铜绿假单胞菌及L型诱导巨噬细胞凋亡的实验研究

目的研究铜绿假单胞菌（PA）及L型诱导巨噬细胞凋亡的能力,比较二者的差异。方法用生物素断端标记（TUNEL）法检测PA及L型感染巨噬细胞2、4、8、12、16和20h后各时间段的细胞凋亡率,Giemsa染色观察细胞凋亡情况,硝酸还原酶法检测培养液中一氧化氮（NO）的浓度变化。结果 PA及L型能诱导巨噬细胞发生凋亡,与对照组比较差异有统计学意义（P〈0.05）;L型诱导细胞的凋亡率弱于原菌（P〈0.05）;PA及L型感染组培养液NO浓度较对照组明显升高（P〈0.05）。结论 PA及L型可诱导巨噬细胞发生凋亡,L型较其原型诱导细胞凋亡的能力弱,NO可能在巨噬细胞凋亡中发挥一定作用。PA及L型可通过诱导巨噬细胞凋亡,发挥致病作用。     

基于分子对接的合欢皂苷J_(8)抑制血管内皮细胞增殖作用靶点及其凋亡相关细胞信号通路研究CSCD

为探讨合欢皂苷J_(8)(julibroside J_(8),J8)抑制肿瘤血管内皮细胞增殖作用靶点及其相关细胞凋亡的信号通路。本文采用HPLC法检测内皮细胞经J8作用后,J8的在细胞内外含量变化;Vina软件将J8与VEGF、FAS、DR3、DR4、DR5、TFR-1进行分子对接分析;Western blot法检测在加药前后,内皮细胞中VEGF、p-JNK、Bax、EnDOG、Caspase-3、Caspase-8以及Caspase-9蛋白表达水平的变化。分子对接结果表明VEGF、FAS对应的靶点蛋白与J8结合性能较好且多位点结合。HUVEC细胞在加入J8作用24 h后,VEGF、p-JNK等蛋白表达明显下调,并可显著上调诱导凋亡相关蛋白Bax和EnDOG的表达,而且对Caspase-3、Caspase-8以及Caspase-9的表达水平无显著的影响。J8可能是通过与血管内皮细胞膜表面的VEGF结合,抑制血管内皮细胞增殖,通过削弱VEGF/JNK通路活性从而引起内皮细胞凋亡。     

葡萄球菌肠毒素B诱导脐静脉内皮细胞凋亡机制的探讨

目的探讨金黄色葡萄球菌(金葡菌)肠毒素B诱导脐静脉内皮细胞凋亡的机制。方法将不同浓度金葡菌肠毒素B感染脐静脉内皮细胞8 h后,用流式细胞术检测细胞凋亡率,同时用比色法检测TNF-α、caspase-3及caspase-8的产生量,并检测加入TNF-α抗体、caspase-3和caspase-8抑制剂后的细胞凋亡率。结果不同浓度肠毒素B作用脐静脉内皮细胞8 h后均可诱导细胞凋亡,且TNF-α、caspase-3和caspase-8的产生量均高于对照组(P0.01);而加入TNF-α抗体、caspase-3和caspase-8抑制剂后凋亡率明显降低。结论金葡菌肠毒素B可以诱导脐静脉内皮细胞凋亡,其凋亡机制可能是通过TNF-α介导的caspase-8及caspase-3激活的外源性死亡因子受体途径。     

