强直性脊柱炎患者外周血T细胞凋亡及凋亡相关蛋白的表达

目的探讨强直性脊柱炎患者外周血T细胞凋亡相关蛋白的表达以及T细胞凋亡状况,分析其在疾病免疫发病机制的作用。方法CT检查患者骶髂关节并分级。尼龙棉柱法分离患者外周血T细胞,PI染色结合流式细胞术检测T细胞凋亡率,免疫组化法检测T细胞促凋亡蛋白FADD、Caspase．3、Caspase-8和抗凋亡蛋白FLIP表达。结果与健康对照相比,强直性脊柱炎患者外周血T细胞凋亡率明显降低（P〈0．05）;促凋亡蛋白FADD、Caspase-3、Caspase-8百分率均明显降低（P〈0．05）,抗凋亡蛋白FLIP百分率明显升高（P〈0．05）。结论强直性脊柱炎患者外周血T细胞凋亡蛋白及抗凋亡蛋白表达异常和T细胞凋亡不足与患者免疫学发病机制相关。     

SLE患者外周血中T、B细胞表面Fas和bcl—2表达的研究

目的 探讨活动期SLE患者外周血中T、B细胞表面Fas和bcl-2的表达水平及其与T、B细胞凋亡的关系。方法 采用流式细胞术,测定活动期SLE患者外周血T、B细胞表面Fas和bcl-2的表达,并同时测定患者血中T、B细胞的凋亡。结果 ①活动期SLE患者外周血中B细胞及CD8T细胞表面bcl-2的表达明显高于正常人（分别为P＜0．05）和P＜0．01）;CD4^ 及CD8^ T细胞表面Fas的表达高于正常人（分别为P＜0．05和P＜0．01）;B细胞表面Fas的表达降低（P＜0．01）;CD4^ T细胞bcl-2的表达下降（P＜0．01）。②活动期SLE患者血中B细胞的凋亡率明显下降,CD8^ T细胞数校正常对照组增加,CD4^ T细胞低于正常人（分别为：P＜0．01、P＜0．05和P＜0．01）。结论 SLE患者体内淋巴细胞凋亡的异常与T、B细胞表面Fas和bcl-2基因的表达的异常有关。     

Caspase-8的研究进展

细胞凋亡依赖一类对天冬氨酸特异的半胱氨酸蛋白酶 (caspases)产生的级联反应。caspase 8是死亡受体介导的凋亡途径中关键的启动子 ,能够通过寡聚而自身切割活化 ,并能激活下游caspase ,产生凋亡效应。除此之外 ,caspase 8在非死亡受体介导的凋亡、细胞增殖等方面发挥作用。caspase 8基因甲基化、基因突变及其它表达异常可导致细胞凋亡和增殖失调 ,从而参与某些疾病的发生。     

SLE患者外周血T细胞亚群凋亡特点及其相关机制的研究

目的:研究系统性红斑狼疮(SLE)患者外周血CD3+、CD4+、CD8+T细胞亚群的凋亡特点及其相关的凋亡机制。方法:采用三色荧光流式细胞术检测高活动性SLE患者、低/非活动性SLE患者以及正常对照者外周血T细胞亚群的百分比例、T细胞亚群膜表面Fas/FasL的表达率、T细胞亚群早期凋亡(AV+PI-)的情况;采用ABC-ELISA法测定各组SLE患者和正常对照者血清IL-10水平;对10例血清IL-10水平异常升高的高活动性SLE患者进行体外PBMCs培养实验,其中分别加入抗FasL抗体和抗IL-10抗体,48小时常规培养后分别检测PBMCs中T细胞亚群百分比例、T细胞亚群膜表面Fas/FasL的表达率、T细胞亚群早期凋亡的变化。结果:SLE患者外周血T细胞凋亡异常增多,其中以高活动性SLE患者CD4+T细胞亚群的凋亡尤为显著(P0.05),CD4+T细胞的异常凋亡与CD4+和CD8+T细胞膜表面表达增高的Fas/FasL密切相关(P0.05);各组SLE患者外周血血清IL-10水平均明显升高(P0.01),高活动性SLE患者更为明显,血清IL-10水平升高不仅与SLE主要临床检验指标相关,而且与CD4+/CD8+比值减少、CD4+T细胞高表达FasL相关(P0.05);随访研究显示,随着SLE病情好转、稳定,血清IL-10水平下降明显、T细胞亚群Fas/FasL的表达率明显减少、T细胞亚群凋亡逐渐减少,以上变化均在CD4+T细胞亚群中体现得最为明显(P0.05)。结论:SLE患者T细胞活动性异常升高,尤其是CD4+T细胞亚群凋亡增加,是SLE疾病发展的重要病理机制,IL-10作为能诱导T细胞高表达Fas/FasL的重要调节因子,参与了SLE患者CD4+T细胞凋亡的免疫调节,SLE患者异常增高的IL-10很可能通过Fas/FasL途径促进了T细胞尤其是CD4+T细胞亚群的凋亡。     

