Tumor necrosis factor-alpha and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells.

BACKGROUND: The glomerular endothelial cell is a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration. During gram-negative sepsis, glomerulonephritis, and acute renal failure, bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) may cause severe cell damage. Our aim was to study and compare the direct effects of TNF-alpha and LPS on the induction of apoptosis in bovine glomerular endothelial cells. METHODS: Primary bovine glomerular endothelial cells were stimulated with TNF-alpha or LPS, and apoptotic cell death was investigated by DNA fragmentation analysis, morphological studies, measurement of cytochrome c efflux and mitochondrial permeability transition, Bak, Bad, Bax, Bcl-2, Bcl-xL protein expression, and caspase-3-like protease activity. RESULTS: TNF-alpha, as well as LPS, elicited apoptotic cell death both time and concentration dependently. Along with DNA ladder formation, we detected the formation of 50 kbp high molecular weight DNA fragments, nuclear condensation, and mitochondrial permeability transition. Concerning all parameters, LPS signaling proved to be more rapid than TNF-alpha. Mechanistically, TNF-alpha-induced cell death was preceded by an efflux of mitochondrial cytochrome c into the cytosol and, subsequently, by a marked increase in the proapoptotic protein Bak and a decrease in the anti-apoptotic Bcl-xL protein content. Comparable but more pronounced effects were seen with LPS. Later, caspase-3-like protease activity was first detectable after 10 hours and was continuously increased up to 24 hours in both TNF-alpha- and LPS-stimulated cells. Correspondingly, we detected an extended cleavage of the nuclear enzyme poly(ADP-ribose) polymerase. Caspase inhibitors Z-Asp-CH2-DCB and Z-VAD-fmk blocked both TNF-alpha- and LPS-induced apoptosis in a comparable manner. Only Z-Asp-CH2-DCB was able to block apoptotic cell death completely. CONCLUSION: Both bacterial LPS and TNF-alpha potently induced apoptotic cell death in glomerular endothelial cells. Direct endotoxin-induced apoptosis may therefore be relevant in the progression of acute renal failure, which is a frequent complication of gram-negative sepsis.     

小鼠骨髓未成熟树突状细胞体外扩增及鉴定

目的建立体外大量扩增小鼠未成熟树突状细胞(DC)的方法，从形态学、免疫表型和细胞功能试验等方面予以鉴定。方法制备小鼠骨髓细胞，分别用不同剂量重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM—CSF)培养，7d后收集悬浮细胞进行扫描电镜观察和免疫表型鉴定，并行混合淋巴细胞反应，观察其诱导未致敏T淋巴细胞增殖的情况。结果小剂量rmGM—CSF培养获得的DC(GM^low DC)具有DC的典型特征，细胞表面高表达CD11c，低表达CD40、I—A／1-E，不表达B7—1，与大剂量rmGM—CSF培养获得的DC(GM^high DC)相比，其体外刺激未致敏T淋巴细胞增殖的能力较弱。结论本实验中获得的GM^low DC形态上具有DC的典型特征，在细胞表型、细胞功能试验上具有未成熟的特性，说明所建立的培养未成熟DC的方法是可行的；rmGM—CSF的剂量与细胞的成熟程度相关，一般说来，较大剂量的rmGM—CSF诱导生成的细胞以成熟。DC为主，小剂量rmGM—CSF诱导生成的细胞以未成熟DC为主。     

前列腺癌细胞凋亡与iNOS、Bcl-2的表达及意义

目的 :探讨细胞凋亡和诱生型一氧化氮合酶 (iNOS)、Bcl 2蛋白表达在前列腺癌中的意义以及两者之间的关系。　方法 :对 2 4例前列腺癌、15例前列腺增生及 5例正常前列腺组织行原位细胞凋亡检测及iNOS、Bcl 2蛋白免疫组化检测。　结果 :前列腺癌组的细胞凋亡率和iNOS的染色强度均显著高于前列腺增生组及正常前列腺组(P <0 .0 1) ,且Bcl 2蛋白表达阳性者的细胞凋亡率显著低于阴性者 (P <0 .0 1)。　结论 :前列腺癌的细胞凋亡率可反映其恶性程度 ;iNOS的表达与前列腺癌的分化程度无关 ,但与Bcl 2蛋白表达呈负相关 ;Bcl 2蛋白表达与前列腺癌细胞凋亡呈负相关。iNOS与Bcl 2均可通过影响前列腺癌细胞凋亡而在前列腺癌病理发生发展中起作用     