二苯乙烯苷对氧化诱导内皮细胞凋亡及Caspase-3和PARP表达的影响

为研究二苯乙烯苷对过氧化氢(H2O2)诱导人脐静脉内皮细胞凋亡的影响并初步探讨二苯乙烯苷抗凋亡的可能机制,采用不同浓度H2O2和二苯乙烯苷处理内皮细胞24 h,以MTT法检测细胞生长活力、Hoechst33258染色观察细胞形态、流式细胞仪检测凋亡率等方法筛选造模内皮细胞凋亡的H2O2浓度和二苯乙烯苷最佳的抗内皮细胞凋亡作用浓度.RT-PCR、Western-blot分别检测Caspase-3、PARP mRNA及蛋白质的表达.结果发现,与空白对照组相比,300μmol/L H2O2作用后,内皮细胞增殖明显受到抑制,Hoechst33258染色可见大量凋亡细胞,细胞凋亡率显著增加,流式细胞仪检测出明显的凋亡峰,Caspase-3和多聚腺苷酸二磷酸核糖基聚合酶(PARP)的表达量显著增加.经二苯乙烯苷处理后,随着二苯乙烯苷浓度增加,细胞的增殖率随之增加,凋亡细胞数减少,凋亡率逐渐降低,与H2O2组比较,10μmol/L的二苯乙烯苷能够显著提高细胞增殖率,降低细胞凋亡率,并显著减少Caspase-3和PARP表达.以上结果表明,二苯乙烯苷能够抑制由H2O2诱导的人脐静脉内皮细胞凋亡,增加细胞生长活性,降低细胞凋亡率,其作用机制可能与下调Caspase-3和多聚腺苷酸二磷酸核糖基聚合酶(PARP)的表达有关.     

脱氧胆酸钠对人脐静脉内皮细胞凋亡的影响

目的:探讨脱氧胆酸钠(SD)对体外培养的人脐静脉内皮细胞(HUVEC)凋亡的影响。方法:(1)以不同终浓度(0、0.015mg/mL、0.05 mg/mL、0.15 mg/mL、0.5 mg/mL、1.0 mg/mL)的脱氧胆酸钠分别作用于人脐静脉血管内皮细胞,使用CCK-8检测细胞活力、TUNEL荧光染色检测细胞凋亡;(2)以终浓度为0.15 mg/mL的SD作用于HUVEC4、8、12 h后用Western blot检测Caspase-3、7、9蛋白及PARP活化情况;(3)观察Caspase-3抑制剂Z-DEVD-FMK对0.15 mg/mL脱氧胆酸钠组的影响。结果:CCK8结果显示随SD浓度(0~1.0 mg/mL)及作用时间(0~12 h)增加,HUVEC活力降低,0.15 mg/mL时活力为80%,1.0 mg/mL时细胞活力仅不到10%;Tunel检测示随着SD浓度的增加HUVEC凋亡明显增多;Western Blot结果示SD作用于HUVEC后Caspase-3、7、9蛋白及PARP活化明显增加;Z-DEVD-FMK明显抑制了0.15 mg/mLSD引起的PARP活化。结论:脱氧胆酸钠(SD)通过启动Caspase级联反应介导了人脐静脉内皮细胞的凋亡。     

环介导等温扩增技术检测化妆品中铜绿假单胞菌的研究

本文建立了环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)快速检测化妆品中铜绿假单胞菌的方法。采用铜绿假单胞菌外膜蛋白oprI基因保守序列引物,评价检测铜绿假单胞菌灵敏度和特异性,并与普通PCR相比,检测人工污染样品中的铜绿假单胞菌。结果显示,LAMP检测铜绿假单胞菌的灵敏度为62. 5 pg/μL,而且特异性高,人工污染样品中的检出限为102cfu/m L,比PCR检测灵敏度高10倍。该方法具有灵敏度高、特异性好、操作简便、耗时短,可用于化妆品中铜绿假单胞菌的快速检测。     