miR-671-5p通过靶向肿瘤坏死因子α诱导蛋白8调控胰腺癌细胞增殖和凋亡

目的：探讨miR-671-5p 靶向肿瘤坏死因子α 诱导蛋白8（ TNFAIP8）对胰腺癌细胞增殖和凋亡的影响。 方法：实时荧光定量PCR（ qRT-PCR）和免疫印迹检测20 例胰腺癌组织和与其配对的癌旁正常组织miR-617-5p、 TNFAIP8 mRNA和TNFAIP8表达水平,验证miR-671-5p 和TNFAIP8的靶向调控关系。将体外培养胰腺癌细胞 capan-1分为miR-NC组、miR-671-5p 组、si-NC 组、si-TNFAIP8 组、miR-671-5p+pcDNA组和miR-671-5p+pcDNATNFAIP8 组。采用四甲基偶氮唑蓝（ MTT）实验检测细胞活力,流式细胞术检测细胞凋亡,免疫印迹检测细胞周 期蛋白D1（ cyclin D1）、p21、B细胞淋巴瘤/ 白血病-2（ Bcl-2）和Bcl-2 相关蛋白（ Bax）的表达水平。结果：与 癌旁正常组织比较,胰腺癌组织miR-617-5p 表达量显著降低,TNFAIP8 的表达量显著升高。miR-671-5p 靶向负向 调控TNFAIP8 表达。与miR-NC组比较,miR-671-5p 组capan-1 细胞活力显著降低,细胞凋亡率显著升高,cyclin D1和Bcl-2 的表达量显著降低,p21 和Bax 的表达量显著升高;与si-NC 组比较,si-TNFAIP8 组capan-1 细胞活力 显著降低,细胞凋亡率显著升高,cyclin D1和Bcl-2 表达量显著降低,p21 和Bax 表达量显著升高;与miR-671- 5p+pcDNA 组比较,miR-671-5p+pcDNA-TNFAIP8 组capan-1 细胞活力显著升高,细胞凋亡率显著降低,cyclin D1和Bcl-2 的表达量显著升高,p21 和Bax 的表达量显著降低。结论：miR-671-5p 通过靶向下调TNFAIP8 抑制胰 腺癌细胞增殖并促进其凋亡。上调miR-671-5p 是胰腺癌潜在治疗靶点。     

热疗降低胶质瘤侵袭性作用过程中caspase 8改变的实验研究

目的:探讨caspase 8在热疗降低胶质瘤侵袭性过程中的作用。方法:利用Transwell构建胶质瘤侵袭模型,随机分为热疗对照组以及热疗30、60、120、180 min和240 min组。采用流式细胞术检测胶质瘤细胞凋亡水平,免疫印迹法检测胶质瘤细胞caspase 8的表达水平。结晶紫染色法测定胶质瘤侵袭性变化。结果:(1)热疗各组胶质瘤细胞凋亡率均明显高于热疗对照组(P0.05);(2)各热疗组胶质瘤细胞的caspase 8表达均高于热疗对照组,有统计学差异(P0.05);(3)各热疗组胶质瘤细胞的侵袭性均较对照组降低(P0.05)。结论:热疗后胶质瘤细胞侵袭性降低,这可能与caspase 8表达增加导致胶质瘤细胞凋亡有关。     