低剂量粒细胞巨噬细胞集落刺激因子诱导的小鼠骨髓未成熟树突状细胞抗成熟特性的研究

目的观察内毒素／脂多糖(LPS)、肿瘤坏死因子α(TNF-α)、干扰素1(IFN-γ)等促成熟物质对低剂量粒细胞巨噬细胞集落刺激因子(rmGM-CSF)诱导的小鼠骨髓未成熟树突状细胞(DC)成熟特性的影响。方法制备小鼠骨髓细胞，分别用不同剂量rmGM-CSF培养，6d后收集悬浮细胞进行检测。用LPS、TNF-α、IFN-1与小剂量rmGM-CSF培养获得的Dc(GM^low DC)共同孵育3d后，行混合淋巴细胞反应，观察其诱导未致敏脾淋巴细胞增殖的情况，并与大剂量rmGM-CSF培养获得的Dc(GM^high DC)进行比较。结果GM^lew DC不能激活未致敏脾淋巴细胞，且在与LPS、TNF-α和IFN-1共同培养3d后，仍不能有效诱导未致敏脾淋巴细胞增殖，刺激指数(SI)均＜2．00；而GM^high DC刺激未致敏脾淋巴细胞增殖的能力较强，SI为4．71。结论GM^lew DC具有对LPS、TNF-α和IFN-1刺激不敏感的抗成熟特性。     

Molecular targeting of Bcl-2 overcomes prostate cancer cell adaptation to XIAP gene downregulation

X-linked inhibitor of apoptosis (XIAP) is a suppressor of apoptosis that supports an increased survival and resistance to chemotherapy of human prostate cancer (PCa) cells. Effects of transient (24 h) and chronic (beyond 1 month) downregulation of XIAP in DU145 hormone refractory prostate cancer (HRPC) cells were studied. We found that transient downregulation of XIAP by siRNAs resulted in an increase of apoptosis and a decrease in Bcl-2 levels and sensitized PCa cells to cisplatin. XIAP downregulation by shRNA vector stable transfection led to upregulation of Bcl-2 protein. Our results identify the adaptability of PCa cells to chronic loss of XIAP in part through upregulation of Bcl-2 and indicate that multitargeting approach is the most effective application in the chemotherapy of human HRPC.     

Human vascular smooth muscle cells from diabetic patients are resistant to induced apoptosis due to high Bcl-2 expression

An emerging body of evidence suggests that vascular remodeling in diabetic patients involves a perturbation of the balance between cell proliferation and cell death. Our aim was to study whether arteries and vascular smooth muscle cells (VSMCs) isolated from diabetic patients exhibit resistance to apoptosis induced by several stimuli. Internal mammary arteries (IMAs) were obtained from patients who had undergone coronary artery bypass graft surgery. Arteries from diabetic patients showed increasing levels of Bcl-2 expression in the media layer, measured by immunofluorescence and by Western blotting. Human IMA VSMCs from diabetic patients showed resistance to apoptosis, measured as DNA fragmentation and caspase-3 activation, induced by C-reactive protein (CRP) and other stimuli, such as hydrogen peroxide and 7beta-hydroxycholesterol. The diabetic cells also exhibited overexpression of Bcl-2. Knockdown of Bcl-2 expression with Bcl-2 siRNA in cells from diabetic patients reversed the resistance to induced apoptosis. Consistent with the above, we found that pretreatment of nondiabetic VSMCs with high glucose abolished the degradation of Bcl-2 induced by CRP. Moreover, cell proliferation was increased in diabetic compared with nondiabetic cells. This differential effect was potentiated by glucose. We conclude that the data provide strong evidence that arterial remodeling in diabetic patients results from a combination of decreased apoptosis and increased proliferation.     

