铜绿假单胞菌密度感应系统自体诱导因子3OC12-HSL对人肠道上皮细胞Caco-2凋亡的影响及其机制

目的 探讨铜绿假单胞菌密度感应系统自体诱导因子3OC12-HSL对肠道上皮细胞Caco-2凋亡的影响及其深入机制.方法 3OC12-HSL干扰Caco-2细胞4 h后,采用MTT法检测Caco-2细胞干扰前后细胞活性变化;Annexin V-FTTC/PI双染法流式细胞术检测细胞凋亡率的变化;Western blot检测Caco-2细胞中凋亡相关蛋白p-p38/MAPK及NF-κB表达变化.结果 MTT结果 显示,3OC12-HSL可以抑制Caco-2细胞活性,并呈剂量依赖性(P<0.05).流式细胞术结果 表明,Caco2细胞凋亡率随3OC12-HSI干扰浓度增加而增加(P<0.05).Western blot结果 显示,3OC12-HSL处理后,细胞p-p38/MAPK及NF-κB蛋白表达水平增加.结论 3OC12-HSL可以抑制肠道上皮细胞Caco-2活性,诱导细胞发生凋亡,这种作用可能和p38/MAPK通路激活以及NF-κB的激活有关.     

铜绿假单胞菌密度感应系统自体诱导因子3OC12-HSL对人肠道上皮细胞Caco-2凋亡的影响及其机制

Objective To investigate the effect of quorum sensing autoinducer 3OC12-HSL of Pseudomonas aeruginosa on the apoptosis of Caco-2 cells and its mechanism. Methods Caco-2 cells were incubated with 3OC12-HSL for 4 h and then examined by MTT method for cytoactivities. Flow cytometry was used to analyze apoptosis rate of Caco-2 cells. Expression of apoptosis associated proteins p-p38/MAPK and NF-κB were detected by Western blot. Results After exposure to 3OC12-HSL for 4 h, cytoactivities of Caco-2 cells was reduced(P<0.05) with dose-dependent pattern, and higher dose of 3OC12-HSL leaded to increasing apoptosis rate of Caco-2 cells(P<0.05). 3OC12-HSL raised expression of apoptosis associated proteins p-p38/MAPK and NF-κB detected by Western blot. Conclusion Quorum sensing autoinducer 3OC12-HSL can effect cytoactivities of Caco-2 cells and may induce its apoptosis by enhancing the expression of p-p38/MAPK and NF-κB protein.     

铜绿假单胞菌密度感应信号分子OdDHL对小鼠肥大细胞作用观察

目的 通过化学合成铜绿假单胞菌密度感应(quorum sensing,QS)信号分子OdDHL,并观察该分子对小鼠肥大细胞生物学作用.方法 通过化学方法 合成OdDHL分子,经质谱、磁共振和高效液相色谱确定其结构和纯度,并采用OdDHL分子感应菌株检测其生物活性.以不同浓度OdDHL分子作用于小鼠肥大细胞系P815,观察不同时间点细胞活力改变、凋亡现象以及胞内钙离子变化.结果 成功合成具有生物活性的OdDHL分子,该分子能以时间、剂量依赖方式抑制细胞活力,并能诱导细胞凋亡,同时可上调胞内钙离子浓度.结论 铜绿假单胞菌OdDHL分子可诱导P815细胞凋亡并上调胞内钙离子浓度.     