淋巴细胞共刺激信号和凋亡信号异常与RA免疫效应亢进的研究

目的：探究T淋巴细胞表面多种细胞信号分子所介导的细胞活化或凋亡信号在RA患者免疫功能紊乱中的作用。方法：采用流式细胞术检测RA患者外周血T细胞亚群及其表面共刺激分子cD154（cD40L）、CD30和凋亡受体CD95（Fas）的表达。结果：RA患者外周血T细胞亚群偏移，CD4^＋T细胞增加，CD8^＋T细胞减少；共刺激分子CD154在CD4^＋和CD8^＋T细胞上的表达均上调，但CD30分子的表达均降低，并以CD4^＋T细胞降低更为明显。同时，凋亡受体CD95分子在T细胞亚群上的表达均明显增加。结论：RA患者T淋巴细胞表面多种信号分子表达异常，共同导致了RA患者免疫功能紊乱。     

CBX8过表达或沉默对人神经胶质瘤细胞增殖和凋亡的影响

目的:探究染色质结构域蛋白8(chromodomain protein 8,CBX8)对人神经胶质瘤细胞增殖与凋亡的作用。方法:Western blot和RT-qPCR检测组织及细胞系中CBX8的表达。构建过表达CBX8和沉默CBX8载体,转染神经胶质瘤细胞T98G和U87MG,分别用MTT法和BrdU实验检测细胞增殖,流式细胞术检测细胞凋亡。结果:与正常脑组织和星形胶质细胞相比,神经胶质瘤组织及细胞中的CBX8蛋白和mRNA水平明显上升。在T98G和U87MG细胞中,过表达CBX8均促进细胞增殖,抑制细胞凋亡,并上调Rb/E2F1的表达水平,而沉默CBX8则作用相反。sh-E2F1转染细胞之后,cyclin D1的表达以及Bcl-2/Bax的比值降低。结论:CBX8可能通过Rb/E2F1通路调节胶质瘤细胞的增殖和凋亡。     

小G蛋白ARL8B是潜在的抗原发性胃低分化黏液样腺癌的药物作用靶标

目的发现更多表达小G蛋白ARL8B而ARL8A无表达的肿瘤细胞系,以扩大ARL8B在抗肿瘤药物研究中的应用范围。方法利用实时荧光定量PCR法测定一些肿瘤细胞系中的ARL8B/ARL8A的相对表达量;以ARL8A和ARL8B都表达的细胞系作对照,对新发现的只表达ARL8B的肿瘤细胞系实施siRNA降低ARL8B表达量,用CCK8法检测ARL8B表达量被降低后的细胞活力变化;用Western blot考察ARL8B表达量降低诱导肿瘤细胞凋亡的原因。结果发现MGC-803细胞表达ARL8B而ARL8A无表达,胃癌细胞系BGC-823和MKN-45均表达ARL8A和ARL8B。ARL8B被敲低后MGC-803细胞出现了明显的凋亡,BGC-823和MKN-45细胞无明显的凋亡出现。细胞自噬水平显著提高是引起细胞凋亡的原因。结论 MGC-803细胞系来源于胃低分化黏液样腺癌患者,是新发现的表达ARL8B而ARL8A无表达的肿瘤细胞系,降低ARL8B表达能导致明显的细胞凋亡,提示ARL8B是抗胃低分化黏液样腺癌潜在的药物作用靶标。     