肿瘤坏死因子α对人肝癌多药耐药逆转作用的实验研究

目的研究肿瘤坏死因子α（TNF-α）对体外培养的人肝癌耐阿霉素细胞系（HepG2／ADM）多药耐药现象的逆转作用。方法不同浓度（100、500及2500U／ml）TNF-α作用于HepG2／ADM细胞72h后进行以下试验：用实时荧光定量聚合酶链反应技术检测各组多药耐药相关基因（MDR1）及脂质过氧化物酶体增殖物激活受体α（PPAR-α）基因的mRNA表达情况;用罗丹明外排法检测各组P-糖蛋白活性;用Annexin V检测0．5mg／L阿霉素诱导的各组细胞凋亡情况;利用MTF法检测各组耐药性的改变。结果TNF-α能诱导HepG2／ADM细胞的MDR1基因表达下调,PPAR-α基因表达上调,且能增加阿霉素诱导的凋亡细胞的比例及细胞毒作用。结论TNF-α可能分别通过抑制MDR1表达及促进PPAR-α表达而逆转HepG2／ADM细胞的耐药性。     

Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy.

OBJECTIVE: The purpose of the study was to characterize the requirements in terms of precursors, developmental pathways, and media for the generation of large numbers of mature dendritic cells (DC) under conditions acceptable for use in adjuvant, active immunotherapy strategies for surgically treated malignancies. SUMMARY BACKGROUND DATA: Although limited previously by the small numbers accessible, DC-based immunotherapies for malignancy have become more realistic with the development of methods for efficiently generating larger numbers of DC from peripheral blood mononuclear cells (PBMC) in vitro, but these methods rely on clinically unacceptable culture conditions (such as inclusion of fetal bovine serum), necessitating the development of methods for generating functionally equivalent DC in serum-free conditions. METHODS: Plastic-adherent PBMC (from healthy donors and patients with cancer) were incubated for 7 days with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) with and without tumor necrosis factor-alpha (TNF-alpha) in fetal bovine serum-containing and serum-free media and were analyzed by Wright's stain for morphology, flow cytometry for phenotype, and mixed lymphocyte reaction for allostimulatory function. RESULTS: Growth in either serum-containing or serum-free media supplemented with GM-CSF and IL-4 yielded a similarly heterogeneous population of cells, 6% to 10% of which had the morphology (large cells with thin projections), immunophenotype (including CD83+), and function of mature DC. Tumor necrosis factor-alpha significantly augmented the number of these mature DC, whereas preculture depletion of CD14+ PBMC virtually eliminated them. CONCLUSIONS: Generation of mature DC in the authors' serum-free clinically applicable conditions is similar to serum-containing conditions and requires CD14+ precursors, differentiation through a CD14-CD83- immature stage under the influence of GM-CSF and IL-4, and maturation into a CD83+ DC under the influence of TNF-alpha.     

Impact of apoptosis in acute rejection episodes after heart transplantation: immunohistochemical examination of right ventricular myocardial biopsies

OBJECTIVE: Acute rejection may lead to cell death following heart transplantation. Programmed cell death (apoptosis) has been described as a cofactor for cell loss in cardiac tissue. The aim of our study was to quantify the amount and extent of apoptotic cells during acute rejection episodes after orthotopic heart transplantation. PATIENTS AND METHODS: Right ventricular biopsies from 27 heart transplant recipients were classified histologically according to rejection grade. Formalin-fixed sections were processed for immunohistochemistry. TUNEL-positive cells were counted and the expression of apoptosis-modulating factors Bax, Bcl-x(L), Bcl-2, and Ki-67 (proliferation marker) was scored. P 相似文献   

Effects of cyclic stretch on prostatic cells in culture

PURPOSE: The fundamental process in the development of benign prostatic hyperplasia (BPH) is a loss of homeostasis between cell proliferation and apoptosis. Prostatic smooth muscle cells contract under adrenergic control. The response of a cell to stretch may have a role in the pathogenesis of BPH. MATERIALS AND METHODS: Monolayer cultures of human prostatic stromal and epithelial cell lines were exposed to cyclic stretch for 48 hours. RESULTS: Cyclic stretch conferred resistance to etoposide induced apoptosis. Underlying this apoptotic resistance was increased expression of the anti-apoptotic Bcl-2 family of proteins. As measured by thymidine incorporation, the rate of proliferation also increased in benign epithelial cells under cyclic stretch conditions. Furthermore, an increase in the production of platelet-derived growth factor by stromal cells and transforming growth factor-beta by epithelial cells occurred under such conditions. CONCLUSIONS: The observed changes in proliferation and apoptosis may contribute to the understanding of BPH, ultimately leading to therapeutic and preventive applications.     