下呼吸道非发酵菌的分布及其耐药性研究

目的了解2000～2003年安微医科大学第一附属医院下呼吸道非发酵菌的分布及其耐药性,为临床合理选用抗生素提供依据。方法细菌鉴定、药敏试验和诱导β-内酰胺酶(IB)检测采用WalkAway-40全自动微生物分析仪,超广谱β-内酰胺酶(ESBb)检测采用纸片确证试验。结果2000～2003年下呼吸道非发酵菌的检出率为28．2％。其构成以铜绿假单胞菌为最多(56．6％),其次为洛菲不动杆菌(9．3％)、嗜麦芽窄食单胞菌(8．6％)和鲍曼或溶血不动杆菌(8．5％)。对复方新诺明,嗜麦芽窄食单胞菌的耐药率最低(14．3％)。对头孢他啶、亚胺培南、哌拉西林或他巴坦,铜绿假单胞菌的耐药性较低,分别为20．3％、20．8％和28．3％;洛菲不动杆菌耐药率比鲍曼或溶血不动杆菌普遍偏低。铜绿假单胞菌ESBLs和IB的检出率分别为15．7％和84．2％。结论下呼吸道非发酵菌的检出率较高,主要为铜绿假单胞菌。非发酵菌耐药现象严重,不同非发酵菌的耐药谱差异较大。铜绿假单胞菌IB发生率高。     

菌蜕搭载的埃博霉素促进Caspase-3介导的HeLa细胞凋亡

[目的]初步探讨菌蜕搭载的埃博霉素抑制HeLa细胞增殖的机制。[方法]用半抑制浓度的游离或菌蜕搭载的埃博霉素B分别处理HeLa细胞4~48 h,应用凋亡检测试剂盒分析细胞凋亡情况,应用Caspase-3分析试剂盒检测Caspase-3的活性,并用透射电镜观察细胞的形态变化。[结果]菌蜕搭载的埃博霉素B能更快地诱导HeLa细胞发生时间依赖的凋亡和坏死,处理24 h时早期凋亡细胞及晚期凋亡和坏死细胞的比例分别达32.72%和18.36%,处理48 h时比例分别达23.14%和51.52%。该药物还能诱导更强的Caspase-3激活,处理24 h时其活性约为游离埃博霉素B的1.5倍。Caspase-3活性的升高程度与细胞的早期凋亡程度相平行。该药物处理的HeLa细胞显示典型的凋亡特征。[结论]菌蜕搭载的埃博霉素B能更有效地促进Caspase-3介导的HeLa细胞凋亡。     

铜绿假单胞菌外膜蛋白Ⅰ的原核表达、纯化及免疫保护作用研究

铜绿假单胞菌外膜蛋白Opr I具有较强的免疫原性,在疫苗上具有开发前景。利用分子方法获得Opr I蛋白的表达菌株。Western blotting验证表明抗体能与Opr I蛋白特异性结合。SDS-PAGE电泳切胶纯化获得Opr I蛋白。将Opr I蛋白免疫小鼠并攻毒铜绿假单胞菌,发现Opr I蛋白激活的特异性免疫对小鼠铜绿假单胞菌感染的保护率达到57.14%,与对照组相比较达到显著性。采用正交试验设计,获得Opr I菌株最佳诱导表达条件为:加IPTG菌夜OD600值0.8,加IPTG终浓度0.3 mmol/L,诱导时间8 h,诱导温度28℃;菌株最佳培养条件为转速230 r/min,葡萄糖浓度0%,装液量50 m L。     

铜绿假单胞菌分泌性蛋白的抗肿瘤效应

目的探讨3株不同来源的铜绿假单胞菌分泌性蛋白的抗肿瘤活性。方法将3株不同来源的铜绿假单胞菌经LB培养基对其过夜静置培养,离心获得上清液,采用硫酸铵盐析沉淀蛋白质,再经PBS透析除盐。然后将蛋白作用于人肝癌细胞Hep-G2、宫颈癌细胞HeLa、肺癌细胞A549和人永生化表皮细胞HaCaT,CCK-8检测其对细胞的毒性作用,吉姆萨染色观察凋亡细胞形态变化。结果 SDS-PAGE证实成功获得不同分子量的分泌蛋白,CCK-8结果显示混合蛋白对3种不同肿瘤细胞的生长均有不同程度的抑制作用,且其抑制作用存在一定的时间和浓度依赖性,但对人正常细胞无明显抑制作用。吉姆萨染色初步显示细胞形态为凋亡状态。结论初步证实该3株铜绿假单胞菌产生的分泌性蛋白具有不同程度的抗肿瘤作用,为进一步分离纯化具有抗肿瘤活性的单一蛋白质及研究其抗肿瘤机制提供依据。     