自然流产中的早孕期蜕膜细胞Bcl-2 /Bax 比例异常

探讨早孕期蜕膜细胞细胞凋亡异常与妊娠失败的关系。应用TUNEL法测定凋亡的发生及凋亡细胞的定位，免疫组化法检测Bcl-2、Bax蛋白的表达和相互关系。结果发现：（1）正常早孕40d蜕膜组织细胞大量凋亡，Bcl-2蛋白表达量较低，Bax蛋白有较强表达。（2）正常早孕50d，凋亡细胞明显减少，Bcl-2的表达显著增强，Bax蛋白表达减弱。（3）早孕50d自然流产组，蜕膜组织大量凋亡，与同时期正常蜕膜组织相比，P＜0．01。Bcl-2蛋白表达明显降低，Bax蛋白表达明显增强，Bcl-2/Bax比例降低。早孕期蜕膜组织凋亡异常可能是自然流产的机制之一，Bcl-2/Bax途径可能是诱导早孕期蜕膜细胞凋亡的重要因素。     

Activated T killer cells induce apoptosis in lung epithelial cells and the release of pro-inflammatory cytokine TNF-alpha

Apoptosis is thought to be involved in lung epithelial cell damage in acute respiratory distress syndrome and interstitial pneumonia. Both the role of apoptosis and its underlying molecular mechanisms in human lung tissue remain unclear. To address these issues, we developed an in vitro assay in which a human lung epithelial cell line and a staphylococcal enterotoxin B (SEB)-reactive human CD8(+) CTL line were co-cultured in the presence of SEB. SEB-stimulated CD8(+) CTL induced apoptosis in the lung epithelial cell line primarily through the perforin/granzyme-mediated pathway. In these cells, apoptosis was initially independent of death receptor pathways. We also tested the effect of IFN-gamma on modulation of apoptosis in lung epithelial cells. In IFN-gamma-pretreated lung epithelial cells, CD95 (APO-1/Fas) activation as well as TNF-related apoptosis-inducing ligand (TRAIL) receptor and TNFR activation led to apoptosis. Furthermore, we found that the interaction of SEB-stimulated CD8(+) CTL with lung epithelial cells induced an increase in TNF-alpha secretion. These results suggest an important role for bacterial superantigen-reactive CD8(+) CTL in induction of lung epithelial cell apoptosis and in modulation of inflammatory processes in lung tissue.     

Costimulatory pathways mediate monocyte-dependent lymphocyte apoptosis in HIV

Examination of annexin V binding, an indicator of early apoptosis, on lymphocytes from HIV+ people immediately after isolation showed that both CD4(+) and CD8(+) T cells were apoptotic, whereas B cell apoptosis was induced mainly after incubation. CD8(+) T cell apoptosis correlated with fewer CD4(+) T cells, but not the level of viremia. To determine potential mechanisms for apoptosis, we examined FasL expression, which was dramatically elevated on CD14(+) monocytes; however, antibody to FasL did not reproducibly inhibit apoptosis. Rather, CD8(+) T cell apoptosis was caused by antigen-presenting cells because removal of monocytes or addition of antibodies to CD80 and CD86 reduced apoptosis. B cell apoptosis also involved costimulatory signals delivered by T cells but not monocytes. A unique CD8(bright)CD28(dim) T cell population died after costimulation by monocytes. Because this population was increased in patients with undetectable viremia, abnormal antigen-presenting cells may contribute to continued CD8(+) T cell exhaustion by inducing apoptosis.     

Infection of mice with Mycobacterium avium primes CD8+ lymphocytes for apoptosis upon exposure to macrophages

Mycobacterial infection is associated with granuloma formation in which the presence of apoptosis has been recognized. The role of CD4+ T and CD8+ T cells in host protection against mycobacterial infections has been demonstrated. Previous studies, however, have shown that CD8+ T cells have a limited role in host defense against Mycobacterium avium infection, and we hypothesize that M. avium infection could lead to T cell apoptosis. To investigate this hypothesis, C57BL/6 mice were infected with M. avium strain 101, and the rate of apoptosis of splenic lymphocytes cultured ex vivo with peritoneal macrophages was determined and compared with that of controls. When exposed to infected macrophages ex vivo, splenic lymphocytes from M. avium-infected mice underwent apoptosis, as determined by the TUNEL assay. This increased T cell apoptosis above the control level was observed after 3 weeks but not after only 1 week of infection in mice. No splenic T cell apoptosis was observed when lymphocytes from Mycobacterium smegmatis-infected mice were cultured in the presence of M. smegmatis-infected peritoneal macrophages. Likewise, macrophages infected in vitro with heat-killed M. avium did not trigger T cell apoptosis. Culture of macrophages in different chamber from lymphocytes, separated by a transwell membrane, was not associated with increase of apoptosis compared with uninfected control, suggesting a requirement for direct cell-cell interactions to trigger lymphocyte apoptosis. Using a double staining TUNEL followed by anti-mouse CD4 or anti-mouse CD8 monoclonal antibodies, it was observed that only CD8+ T cells but not CD4+ T cells underwent apoptosis at 3 weeks of infection. In conclusion, M. avium infection in C57/BL6 mice for 3 weeks renders CD8+ T cells prone to apoptosis when exposed ex vivo to macrophages infected with M. avium.     