Bcl-2 Protects Tubular Epithelial Cells From Ischemia/Reperfusion Injury by Dual Mechanisms

Ischemia/reperfusion (I/R) injury, which induces extensive loss of tubular epithelial cells, is associated with delayed graft function following kidney transplantation. Recent reports have suggested that cell death by I/R injury occurs by autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, as well as by apoptosis. Recently, we demonstrated that overexpression of the anti-apoptotic factor, Bcl-2, inhibited tubular apoptosis and subsequent tubulointerstitial damage after I/R injury. Autophagy is also observed in cells undergoing cell death in several diseases. Therefore, we hypothesized that increased Bcl-2 protein may protect tubular epithelial cells by suppressing autophagy and inhibiting apoptosis. In the present study, a transgenic mouse model (LC3-GFP TG) in which autophagosomes are labeled with LC3-GFP and Bcl-2/LC3-GFP double transgenic mice (Bcl-2/LC3-GFP TG) were used to examine the effect of Bcl-2 on I/R-induced autophagy. I/R injury, which is associated with marked disruption of normal tubular morphology, promoted the formation of LC3-GFP dots, representing extensively induced autophagosomes. On electron microscopy, the autophagosomes contained mitochondria in I/R-injured tubular epithelial cells. In contrast, Bcl-2 augmentation suppressed the formation of autophagosomes and there was less tubular damage. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R injury by suppressing autophagosomal degradation and inhibiting tubular apoptosis.     

Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.     

Increased apoptosis of immunoreactive host cells and augmented donor leukocyte chimerism, not sustained inhibition of B7 molecule expression are associated with prolonged cardiac allograft survival in mice preconditioned with immature donor dendritic cells plus anti-CD40L mAb.

BACKGROUND: We previously reported the association among donor leukocyte chimerism, apoptosis of presumedly IL-2-deficient graft-infiltrating host cells, and the spontaneous donor-specific tolerance induced by liver but not heart allografts in mice. Survival of the rejection-prone heart allografts in the same strain combination is modestly prolonged by the pretransplant infusion of immature, costimulatory molecule-(CM) deficient donor dendritic cells (DC), an effect that is markedly potentiated by concomitant CM blockade with anti-CD40L (CD154) monoclonal antibody (mAb). We investigated whether the long survival of the heart allografts in the pretreated mice was associated with donor leukocyte chimerism and apoptosis of graft-infiltrating cells, if these end points were similar to those in the spontaneously tolerant liver transplant model, and whether the pretreatment effect was dependent on sustained inhibition of CM expression of the infused immature donor DC. In addition, apoptosis was assessed in the host spleen and lymph nodes, a critical determination not reported in previous studies of either spontaneous or "treatment-aided" organ tolerance models. METHODS: Seven days before transplantation of hearts from B10 (H-2b) donors, 2x10(6) donor-derived immature DC were infused i.v. into C3H (H-2k) recipient mice with or without a concomitant i.p. injection of anti-CD40L mAb. Donor cells were detected posttransplantation by immunohistochemical staining for major histocompatibility complex class II (I-Ab) in the cells of recipient lymphoid tissue. CM expression was determined by two-color labeling. Host responses to donor alloantigen were quantified by mixed leukocyte reaction, and cytotoxic T lymphocyte (CTL) assays. Apoptotic death in graft-infiltrating cells and in areas of T-dependent lymphoid tissue was visualized by terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick-end labeling and quantitative spectrofluorometry. Interleukin-2 production and localization were estimated by immunohistochemistry. RESULTS: Compared with control heart transplantation or heart transplantation after only DC administration, concomitant pretreatment with immature donor DC and anti-CD40L mAb caused sustained elevation of donor (I-Ab+) cells (microchimerism) in the spleen including T cell areas. More than 80% of the I-Ab+ cells in combined treatment animals also were CD86+, reflecting failure of the mAb to inhibit CD40/ CD80/CD86 up-regulation on immature DC in vitro after their interaction with host T cells. Donor-specific CTL activity in graft-infiltrating cells and spleen cell populations of these animals was present on day 8, but decreased strikingly to normal control levels by day 14. The decrease was associated with enhanced apoptosis of graft-infiltrating cells and of cells in the spleen where interleukin-2 production was inhibited. The highest levels of splenic microchimerism were found in mice with long surviving grafts (>100 days). In contrast, CTL activity was persistently elevated in control heart graft recipients with comparatively low levels of apoptotic activity and high levels of interleukin-2. CONCLUSION: The donor-specific acceptance of rejection-prone heart allografts by recipients pretreated with immature donor DC and anti-CD40L mAb is not dependent on sustained inhibition of donor DC CM (CD86) expression. Instead, the pretreatment facilitates a tolerogenic cascade similar to that in spontaneously tolerant liver recipients that involves: (1) chimerism-driven immune activation, succeeded by deletion of host immune responder cells by apoptosis in the spleen and allograft that is linked to interleukin-2 deficiency in both locations and (2) persistence of comparatively large numbers of donor-derived leukocytes. These tolerogenic mechanisms are thought to be generic, explaining the tolerance induced by allografts spontaneously, or with the aid of various kinds of immunosuppression.     