Pseudomonas aeruginosa-induced human mast cell apoptosis is associated with up-regulation of endogenous Bcl-xS and down-regulation of Bcl-xL

Mast cells play a critical role in the host defense against bacterial infection. Recently, apoptosis has been demonstrated to be essential in the regulation of host response to Pseudomonas aeruginosa. In this study we show that human mast cell line HMC-1 and human cord blood-derived mast cells undergo apoptosis as determined by the ssDNA formation after infection with P. aeruginosa. P. aeruginosa induced activation of caspase-3 in mast cells as evidenced by the cleavage of D4-GDI, an endogenous caspase-3 substrate and the generation of an active form of caspase-3. Interestingly, P. aeruginosa treatment induced up-regulation of Bcl-x(S) and down-regulation of Bcl-x(L). Bcl-x(S), and Bcl-x(L) are alternative variants produced from the same Bcl-x pre-mRNA. The former is proapoptotic and the latter is antiapoptotic likely through regulating mitochondrial membrane integrity. Treatment of mast cells with P. aeruginosa induced release of cytochrome c from mitochondria and loss of mitochondrial membrane potentials. Moreover, P. aeruginosa treatment reduced levels of Fas-associated death domain protein-like IL-1beta-converting enzyme-inhibitory proteins (FLIPs) that are endogenous apoptosis inhibitors through counteraction with caspase-8. Thus, human mast cells undergo apoptosis after encountering P. aeruginosa through a mechanism that likely involves both the Bcl family protein mitochondrial-dependent and the FLIP-associated caspase-8 pathways.     

AREL1 resists the apoptosis induced by TGF-β by inhibiting SMAC in vascular endothelial cells

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-β (TGF-β), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-β treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-β. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-β treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-β induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.     

Apoptotic cell death of vascular endothelial cells and renal tubular cells in the generalized Shwartzman reaction

Abstract The participation of apoptotic cell death in the generalized Shwartzman reaction was examined. The generalized Shwartzman reaction was induced in mice by two consecutive injections of lipopolysaccharide. Vascular endothelial cells in various organs of those mice were stained positively by the in situ specific labeling of fragmented DNA. Renal tubules were also stained focally. It was suggested that apoptotic cell death might participate in the development of vascular endothelial cell damage and acute tubular necrosis in the generalized Shwartzman reaction. Simultaneous administration of anti-γ-interferon antibody in the preparative injection of lipopolysaccharide completely blocked apoptosis of vascular endothelial cells. Priming with recombinant γ-interferon instead of lipopolysaccharide could produce apoptosis of vascular endothelial cells. It was suggested that γ-interferon might play a critical role on sensitization of endothelial cells for apoptosis.     

A possible involvement of ion transporter in tumor necrosis factor alpha and cycloheximide-induced apoptosis of endothelial cells

We examined the tumor necrosis factor alpha (TNFalpha)-induced apoptosis of vascular endothelial cells from the standpoint of ion channels. Cultured vascular endothelial cells from bovine carotid artery were used. Apoptosis was determined by a propidium iodide assay. Treatment of the endothelial cells with TNFalpha and cycloheximide for 6 h induced nuclear fragmentation in a TNFalpha dose-dependent manner (1-10 ng/ml). Concomitant treatment of endothelial cells with TNFalpha at a dose of 10 ng/ml and cycloheximide at a dose of 10 microg/ml elicited endothelial cell apoptosis as high as 23.4+/-4.1% at 6 h after administration. However, 10 ng/ml TNFalpha alone elicited a little apoptosis at 6 h after its administration (% apoptosis=4.1+/-0.8%). Cycloheximide (10 microg/ml) did not induce apoptosis at all. Concomitant treatment of endothelial cells with 1 mmol/l of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, which is a chloride bicarbonate exchanger blocker, partially inhibited the TNFalpha and cycloheximide-induced endothelial cell apoptosis. On the other hand, endothelial cell apoptosis due to TNFalpha and cycloheximide was completely inhibited by benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (50 micromol/l), an inhibitor of caspase. Moreover, pyrrolidine dithiocarbanate, an inhibitor of nuclear factor kappa B (NF-kappaB), also suppressed endothelial cell apoptosis induced by TNFalpha and cycloheximide completely. These findings suggest that the endothelial cell apoptosis induced by TNFalpha and cycloheximide is closely related to not only chloride ions, but also both NF-kappaB and caspase activation. That is to say, there is a possibility that chloride ions or bicarbonate (pH) may play an important role in signal transduction such as NF-kappaB and caspase activation in the apoptosis induced by TNFalpha and cycloheximide.     