AiiA蛋白对铜绿假单胞菌毒力因子生成的影响

目的 构建苏云金杆菌AiiA基因的真核表达载体并转染A549细胞,观测真核细胞表达的AiiA蛋白对铜绿假单胞菌(Pseudomonas aeruginosa,tgiaosa,Pa)密度感应(quorum sensing)系统酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL)信号分子及毒力因子合成的影响.方法 质粒pET-AiiA经Nhe Ⅰ与Xho Ⅰ酶切后,将AiiA基因片段连接入真核表达质粒pEGFP-N2,脂质体转染A549细胞,提取细胞蛋白,Western blot检测AiiA蛋白表达.将蛋白提取物加入Pa培养物中,观察Pa AHL信号分子及毒力因子绿脓素、弹性蛋白酶的生成情况.结果 AiiA基因片段成功克隆入真核表达质粒pEGFP-N2,转染该质粒后的A549细胞能够表达AiiA蛋白,且该蛋白能够降低Pan培养物中AHL信号分子水平,且绿脓素和弹性蛋白酶的牛成减少.结论 本研究成功构建了真核表达质粒pEGFP-N2-AiiA,转染细胞后表达的AiiA蛋白能够水解AHL信号分子,减少Pa毒力因子的生成,故具有抗Pa感染的潜在价值.     

姜黄素抑制顺铂诱导人肾小管上皮细胞系HK-2凋亡

目的探讨姜黄素对顺铂诱导人肾小管上皮细胞系HK-2凋亡的影响。方法用MTT法观察HK-2增殖;用Western blot检测HK-2的凋亡蛋白。结果 1)顺铂呈剂量依赖性诱导HK-2凋亡(P<0.05)。2)在姜黄素和顺铂联合作用下,凋亡的HK-2明显减少,细胞存活率提高(P<0.05);HK-2中Bax蛋白表达降低(P<0.05);Bcl-2蛋白表达增加(P<0.05)。结论姜黄素可以通过调控Bax和Bcl-2蛋白的表达抑制顺铂引起的细胞凋亡。     

铁离子对Caco-2细胞内钙离子转运的影响及钙离子浓度变化与细胞凋亡关系的研究

目的研究细胞外铁离子(Fe^3 )浓度变化对钙离子(Caco-22)细胞内Ca^2 转运的影响及细胞内钙离子浓度升高与Caco-2细胞凋亡的关系。方法采用激光共聚焦扫描显微镜和流式细胞技术进行观察与检测。结果(1)利用激光共聚焦扫描显微镜(cLSM)观察发现,随着Caco-2细胞外Fe^3 浓度的减少(DFO终浓度为1130～300μmol／L),Ca^2 向细胞内的转运量增加;而随着细胞外Fe^3 浓度的增加(FAC终浓度为10～100μmol／L),Ca^2 向细胞内的转运呈降低趋势;(2)经A23187,和Fluo-3,AM处理后Caco-2细胞的存活率较高,达90％以上(用荧光素二醋酸酯检测);(3)A23187升高Caco-2细胞的胞内Ca^2 浓度,呈剂量依赖性;经流式细胞技术(FCM)检测发现胞内Ca^2 浓度增加具有诱导Caco-2细胞凋亡的作用。结论Caco-2细胞外Fe^3 浓度的减少有促进Ca^2 向细胞内转运的作用。但胞外Fe^3 浓度增加时有抑制Ca^2 向胞内转运的作用;胞内Ca^2 浓度增加对Caco-2细胞凋亡具有诱导作用。     

KIM-1对AGEs诱导的肾小管上皮细胞EMT和凋亡的影响及其机制

目的：探讨肾损伤分子1(KIM-1)对晚期糖基化终末产物（AGEs）诱导的人近端肾小管上皮HK-2细胞上皮-间充质转化（EMT）和凋亡的影响及其可能的机制。方法：将HK-2细胞分为对照组、AGEs组、AGEs+阴性对照小干扰RNA(NC siRNA)组和AGEs+KIM-1 siRNA组。免疫荧光检测KIM-1蛋白的表达;Western blot检测KIM-1、α-平滑肌肌动蛋白（α-SMA）、E-cadherin、Bcl-2、Bcl-2相关X蛋白（Bax）、caspase-3、细胞外信号调节激酶1/2(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)蛋白水平;RT-qPCR检测KIM-1 mRNA的表达;MTS检测细胞活力;TUNEL染色和annexin V-FITC/PI双染流式细胞术检测细胞凋亡;流式细胞术检测细胞活性氧（ROS）水平。结果：与对照组比较,AGEs处理后的HK-2细胞中KIM-1蛋白、KIM-1 mRNA和α-SMA蛋白表达呈时间依赖性升高,E-cadherin蛋白表达和细胞活力呈时间依赖性降低,凋亡率呈时间依赖性升高,Bcl-2蛋白表达降低,Bax和cas...     