睾丸去神经支配诱发睾丸组织内Bax、Bcl-2基因表达

目的:观察切除精索神经后睾丸组织内Bax、Bcl-2基因表达的变化,探讨睾丸去神经支配诱发生殖细胞凋亡的基因调控机制。方法:健康成年雄性SD大鼠18只,随机分为假手术组(n=6)、精索上神经切除组(n=6)、精索下神经切除组(n=6)。解剖显微镜下建立睾丸去神经支配大鼠模型,于术后1个月采用免疫组化法检测睾丸组织中Bax、Bcl-2蛋白的表达。结果:术后1个月各手术组大鼠睾丸组织内Bax蛋白表达均较假手术组显著升高(P<0.05),Bcl-2蛋白水平无显著变化。结论:Bax基因可能参与对睾丸去神经支配诱发生殖细胞凋亡过程的调控。     

MicroRNA-503 regulates high-glucose induced apoptosis of renal tubular epithelial cells by targeting Bcl-2

Objective To investigate the expression vibration of microRNA-503(miR-503) and its effect on target gene Bcl-2, caspase enzyme activity and apoptosis of human renal tubular epithelial cells (HK-2) induced by high glucose, and to clarify the pathogenesis of renal tubular injury induced by high glucose. Methods HK-2 cells were cultured in normal glucose group (NG), mannitol hypertonic control group (MA), and high glucose group (HG). The morphology of apoptotic cells was observed using inverted microscope. The expression of miR-503 was determined using real-time quantitative PCR. The apoptosis rate of HK-2 cells was detected by Annexin Ⅴ-FITC double dye using flow cytometry instrument. The expression of Bcl-2 and cleaved caspase-9 were detected by Western blotting. Results In the high glucose and mannitol groups HK-2 cell, an obviously increased apoptotic rate was observed under inverted microscope compared with normal glucose group (P＜0.05). MA and HG up-regulated miR-503 expression (P＜0.01), down-regulated anti-apoptotic protein Bcl-2 expression (P＜0.05) and up-regulated cleaved caspase-9 (P＜0.05). Conclusions The expression of miR-503 increases in HK-2 cells cultured by high glucose and mannitol. MiR-503 promotes apoptosis of HK-2 cells via activating mitochondrial apoptotic pathways and enhancing cleaved caspase-9 for Bcl-2 insufficiency. The tubular toxicity of high glucose is partly due to osmotic pressure. The miR-503 may be involved in diabetic tubular injury and may be a new therapeutic target of DN.     

大鼠未成熟树突状细胞体外扩增及功能鉴定

摘要：目的 探讨建立大鼠体外大量扩增未成熟树突状细胞(DC) 的方法, 以及不同剂量粒细胞巨噬细胞集落刺激因子(GM CSF)对大鼠DC分化成熟的影响。方法 分离纯化并扩增大鼠骨髓细胞，用不同剂量GM CSF培养,6 d 和10 d后收集悬浮细胞进行扫描电镜观察和免疫表型鉴定,并行混合淋巴细胞反应,观察其诱导未致敏T 淋巴细胞增殖的情况。结果 小剂量GM CSF 培养获得的DC(GMlowDC) 形态上具有DC 的典型特征,在细胞表型、细胞功能试验上具有未成熟的特性,具有DC 的典型特征,细胞表面高表达CD11c,低表达CD80，CD86及MHC II类分子,与大剂量GM CSF加IL 4的联合组培养获得的DC( GMhighDC) 相比,其体外刺激未致敏T 淋巴细胞的增殖能力较弱.结论 笔者所建立的培养未成熟DC 的方法是可行的；GM CSF的剂量与细胞的成熟程度相关。