NF-κB介导哮喘过敏原刺激后支气管上皮细胞的凋亡

目的研究核转录因子κB(NF-κB)在哮喘过敏原刺激后支气管上皮细胞中的表达及对细胞凋亡的影响。方法以300U/L尘螨抗原提取物作为过敏原刺激支气管上皮细胞系16HBE细胞,应用RT-PCR和Western blot检测尘螨抗原提取物刺激对16HBE细胞NF-κB(p65)表达水平的影响;用NF-κB(p65)sh RNA沉默16HBE细胞内NF-κB表达后,应用流式细胞术和Western blot检测NF-κB(p65)在过敏原刺激诱导16HBE细胞凋亡中的作用。结果过敏原刺激后使16HBE细胞内NF-κB(p65)m RNA和蛋白水平明显上调,明显诱导16HBE细胞凋亡和活化型caspase-3水平上调,沉默NF-κB可使过敏原刺激诱导的16HBE细胞凋亡率降低和活化型caspase-3水平上调减少。结论 NF-κB可介导哮喘过敏原刺激后的支气管上皮细胞凋亡。     

bc1-2和bax及NF-kB在白藜芦醇诱导肝癌细胞凋亡中的作用

目的探讨白藜芦醇诱导肝癌细胞凋亡的途径.方法白藜芦醇体外处理HepG2肝癌细胞24 h后,以免疫组化检测凋亡调控基因bcl-2和bax及NF-kB的表达.结果白藜芦醇处理组HepG2细胞bcl-2的阳性积分和NF-kB的阳性细胞密度均明显低于对照组(P＜0.01);而bax阳性积分明显高于对照组(P＜0.01).结论白藜芦醇能下调HepG2细胞bcl-2基因的表达,上调bax的表达,同时抑制NF-kB的活化,这可能是其诱导HepG2细胞凋亡的途径之一.     

NF-κB、p53和bax在顺铂所致肾损伤中的作用

目的探讨NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)和顺铂治疗对Lewis肺癌小鼠肾脏组织中p53和bax表达的影响。方法给小鼠皮下接种Lewis肺癌细胞用顺铂和/或PDTC治疗,15 d后处死小鼠。取肾脏并制成切片,应用免疫组织化学技术检测p53和bax蛋白的表达。结果对照组和PDTC组小鼠肾脏未检测到p53和bax表达;顺铂治疗小鼠肾小管上皮细胞表达p53和bax;合用PDTC治疗组小鼠p53和表达水平较单用顺铂治疗组明显减低。结论合用PDTC下调顺铂诱导的p53和bax表达上调,减少肾细胞凋亡。     

甘草次酸对大肠癌LoVo细胞增殖和侵袭的影响*

目的: 研究不同浓度的甘草次酸对大肠癌LoVo细胞增殖和侵袭的影响。方法: 将大肠癌LoVo细胞分为对照组,甘草次酸低、中、高剂量组(甘草次酸浓度分别为50, 100, 200 μmol/L)和5氟尿嘧啶组(5氟尿嘧啶浓度为100 μmol/L ),各组细胞经过药物孵育24和48 h后进行检测。通过四氮唑蓝试验检测甘草次酸对各组细胞增殖率的影响;通过Annexin V/PI双标流式细胞术检测各组细胞的凋亡率;Transwell 小室法检测各组细胞的侵袭能力;通过蛋白质印迹法检测检各组细胞的NF-κB蛋白表达。结果: 与对照组相比,甘草次酸中、高剂量组和5氟尿嘧啶组中细胞抑制率均显著降低(P＜0.05);甘草次酸中、高剂量组和5氟尿嘧啶组中细胞凋亡率均显著升高(P＜0.05);甘草次酸中、高剂量组和5氟尿嘧啶组中大肠癌LoVo 细胞侵袭能力显著降低(P＜ 0.05);甘草次酸中、高剂量组和5氟尿嘧啶组中NF-κB相对表达量均显著降低(P＜0.05)。结论: 浓度为100 μmol/L和200 μmol/L甘草次酸可以抑制大肠癌LoVo细胞的增殖,降低侵袭能力,这些作用的机制与抑制NF-κB蛋白的表达有关。     