双歧杆菌BB12对肠炎沙门氏菌诱导Caco-2细胞分泌IL-8 的影响

目的：体外研究双歧杆菌对肠炎沙门氏菌诱导Caco-2细胞分泌IL-8的影响。方法：建立双歧杆菌、肠炎沙门氏菌和结肠癌上皮细胞系Caco-2细胞共培养，逆转录PCR和实时荧光定量PCR和EMSA等方法检测细胞内IL-8mRNA和NF-κBp65，ELISA法检测培养上清中IL-8。结果：对照组的Caco-2细胞低表达IL-8mRNA，细胞核中很难检测出NF-κBp65，培养上清中IL-8的质量浓度为126ng／L。经肠炎沙门氏菌刺激后，IL-8mRNA和NF-κBp65以及培养上清中IL-8的质量浓度（536ng／L）明显高于对照组（分别为P〈0．01，P〈0．01和P〈0．01）。双歧杆菌和肠炎沙门氏菌共培养的Caco-2细胞中，IL-8mRNA和NF-κBp65以及培养上清中IL-8质量浓度（336ng／L）显著低于肠炎沙门氏菌组（分别为P〈0．01，P〈0．01和P〈0．01）。结论：双歧杆菌能明显抑制肠炎沙门氏菌诱导Caco-2细胞IL-8的分泌．其调节方式是抑制NF-κB065的活化．     

As2O3诱导喉鳞癌Hep-2细胞凋亡及其机制的研究

目的 探讨三氧化二砷( As2O3)诱导喉鳞癌Hep-2细胞凋亡及其作用机制.方法 体外培养喉鳞癌Hep-2细胞株,MTT法检测不同作用时间及不同浓度的As2O3对Hep-2细胞生长的作用;TUNEL染色、电镜观察Hep-2细胞凋亡的发生和形态学改变;Western印迹检测As2O3诱导Hep-2细胞凋亡时Survivin蛋白的表达改变.结果 As2O3可明显抑制喉鳞癌Hep-2细胞的生长(P＜0.05),并随药物浓度及作用时间的增加而增强.TUNEL、电镜结果显示As2O3作用后,Hep-2细胞发生凋亡.As2O3作用后,Survivin基因的表达降低.结论 As2O3可诱导喉鳞癌Hep-2细胞凋亡,其生长抑制作用呈时间及剂量依赖性;其机制可能是As2O3通过抑制凋亡抑制因子Survivin蛋白的表达,促进细胞凋亡,从而发挥抗肿瘤的作用.     

抗HER-2抗体chA21对人乳腺癌SKBR3细胞凋亡的诱导作用及其机制

目的探讨抗HER-2工程抗体chA21在体外对高表达HER-2的人乳腺癌SKBR3细胞凋亡的诱导作用及其分子机制。方法采用透射电镜和原位末端标记技术(TUNEL)观察和检测chA21对SKBR3细胞凋亡的诱导,采用免疫细胞化学技术检测凋亡相关基因bc l-2、bax、Fas及caspase-3表达的改变。结果chA21作用72 h,可见SKBR3细胞凋亡,chA21高浓度组(5.4mg/L)凋亡指数显著高于低浓度组(0.2 mg/L)(P<0.01);chA21处理组SKBR3细胞的bax、Fas及caspase-3表达增加,而bc l-2表达及bc l-2/bax比值降低,上述改变在chA21高、低两个浓度组间有显著差异(P<0.01)。结论chA21在体外可诱导SK-BR3细胞凋亡,其分子机制与调节凋亡相关基因bax、bc l-2、Fas及caspase-3的表达有关。     