RIP1 has a role in CD40-mediated apoptosis in human follicular lymphoma cells

CD40 is a cell surface receptor which belongs to tumor necrosis factor receptor (TNFR) family members. It transmits signals that regulate diverse cellular responses such as proliferation, differentiation, adhesion molecule expression and apoptosis. Unlike other TNFR family members (TRAIL-R, Fas-R and TNFR1), the CD40 cytoplasmic tail lacks death domain. However, CD40 is capable of inducing apoptosis in different types of cancer cells including lymphoma. The apoptotic effect of CD40 is linked to the involvement of Fas, TRAIL or receptor interacting protein 1 (RIP1) kinase. We have previously shown that CD40 activation has anti-apoptotic or apoptotic effect in follicular lymphoma (FL) cell lines. In this study, we investigated the mechanism by which CD40 mediates apoptosis in a follicular lymphoma cell line, HF4.9. We show here that CD40-induced apoptosis was dependent on caspase-8 activation because caspase-8 specific inhibitor, Z-IETD-FMK completely prevented apoptosis. Therefore, the involvement of TRAIL, Fas and RIP1 in caspase-8 activation was examined. The exogenous TRAIL-induced apoptosis was fully prevented by anti-TRAIL neutralizing antibody. However, the antibody had no effect on CD40-induced apoptosis indicating that CD40 did not induce the expression of endogenous TRAIL in HF4.9 cells. Moreover, the cells were not sensitive to Fas-mediated apoptosis. Interestingly, RIP1 specific inhibitor, necrostatin-1 decreased CD40-induced apoptosis, which showed that RIP1 has a role in caspase-8 activation. In conclusion, the survival or apoptotic effects of CD40-mediated signaling might be related to the differentiation stages of FL cells.     

Comparison of signaling pathways involved in apoptosis of a thymocyte hybridoma triggered by a rat thymic medullary epithelial cell line, dexamethasone or T-cell receptor cross-linking

Using an in vitro co-culture assay we found that a rat medullary thymic epithelial cell (TEC) line (TE-R2.5) induces apoptosis of the BWRT8 thymocyte hybridoma (TH) (CD4(hi)CD8(low) alphabetaTCR(hi)). TH apoptosis induced by this TEC line was predominantly mediated by direct cell-cell contacts and was potentiated by cross-linking of the T cell receptor (TCR) by R73 monoclonal antibody (mAb). Dexamethasone (Dx) also triggered TH apoptosis but inhibited death of these cells induced by TE-R2.5 cells or immobilized R73 mAb. The TEC-induced apoptosis was independent of the LFA-1/ICAM-1 interaction but partly depended on a novel 29 kDa molecule expressed on TE-R2.5 cells. All three types of TH apoptosis were followed by the cleavage of poly-(ADP-ribose)-polymerase and were blocked by a caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F.PKC stimulation by phorbol myristate acetate interfered with the TH apoptosis induced by TE-R2.5 and Dx, but did not modulate the effect of R73 mAb. On the contrary, inhibition of calcineurin with cyclosporine A did not influence the apoptosis induced by TE-R2.5 and Dx, but completely prevented the R73-triggered TH cell death. The TE-R2.5-mediated BWRT8 apoptosis was suppressed by Na-orthovanadate, an inhibitor of protein tyrosine phosphatases (PTP) as well as by genistein, a protein tyrosine kinase (PTK) inhibitor, while both compounds potentiated the effect of Dx. Blocking PTP, but not PTK decreased the proapoptotic effect of R73 mAb. These results, including those using a BWRT8 subclone (BWRT8-MDP.2) which is resistant to TCR-triggered apoptosis, but sensitive to apoptosis stimulated by TE-R2.5 and Dx, indicate that TE-R2.5-induced TH apoptosis in our model is different from apoptosis in other TEC co-culture models, published so far.     