NF-κB p65在高糖诱导3T3-L1脂肪细胞胰岛素抵抗中的作用

建立高糖诱导胰岛素抵抗的细胞模型,研究高糖对3T3-L1脂肪细胞NF-κB p65表达及转位的影响。诱导成熟的3T3-L1脂肪细胞与5.0mmol/L的葡萄糖含或不含0.6nmol/L的胰岛素(LGIns 组与LGIns-组)或者与25.0mmol/L葡萄糖含或不含0.6nmol/L的胰岛素(HGIns 组与HGIns-组)培养18h,以2-脱氧-[3H]-D-葡萄糖摄入法观察葡萄糖的转运率,用Western印迹检测总NF-κBp65及核NF-κB p65的表达,用激光扫描共聚焦(CLSM)对NF-κB p65进行定位显示。结果显示,仅HGIns 组,即3T3-L1脂肪细胞与25.0mmol/L葡萄糖含0.6nmol/L的胰岛素培养18h后,胰岛素刺激的葡萄糖转运减少55%(P<0.01),同时Western印迹和CLSM均显示NF-κB p65核转位增加(P<0.01),但对3T3-L1脂肪细胞总NF-κB p65的表达无明显影响(P>0.05)。研究结果表明,只有在胰岛素(0.6nmol/L)存在的条件下,高糖(25.0mmol/L)才可以诱导胰岛素抵抗,其分子机制可能与其刺激NF-κB p65的核转位,调节相关基因的表达有关。     

B3型柯萨奇病毒上调NF-κB引起胰岛β细胞凋亡的作用及机制研究

B3型柯萨奇病毒（Coxsackie virus B3,CVB3）与1型糖尿病发病、胰岛功能破坏有关,但CVB3对胰岛β细胞凋亡的影响及机制尚不清楚。本研究的目的是观察CVB3上调NF-κB p-p65引起胰岛β细胞凋亡的作用及机制,进而初步探究CVB3感染引起胰岛β细胞损伤的分子机制。本研究培养了人胰岛β细胞株,分为对照组、CVB3组、阴性对照（NC）质粒组、NF-κB p65质粒组、si-NC组、si-NC+CVB3组、si-NF-κB p65+CVB3组,检测细胞活力A490水平、细胞凋亡率及细胞中cleaved caspase-3、NF-κB p-p65、IκB α的表达水平。结果显示与对照组比较,CVB3组的A490水平及细胞中IκB α的表达水平降低,细胞凋亡率及细胞中cleaved caspase-3、NF-κB p-p65的表达水平增加（P<0.05）;与NC质粒组比较,NF-κB p65质粒组的A490水平降低,细胞凋亡率及细胞中cleaved caspase-3、NF-κB p-p65的表达水平增加（P<0.05）;与si-NC+CVB3组比较,si-N...     

过表达MicroRNA-199a促进TNFα诱导的脂肪细胞凋亡

旨为探明micro RNA-199a(mi R-199a)对脂肪细胞凋亡的影响及核转录因子κB(nuclear factorκB,NF-κB)在其中的作用。培养3T3-L1脂肪细胞,使用肿瘤坏死因子α(tumor necrosis factorα,TNFα)诱导细胞凋亡。在此基础上分别使用NF-κB阻断剂PDTC和mi R-199a mimic处理细胞,判断过表达mi R-199a对TNFα诱导的脂肪细胞凋亡的影响及NF-κB在其中的作用。使用流式细胞仪分析细胞凋亡率,试剂盒检测Caspase3/7酶活,双荧光素酶报告系统检测NF-κB活性,q RT-PCR检测mi R-199a的表达水平,Western blotting检测NF-κB相关蛋白变化。结果显示,TNFα可以诱导分化的脂肪细胞凋亡并同时激活NF-κB,激活的NF-κB在TNFα诱导的脂肪细胞凋亡中发挥负调控作用。在脂肪细胞过表达mi R-199a明显抑制NF-κB的激活进而显著促进TNFα诱导的凋亡。mi R-199a通过调控NF-κB活性参与TNFα诱导的脂肪细胞凋亡。     

SLE患者PBMC凋亡状态及相关基因表达的研究

探讨外周血单个核细胞(PBMC)凋亡及其基因调控在系统性红斑狼疮(systemic lupus erythematosus,SLE)发病机制中的作用.用流式细胞仪(FCM)检测PBMC凋亡百分率及T细胞亚群的凋亡状态;用RT-PCR检测PBMC bcl-2和bax的mRNA表达;用FCM检测凋亡相关基因bcl-2,bax,fas,p53和c-myc的蛋白表达.结果显示,SLE患者PBMC凋亡百分率明显高于正常人,且活动期患者高于非活动期患者.SLE活动期患者CD4+,CD8+T细胞数明显低于正常人;非活动期患者CD8+T细胞数明显低于正常人,而CD4+T细胞数与正常人比较无统计学差异;SLE患者PBMC bcl-2和bax mRNA表达与正常人比较无统计学差异;SLE患者PBMC bcl-2,bax和fas蛋白表达明显高于正常人,p53和c-myc蛋白表达在各组之间无统计学差异.SLE患者PBMC凋亡百分率增高、外周血T细胞亚群的异常及bcl-2,bax和fas蛋白表达增高,在SLE发病机制中可能起了一定的作用.     