3-O-C12-HSL通过阻碍人树突状细胞成熟介导Th细胞极性分化

目的 探讨铜绿假单胞菌分泌的信号分子3-O-C12-HSL对脂多糖诱导的人外周血单核细胞来源树突状细胞(Mo-DCs)成熟及Th细胞极化的干预作用.方法 采用免疫磁珠法分选人外周血CD14+单核细胞,经重组人粒-单核细胞集落刺激因子和重组人IL-4诱导其分化为Mo-DCs.CCK-8法检测3-O-C12-HSL对Mo-DCs活性影响;流式细胞术检测3-O-C12-HSL对Mo-DCs表型(CD11c、CD40、CD80、HLA-DR)的影响;ELISA检测Mo-DCs培养上清IFN-γ和IL-12浓度;混合淋巴细胞反应观察3-O-C12-HSL对Th细胞增殖和细胞因子分泌的影响.结果 3-O-C12-HSL终浓度200μmol/L时可影响Mo-DCs活性.与脂多糖对照组比较,终浓度50 μmoL/L和100 μmoL/L的3-O-C12-HSL明显下调脂多糖诱导的Mo-DCs表面分子CD40、CDS0和HLA-DR分子表达水平,抑制Mo-DCs分泌IL-12,促进Mo-DCs分泌IL-10,抑制Th细胞增殖和IFN-γ、IL-12分泌,促进Th细胞分泌IL-10.结论3-O-C12-HSL可抑制脂多糖诱导的Mo-DCs成熟,诱导Th2细胞极性分化,影响机体免疫系统对铜绿假单胞菌的清除作用.     

Rotavirus induces apoptosis in fully differentiated human intestinal Caco-2 cells

Rotaviruses, which are the main cause of viral gastroenteritis in young children, induce structural and functional damages in infected mature enterocytes of the small intestine. To investigate a relationship between rotavirus infection and cell death by apoptosis, we used the human intestinal Caco-2 cell line. We demonstrated by several methods including TUNEL and ELISA detection of cytoplasmic histone-associated DNA fragments that the infection of fully differentiated Caco-2 cells by the RRV rotavirus strain induces apoptosis. Rotavirus infection leads to the loss of mitochondrial membrane potential and the release of cytochrome C from mitochondria. We showed that rotavirus-induced apoptosis was dependent of the multiplicity of infection and increased with time from 4 h to 24 h of infection. Flow cytometric analysis showed that DNA fragmentation occurs in productively infected cells, suggesting that rotavirus induces apoptosis by a direct mechanism. We also demonstrated that non-replicative RRV particles are not sufficient to induce apoptosis and viral gene expression seems required. Intracellular calcium plays a role in RRV-induced apoptosis because treatment with an intracellular calcium ion chelator (BAPTA-AM) partially inhibited apoptosis.     

二硫苏糖醇通过激活calpain-2/caspase-12信号通路诱导BRL-3A细胞凋亡

目的:探讨二硫苏糖醇(DTT)诱导大鼠正常肝细胞株BRL-3A发生内质网应激时细胞内葡萄糖调节蛋白78(GRP78)、钙蛋白酶2 (calpain-2)、caspase-12及caspase-3的表达变化及对细胞凋亡的影响。方法:采用2. 5 mmol/L DTT分别处理BRL-3A细胞12 h和24 h,应用real-time PCR检测细胞内GRP78、calpain-2、caspase-12及caspase-3的mRNA水平;采用细胞免疫荧光检测细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达;应用Western blot检测cleaved caspase-12及cleaved caspase-3的表达变化;采用流式细胞术检测细胞凋亡情况。结果:BRL-3A细胞经DTT处理12 h及24 h后,GRP78、calpain-2及caspase-12的mRNA表达较正常对照组显著升高(P 0. 01),而caspase-3的mRNA水平与正常对照组比较无显著变化;细胞免疫荧光及Western blot检测发现,DTT处理细胞12 h及24 h后,BRL-3A细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达均较正常对照组显著增高,同时,cleaved caspase-12及cleaved caspase-3的表达也较正常对照组明显增多(P 0. 05);流式细胞术检测发现,经DTT处理后BRL-3A细胞的凋亡率较正常对照组显著增加(P 0. 05)。结论:二硫苏糖醇诱导BRL-3A细胞凋亡可能与calpain-2/caspase-12信号通路激活有关。     

