, and then the entire chamber was gradually covered with increased culture time. Adhesion densities in the side zones were larger than those in the center zones because of the lower shearing force in the side zone. Also, the adhesion densities in the complex chambers were larger than those in the simple chambers. At low flow rates, the orientation of adhered bacteria was random and disorderly. At high flow rates, bacterial orientation became close to the streamline and oriented toward the flow direction; All these results implied that bacterial adhesion tended to occur in complicated aqueous flow areas.The present study provided an on-chip flow system for physiological behavior of biological cells, as well as provided a strategic cue for the prevention of bacterial infection and biofilm formation.     

TGF-β1对成人骨髓CD105~+间充质干细胞增殖与分化的影响

目的分离并鉴定成人骨髓间充质干细胞(mesenchymalstemcells,MSCs),研究TGFβ1对骨髓MSCs增殖与分化的影响。方法分离成人骨髓单个核细胞;MACS磁珠分选CD105+细胞;FACS检测其免疫表型;诱导分选的CD105+细胞向脂肪及成骨细胞分化,油红O染色、VonKossa染色及RTPCR检测脂肪细胞及成骨细胞;观察TGFβ1对分选出的CD105+细胞分化与增殖的影响;免疫荧光染色检测诱导后的脂肪细胞CD105的表达情况;MTT法检测TGFβ1对CD105+细胞增殖的影响。结果成人骨髓来源的CD105+细胞体外可向脂肪及成骨细胞分化;在向脂肪细胞分化过程中,加入1～50ng/mLTGFβ1后,成脂诱导体系中无脂肪细胞出现;在向成骨诱导过程中,加入20ng/mLTGFβ1后,钙化基质沉淀明显降低;在脂肪诱导体系中,MTT染色检测显示TGFβ1对CD105+细胞的增殖有明显促进作用(P<0.05)。结论TGFβ1抑制成人骨髓源CD105+MSCs向脂肪及成骨细胞分化,在向脂肪诱导过程中,TGFβ1促进MSCs的增殖。     

Alendronate affects osteoprotegerin/receptor of activator of nuclear factor κB-ligand expression in human marrow stroma cells in vitro

Objective To evaluate the effect of alendronate on osteoprotegerin (OPG) and receptor of activator of nuclear factor κB-ligand (RANKL) expression in human marrow stroma cells (hMSCs) in vitro. Methods hMSCs were isolated from haman marrow, cultured in vitro, and randomly divided into two groups: alendronate group, hMSCs culture fluid containing 1×10-7 mol/ L alendronate; control group, no special treatment but culturing hMSCs in DMEM. Two weeks after treatment, the expressions of OPG and RANKL were evaluated by RT-PCR and Western blot.Results hMSCs became uniform spindle-shaped fibroblasts. As cells proliferated, they formed colonies and showed whirlpool arrangement. After one week's treatment, hMSCs in alendronate group had reduced processes and gradually showed disc shape, which did not happen in control group but kept fibroblast shape and just increased in density. In RT-PCR, the ratio of OPG/RANKL in alendronate group and control group was 8.77±1.16 and 4.58±1.27, respec-tively. In Western blot, the ratio of OPG/RANKL in alendronate group and control group was 2.58±0.47 and 1.52±0.32, respectively. The ratio of OPG/RANKL was higher in alendronate group than in control group (P<0.01).Conclusion Alendronatc enhances OPG expression and inhibits RANKL expression of hMSCs in vitro.     

GROWTH DIFFERENTIATION FACTOR-5 STIMULATES THE GROWTH AND ANABOLIC METABOLISM OF ARTICULAR CHONDROCYTES

Growthdifferentiationfactor5(GDF5),also knownascartilage derivedmorphogeneticprotein1(CDMP1),isamemberofthebonemorphogenet icprotein(BMP)familywhichbelongstothe transforminggrowthfactorβ(TGFβ)superfami ly[1].GDF5isarelativelynewmemberofthe BMPfamily.LikeotherBMPs,implantationofre combinantGDF5caninduceectopiccartilagefor mationinmusculartissues.Thephysiologicalrole ofGDF5hasbeenreportedincludingregulationof myogenesis,regulationofchondrogenesis,bone morphogenesis,andneurondifferen…