乳酸堆积和二氯乙酸钠对肝癌细胞凋亡及bax、bcl-2 表达 和caspase-3 活性的影响

目的：探讨乳酸堆积和二氯乙酸钠(DCA)对肝癌细胞(HepG2)凋亡和bax、bcl-2 表达及caspase-3 活性的影响。方法：通过体 外培养HepG2,建立稳定的体外培养模型,配制成终浓度分别为0 mmol/L、1.0 mmol/L、2.0 mmol/L、4.0 mmol/L、8.0 mmol/L的乳 酸培养液以及在不同浓度乳酸组中加入终浓度为10-3mmol/L DCA 培养液与HepG2共同培养,其中以0 mmol/L 乳酸组为对照 组。采用MTT法检测乳酸对HepG2 的抑制率,流式细胞仪检测乳酸和DCA 对HepG2的凋亡百分率,用Real-time PCR法测定 bax 及bcl-2 mRNA的表达,用免疫荧光法检测caspase-3 的活性。结果：乳酸对HepG2 的IC50值为13.6 mol/L,与对照组比较,随 着乳酸浓度的增加,HepG2 凋亡率增加,bax mRNA 表达升高,bcl-2 mRNA 的表达降低,caspase-3活性增加,其中1.0 mmol/L 乳 酸组与对照组比较(P>0.05),2.0 mmol/L,4.0 mmol/L 和8.0 mmol/L乳酸组与对照组比较差异有统计学意义(P<0.05)。加入DCA 后,HepG2 凋亡减少,2.0 mmol/L 乳酸+DCA 组、4.0 mmol/L乳酸+DCA 组、8.0 mmol/L乳酸+DCA 组与同浓度的乳酸组比较, bax mRNA 表达减少（P<0.05）,bcl-2 mRNA 表达增加（P<0.05）,caspase-3 活性减低（P<0.05）。结论：乳酸可诱导HepG2凋亡,且随 着乳酸浓度的增高,HepG2 的凋亡率增加,其机制可能是通过对bcl-2 及bax mRNA 表达的改变以及激活caspase-3 活性而实现, DCA可以降低HepG2 凋亡,对乳酸堆积造成的HepG2凋亡有抑制作用。     

Ad-IL-24对人胶质瘤细胞生长抑制效应的体外实验

研究携带人白介素24(IL-24)的腺病毒表达载体(Ad-IL-24)对人U251胶质瘤细胞生长的影响和抗肿瘤分子机制。将不同MOI Ad-IL-24感染U251人胶质瘤细胞后, MTT法检测Ad-IL-24对U251细胞生长的抑制作用, 流式细胞仪和Hochest 染色法检测细胞的凋亡率。RT-PCR检测bcl-2、bax、ICE、C-myc、HIF-1a和p53等基因的转录表达水平, Western blotting检测Cleaved Caspase-3的表达。结果表明100 MOI Ad-IL-24感染U251细胞后能明显抑制细胞生长, 并能明显诱导细胞凋亡, 感染72 h后细胞凋亡率可达42%, 感染4 d后细胞生长抑制率可达50%。RT-PCR检测发现Ad-IL-24能引起与细胞凋亡和血管形成相关基因bax/bcl-2、ICE、C-myc、p53的上调和HIF-1a的下调, 并促进Caspase-3的活化。本研究结果显示Ad-IL-24能明显抑制人胶质瘤细胞U251生长和诱导细胞凋亡, 其抗肿瘤机制可能与通过bax/ bcl-2、ICE、c-myc、p53的上调和HIF-1a的下调, 进而导致Caspase-3的活化而诱导肿瘤细胞发生凋亡有关。     

A novel copper (II) complex activated both extrinsic and intrinsic apoptotic pathways in liver cancerous cells