米诺环素通过调控PI3K/Akt通路抑制硝普钠诱导的PC12细胞凋亡

 目的： 探讨PI3K/Akt信号通路在米诺环素（minocycline,MC）抑制硝普钠(sodium nitoprusside,SNP)诱导的PC12细胞凋亡中的作用。方法：将体外培养的PC12细胞分为4组：空白对照组、SNP组、MC+SNP组和PI3K抑制剂LY294002+ MC+SNP组。用四甲基偶氮唑盐（MTT）法检测细胞活力,流式细胞术检测细胞凋亡;Western blotting检测不同时点（0.5、1、2、3 h）各处理组PI3K/Akt通路蛋白p-Akt和Akt的表达。结果：SNP处理PC12细胞24 h能抑制细胞生长,加入10 μmol/L MC预处理30 min可明显提高细胞活力,降低细胞凋亡率（P<0.05）,抑制SNP诱导的PC12细胞凋亡。MC组的p-Akt表达高于其它组,而加入LY294002后可阻断MC的上述效应。结论：MC可通过调控PI3K/Akt通路抑制SNP诱导的PC12细胞凋亡。     

Visfatin induces neurite outgrowth in PC12 cells via ERK1/2 signaling pathway

The angiogenic and inflammatory functions of visfatin and its effect on vascular cells, are fairly well known. However, its role within the nervous system remains largely unclear. To gain insight into this area, we studied the neuritogenic effect of visfatin on PC12 rat pheochromocytoma cells. We investigated whether visfatin gene expression, which is upregulated by hypoxia in cancer cells, is associated with neuritogenesis in PC12 cells. Using RT-PCR, Western blot analysis, ELISA, morphological observations, and immunostaining, we initially showed that CoCl₂, a hypoxic mimetic agent, upregulated visfatin gene expression along with neurite outgrowth in PC12 cells. We also showed that visfatin stimulated neurite outgrowth in PC12 cells. Moreover, in PC12 cells, visfatin evoked the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), which is closely linked to neuritogenesis. Visfatin-induced outgrowth of neurites was prevented by inhibition of the ERK1/2 pathway. Taken together, our results demonstrate for the first time that visfatin induces neurite outgrowth in PC12 cells via the activation of an ERK-dependent pathway, and suggest that visfatin may exert various biological, physiological, and pathological functions in not only the vascular system but also the nervous system.     

Thapsigargin-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells

Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases, including ischemia, excitotoxicity and Alzheimer's disease. Thapsigargin, which increases [Ca²⁺]_i, induces apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by caspase-3 activation in PC12 cells. We examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors in PC12 cells. Cells treated with 0.1 μM thapsigargin for 24 h shrank. The injured cells underwent chromatin condensation and nuclear fragmentation, indicating apoptotic cell death. We assayed the effects of selective GSK-3 inhibitors, SB216763, azakenpaullone and alsteropaullone on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. Alsterpaullone did not reduce the GRP78 protein expression induced by thapsigargin, suggesting that GSK-3 activation is not involved in induction of GRP78. In addition, GSK-3 inhibitors inhibited caspase-3 activation accompanied by thapsigargin-induced apoptosis. We showed in this report that thapsigargin-induced apoptosis is prevented by GSK-3 inhibitors, suggesting that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3 activation in PC12 cells.