Shiga toxin 1 induces apoptosis in the human myelogenous leukemia cell line THP-1 by a caspase-8-dependent, tumor necrosis factor receptor-independent mechanism

Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-alpha) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.     

Peptide-induced deletion of CD8 T cells in vivo occurs via apoptosis in situ

The ultimate fate of T cells undergoing antigen-induced cell death in vivo remains controversial. Whereas apoptosis of CD4+ T cells driven by superantigen is readily detectable in lymphoid organs, CD8+ T cells have been reported to disappear from the lymphoid organs and accumulate in the liver where they undergo apoptosis. Using transgenic mice that produce large numbers of ovalbumin-specific CD8+ T cells (OT-I cells), we were able to investigate the events that follow soluble peptide administration in an independent CD8+ T cell system. Here we show that the OT-I cells undergo proliferation and apoptosis in situ in lymphoid organs in response to antigenic stimulation with no evidence for liver involvement. This is similar to the course of events found for CD4+ T cell activation and counters the view that the liver is a general site for CD8+ T cell clearance following antigen-specific activation.      

Kinetics of Lymphocyte Apoptosis in Macaques Infected with Different Simian Immunodeficiency Viruses or Simian/Human Immunodeficiency Hybrid Viruses

Increased rates of T-cell apoptosis have been detected in human immunodeficiency virus (HIV)-infected individuals and in the simian immunodeficiency model (SIV) for AIDS research. We have infected macaques with virulent SIV or SIV/HIV hybrid viruses (SHIV) of different pathogenic potentials to study the early kinetics of apoptosis in this model. Animals infected with SIV showed an increased degree of apoptosis in their peripheral blood mononuclear cells as early as 8 weeks after virus inoculation. Apoptotic cells were detected in the CD4 and CD8 cell populations of infected animals. In contrast, apathogenic SHIV did not lead to increased lymphocyte apoptosis and moderately pathogenic SHIV induced only transient apoptosis. T-cell death was temporally linked to viral replicationin vivo.Furthermore, lymphocyte apoptosis in infected macaques was associated with impaired proliferative responses of helper T-cells and with CD4 cell depletion. The monkey model described here provides the opportunity for testing early therapeutic interventions to prevent virus-induced programmed cell death and the subsequent onset of AIDS.     

A new function for LAT and CD8 during CD8‐mediated apoptosis that is independent of TCR signal transduction

The majority (>95%) of thymocytes undergo apoptosis during selection in the thymus. Several mechanisms have been proposed to explain how apoptosis of thymocytes that are not positively selected occurs; however, it is unknown whether thymocytes die purely by “neglect” or whether signaling through a cell‐surface receptor initiates an apoptotic pathway. We have previously demonstrated that on double positive thymocytes the ligation of CD8 in the absence of TCR engagement results in apoptosis and have postulated this is a mechanism to remove thymocytes that have failed positive selection. On mature single positive T cells CD8 acts as a co‐receptor to augment signaling through the TCR that is dependent on the phosphorylation of the adaptor protein, linker for activation of T cells (LAT). Here, we show that during CD8‐mediated apoptosis of double positive thymocytes there is an increase in the association of CD8 with LAT and an increase in LAT tyrosine phosphorylation. Decreasing LAT expression and mutation of tyrosine residues of LAT reduced apoptosis upon crosslinking of CD8. Our results identify novel functions for both CD8 and LAT that are independent of TCR signal transduction and suggest a mechanism for signal transduction leading to apoptosis upon CD8 crosslinking.