Recent advances have put fundamental focus on the application of copper (II) (Cu [II]) complexes as agents for fighting against cancer. To determine whether [Cu(L)(2imi)] complex as a novel Cu complex can induce apoptosis in HepG2 as cancerous cells and L929 as normal cells via extrinsic or intrinsic apoptotic pathways, both cell lines were treated for 24 and 48 hours at IC₅₀ concentrations of [Cu(L)(2imi)] complex. Then, the expression of some apoptosis-related genes including p53, caspase-8, bcl-2, and bax were assayed by real-time polymerase chain reaction. The [Cu(L)(2imi)] complex seems to inhibit the expression of bcl-2 in complex-treated HepG2 cancerous cells following the 24- and 48-hour treatment. The complex upregulated the p53, bax, and caspase-8 genes, therefore treatment of HepG2 cancerous cells with [Cu(L)(2imi)] complex induces programmed cell death via the upregulation of relative bax/bcl-2 ratio. Finally, this copper complex triggered apoptosis in HepG2 cells via both intrinsic and extrinsic pathway, whereas treatment of normal L929 cells with this complex induce apoptosis only via intrinsic pathway with the upregulation of relative bax/bcl-2 ratio and does not affect the expression level of caspase-8 gene and does not trigger the extrinsic pathway. Finally, these results obtained from present study confirm the role of a novel Cu complex on the induction of apoptosis process in HepG2 and L929 cells by overexpression of bax, inhibition of bcl-2 and increase of the relative bax/bcl-2 ratio. These results support that the [Cu(L)(2imi)] complex is able to induce apoptosis in cancerous cells, therefore, it has a potential for development as a novel anticancer drug.     

pGhrelin对离体猪脂肪细胞Caspase-3活性及基因表达的影响

研究新近发现的猪Ghrelin (porcine ghrelin,pGhrelin)对猪前体脂肪细胞Caspase-3活性及其基因表达的影响．采用细胞培养技术,以仔猪背部皮下前体脂肪细胞为靶细胞,经0、1、10和100 nmol/L pGhrelin处理细胞48 h后,于倒置生物显微镜下进行脂肪细胞形态学观察．利用MTT法测定pGhrelin对细胞增殖的影响．采用分光光度法检测Caspase-3活性．以实时荧光定量RT-PCR方法测定Caspase-3的基因表达．结果显示,10 nmol/L pGhrelin可以显著降低脂肪细胞Caspase-3的活性与mRNA的表达水平(P < 0.05),100 nmol/L pGhrelin对猪脂肪前体细胞增殖有极显著促进作用(P < 0.01)．上述结果表明,pGhrelin可以下调Caspase-3的活性与基因表达,促进脂肪细胞增殖,抑制脂肪细胞凋亡,其机制可能与Caspase-3依赖性凋亡调节信号通路有关．     

乳酸堆积和二氯乙酸钠对肝癌细胞凋亡及bax、bcl-2表达和caspase-3活性的影响

目的：探讨乳酸堆积和二氯乙酸钠（OCA）对肝癌细胞（HepG2）凋亡和bax、bcl-2表达及caspase-3活性的影响。方法：通过体外培养HepG2,建立稳定的体外培养模型,配制成终浓度分别为0mmol／L、1．0mmol／L、2．0mmol／L、4．0mmol／L、8．0mmol／L的乳酸培养液以及在不同浓度乳酸组中加入终浓度为10^-3mmol／LDCA培养液与HepG2共同培养,其中以0mmol／L乳酸组为对照组。采用MTT法检测乳酸对HepG2的抑制率,流式细胞仪检测乳酸和DCA对HepG2的凋亡百分率,用Real-time PCR法测定bax及bcl-2mRNA的表达,用免疫荧光法检测caspase-3的活性。结果：乳酸对HepG2的IC50值为13．6mol／L,与对照组比较,随着乳酸浓度的增加,HepG2凋亡率增加,baxmRNA表达升高,bcl-2mRNA的表达降低,caspase-3活性增加,其中1．0mmol／L乳酸组与对照组比较（P〉0．05）,2．0mmol／L,4．0mmol／L和8．0mmol／L乳酸组与对照组比较差异有统计学意义（P〈0．05）。加入DCA后．HepG2凋亡减少,2．0mmol／L乳酸＋DCA组、4．0mmol／L乳酸＋DCA组、8．0mmol／L乳酸＋DCA组与同浓度的乳酸组比较,baxmRNA表达减少（P〈0．05）,bcl-2mRNA表达增加（P〈0．05）,caspase-3活性减低（P〈0．05）。结论：乳酸可诱导HepG2凋亡,且随着乳酸浓度的增高,HepG2的凋亡率增加,其机制可能是通过对bcl-2及baxmRNA表达的改变以及激活caspase-3活性而实现,DCA可以降低HepG2凋亡,对乳酸堆积造成的HepG2凋亡有抑制作用。     

Antiapoptotic effect of interferon-alpha on hepatic stellate cells (HSC): a novel pathway of IFN-alpha signal transduction via Janus kinase 2 (JAK2) and caspase-8