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.     

Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells

Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 micromol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.     

Chronic effect of doxorubicin on vascular endothelium assessed by organ culture study.

We have attempted to determine the chronic effects of doxorubicin, a commonly used anticancer agent, on vascular endothelium using an organ culture system. In rabbit mesenteric arteries treated with 0.3 microM doxorubicin for 7 days, rounding and concentrated nuclei and TUNEL-positive staining were observed in endothelial cells, indicating DNA damage and the induction of apoptosis. However, the endothelium-dependent relaxation induced by substance P and the expression of mRNA encoding endothelial NO synthase (eNOS) did not differ from those in control arteries. In arteries treated with a higher concentration (1 microM) of doxorubicin, apoptosis and damage to nuclei occurred in the endothelial cells at the third day of treatment, and the detachment and excoriation of endothelium from the tunica interna of the vascular wall were also observed. The impairment of endothelium-dependent relaxation was observed at the fifth day of the treatment with 1 microM doxorubicin. Additionally, apoptotic change in the smooth muscle layer was observed at this concentration of doxorubicin. Apoptotic phenomena were further confirmed by DNA fragmentation using isolated bovine aortic endothelial cells (BAECs) and A7r5 vascular smooth muscle cells, and it was revealed that BAECs are more sensitive than A7r5 to the apoptotic effect of doxorubicin. These results suggest that chronic treatment with doxorubicin at therapeutic concentrations induces apoptosis and excoriation of endothelial cells, which diminishes endothelium-dependent relaxation.     

Possible involvement of a chloride-bicarbonate exchanger in apoptosis of endothelial cells and cardiomyocytes

We examined the role of ion movement in staurosporine-induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by propidium iodide assay. Treatment of the endothelial cells with staurosporine (10 nmol/l-1 micromol/l) for 6 h induced nuclear fragmentation in a dose-dependent manner. Staurosporine (1 micromol/l) elicited apoptosis in 70.5+/-1.5% of cells. Concomitant treatment of endothelial cells with 1 mmol/l of 4, 4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a chloride-bicarbonate exchange blocker, completely inhibited staurosporine-induced apoptosis. Other ion transporter inhibitors such as dimethyl amiloride and anthracene-9 carboxylic acid were less effective inhibitors of staurosporine-induced apoptosis of endothelial cells. DIDS prevented staurosporine-induced apoptosis of endothelial cells as well as cardiomyocytes. Next, we determined whether chloride ions or bicarbonate are involved in apoptosis. Incubation with a chloride ion removal buffer did not inhibit staurosporine-induced apoptosis of endothelial cells. However, endothelial cell apoptosis was completely suppressed by an inhibitor of caspase, benzyloxycarbonyl-Asp-CH(2)-O(C)O-dichlorobenzene (zD-dcb, 50 micromol/l). Staurosporine (1 micromol/l) increased the intracellular pH of endothelial cells, and DIDS (1 mmol/l), but not a caspase inhibitor, inhibited this increase in pH caused by staurosporine. Our findings suggest that endothelial cell apoptosis induced by staurosporine may be associated with the Cl(-)and bicarbonate (HCO-3) ions. Thus, Cl(-)efflux from cells or HCO-3 influx to cells (which increases pH) may play an important role in signal transduction leading events such as activation of caspase in staurosporine-induced apoptosis.