The hepatic stellate cell (HSC), the pericyte of the liver sinusoids belongs to the mesenchymal cells of the liver. Damaging noxae induce a transformation from the quiescent (vitamin A-storing cell) to the activated (connective tissue-producing cell) state. The balance between proapoptotic and surviving factors decides about the fate of the activated HSC. Interferon-alpha (IFN-alpha) has been shown to elicit antiproliferative and/or antifibrogenic effects in various cell types of mesenchymal origin. We therefore investigated the effect of IFN-alpha on primary cultured rat HSC in their quiescent (day 2) and activated state (day 7). IFN-alpha significantly inhibited spontaneous apoptosis in activated HSC in vitro and simultaneously inhibited cell cycle progression by inducing a G1 arrest. The effect of IFN-a is not accompanied by a modulation of CD95, CD95L, p53, p21(WAF1), p27, bcl-2, bcl-xL, bax, NFkappaB, or IkappaB gene expression. Surprisingly, the IFN-alpha effect could be abolished completely by blocking JAK2 activity or JAK2 translation. The downregulating effect of IFN-alpha on the activity of caspase-8 and caspase-3 could also be neutralized using tyrphostin AG490 or JAK-2 antisense. Taken together IFN-alpha inhibits apoptosis of activated HSC by activation of JAK2 which inhibits the caspase-8 apoptosis pathway.     

Persian shallot, <Emphasis Type="Italic">Allium hirtifolium</Emphasis> Boiss,induced apoptosis in human hepatocellular carcinoma cells

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC₅₀ value (inhibiting cell growth by 50%) was 149 μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24 h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.     

Lentiviral-mediated RNAi targeting p38MAPK ameliorates high glucose-induced apoptosis in osteoblast MC3T3-E1 cell line

The p38 mitogen activated protein kinase (p38MAPK) pathway is an important signaling cascade involved in cell growth, differentiation and apoptosis. High glucose activates p38MAPK pathway in different cells, including osteoblasts. In the present study, role of p38MAPK in high glucose induced osteoblast apoptosis and potential of RNA interference (RNAi) targeting p38MAPK as a therapy strategy have been reported. Lentiviral-mediated RNAi effectively reduced p38MAPK and p-p38MAPK expressions in osteoblastic cell line (MC3T3-E1) following high glucose (22 mM) induction. Inhibition of p38MAPK activity significantly suppressed high glucose induced apoptosis of MC3T3-E1 cell and was confirmed by flow cytometry and ultra-structural examination by transmission electronic microscope. Inhibition of p38MAPK also significantly attenuates caspase-3 and bax protein expressions, but increased significantly bcl-2 expression as determined by Western blot analysis. The results suggested that p38MAPK mediates high glucose induced osteoblast apoptosis, partly through modulating the expressions of caspase-3, bax and bcl-2. Inhibition of p38MAPK with lentiviral-mediated RNAi or its specific inhibitor provides a new strategy to treat high glucose induced osteoblast apoptosis.     

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis

In order to determine the ability of bovine herpesvirus type 1 (BHV-1) to suppress apoptosis, we examined the effects of BHV-1 infection on sorbitol-induced apoptosis on Madin-Darby bovine kidney (MDBK) cells. BHV-1 suppresses sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that BHV-1 has one or more anti-apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis-inhibiting and -promoting factors were analyzed on BHV-1 infected cells during the process of sorbitol-induced apoptosis. Our results revealed that the expression of bcl-2 and bcl-x(L) decreased after 5 and 3 h p.i., respectively; while bax and procaspase-3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV-1 has one or more genes encoding apoptosis-inhibiting factors which interfere with the involvement of bcl-2 gene family members and apoptotic pathway, depending upon caspase-3, triggered by sorbitol.     

CCL21/CCR7 prevents apoptosis via the ERK pathway in human non-small cell lung cancer cells

Previously, we confirmed that C-C chemokine receptor 7 (CCR7) promotes cell proliferation via the extracellular signal-regulated kinase (ERK) pathway, but its role in apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. A549 and H460 cells of NSCLC were used to examine the effect of CCL21/CCR7 on apoptosis using flow cytometry. The results showed that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant decline in the percent of apoptosis. Western blot and real-time PCR assays indicated that activation of CCR7 significantly caused upregulation of anti-apoptotic bcl-2 and downregulation of pro-apoptotic bax and caspase-3, but not p53, at both protein and mRNA levels. CCR7 small interfering RNA significantly attenuated these effects of exogenous CCL21. Besides, PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished these effects of CCL21/CCR7. Coimmunoprecipitation further confirmed that there was an interaction between p-ERK and bcl-2, bax, or caspase-3, particularly in the presence of CCL21. These results strongly suggest that CCL21/CCR7 prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax and caspase-3 potentially via the ERK pathway in A549 and H460 cells of NSCLC.