雷公藤内酯醇诱导淋巴细胞凋亡的作用与细胞活化程度的关系

目的　通过观察雷公藤内酯醇诱导 Ｂ Ａ Ｌ Ｂ／ｃ 小鼠脾细胞凋亡与细胞活化程度的关系,探讨该化合物免疫抑制的作用机理。方法　 以脾淋巴细胞为研究对象, Ｐ Ｈ Ａ Ｐ、 Ｐ Ｍ Ａ 和ｒｈ Ｉ Ｌ２ 为有丝分裂原活化淋巴细胞。结合荧光染色法, Ｄ Ｎ Ａ 凝胶电泳及 Ｄ Ｎ Ａ 片段测定等方法,检测雷公藤内酯醇诱导凋亡的作用。结果　（１） 雷公藤内酯醇能够造成活化的淋巴细胞凋亡,并呈浓度依赖性;（２） 雷公藤内酯醇不能使处于静止状态的脾淋巴细胞凋亡;（３） 雷公藤内酯醇诱导淋巴细胞凋亡的作用,与淋巴细胞的活化程度和增殖反应程度密切。结论　雷公藤内酯醇不能造成静止状态的淋巴细胞发生细胞凋亡,只能够诱导已活化淋巴细胞发生细胞凋亡,并以此影响机体的免疫功能。     

雷化藤内酯醇诱导淋巴细胞凋亡的作用与细胞活化 …

通过观察雷公藤内酯醇诱导ＢＡＬＢ／ｃ小鼠脾细胞凋亡与细胞活化程度的关系,探讨该化合物免疫抑制的作用机理。方法以脾淋巴细胞为研究对象,ＰＨＡ－Ｐ、ＰＭＡ和ｒｈＩＬ－２为有丝分裂原活化淋巴细胞。     

雷公藤内酯醇抑制Raji细胞增殖和迁移的体外实验研究

 目的： 研究雷公藤内酯醇在体外是否具有抗淋巴瘤细胞增殖和迁移的效应。方法： 采用四甲基偶氮唑蓝（MTT）比色法检测雷公藤内酯醇对Raji细胞增殖的作用；采用Annexin V/PI双标流式细胞术检测雷公藤内酯醇对Raji细胞凋亡的影响；采用流式细胞仪检测雷公藤内酯醇对Raji细胞表面CXCR4受体表达的影响；并采用Transwell微孔隔离室迁移实验观察雷公藤内酯醇对Raji细胞体外迁移作用的影响。结果： ①雷公藤内酯醇能以时间和剂量依赖方式抑制Raji细胞的增殖，24 h和36 h的 IC50值分别为43.06 nmol/L和25.08 nmol/L。② 随雷公藤内酯醇药物浓度的增加和作用时间的延长，Raji细胞凋亡率逐渐增高，且呈时效和量效关系。③ Raji细胞经不同浓度雷公藤内酯醇（12.5、25、50 nmol/L）处理后，CXCR4表达率（34.66%±4.34%、23.74%±1.87%、18.10%±1.18%）明显低于对照组（72.47%±7.28%，P＜0.01），此抑制效应呈剂量依赖性；④ Transwell趋化活性分析显示雷公藤内酯醇能抑制rhSDF-1α对Raji细胞的趋化作用，此抑制效应也呈量效关系。结论： 雷公藤内酯醇具有抗淋巴瘤细胞增殖和诱导凋亡的效应，并能阻断Raji细胞的体外迁移，其机制与其对SDF-1/CXCR4生物学轴的抑制效应有关。     

超抗原SEB激活诱导CD4^＋T细胞凋亡模型的建立

目的 为了探讨超抗原活化诱导ＣＤ４＾＋Ｔ淋巴细胞凋亡分子机理及其信号传导途径,有必要建立超抗原活化诱导ＣＤ４＾＋Ｔ细胞凋亡模型。方法 采用电镜观察细胞凋亡的形态学特征,借助流式细胞仪ＰＩ染色观察细胞凋亡的光散射特征及亚二倍体核型峰特征,琼脂糖凝胶电泳分析细胞凋亡的ＤＮＡ片段化图谱,最后采用改进的二苯胺（ＤＰＡ）法定量分析细胞凋亡ＤＮＡ片段化百分率。结果 ４ｕｇ／ｍＬ ＳＥＢ刺激静息的ＳＥＢ应答ＣＤ４＾＋Ｔ细胞１２ｈ后,电镜下观察呈现典型的凋亡形态学特征;流式细胞仪ＰＩ染色分析表明,在二倍体峰的左侧出现亚二倍体核型峰典型凋亡特征,在光散射图谱上呈现低于正常细胞的前向散射和高于正常细胞的侧向散射;琼脂糖凝胶电泳分析呈现典型的凋亡ＤＮＡ梯状图谱;ＤＮＡ片段化百分率分析表明,超抗原ＳＥＢ活化诱导ＣＤ４＾＋Ｔ细胞凋亡率的     

雷公藤多甙诱导人前骨髓白血病细胞的凋亡

探讨雷公藤多甙诱导人前骨髓白血病细胞凋亡的作用。方法将ＨＬ－６０与雷公藤多甙、泼尼松共同培养,观察细胞的细胞凋亡形态学变化,如细胞膜泡样变、细胞浆及细胞核内染色质固缩、凋亡小体出现以及ＤＮＡ规律性断裂。（２）细胞周期分析提示作用首先影响增殖期细胞,并且呈剂量、时间依赖性。（３）雷公藤多甙诱导ＨＬ－６０细胞凋亡的作用较泼尼松强;两种药物间无协同作用。结论体外实验发现雷公藤多甙具有诱导细胞凋亡的作用,     

雷公藤甲素对诱导CD4+、CD8+T细胞凋亡的作用

目的 探讨雷公藤甲素对ＣＤ４＾＋、ＣＤ８＾＋Ｔ细胞凋亡的作用。方法 分离后的正常人外周血ＣＤ４＾＋、ＣＤ８＾＋Ｔ细胞在外培养条件下,加入雷公藤甲素处理,然后用３末端脱氧核苷酸基（ＴｄＴ）介导的ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ）检测凋亡细胞。结果 雷公藤甲素处理后ＣＤ４和ＣＤ８＾＋细胞凋亡率比对照组显著增加,但ＣＤ４＾＋、ＣＤ８＾＋Ｔ细胞凋亡率则针显著差别。结论 雷公藤甲素诱导诱活化的ＣＤ４＾＋、Ｃ     

雷公藤内酯醇对AD细胞模型海马神经元凋亡的影响

目的:研究雷公藤内酯醇对阿尔茨海默病(AD)细胞模型海马神经元凋亡的影响,探讨雷公藤内酯醇治疗AD的可能机制。方法:用凝聚态Aβ1-40(20μg/ml)刺激的小胶质细胞条件培养液(MCM)作用于培养的大鼠海马神经元,建立AD细胞模型,应用MTT法和TUNEL染色,观察不同剂量的雷公藤内酯醇(5μg/ml和25μg/ml)在不同时程(2 h和24 h)内对AD细胞模型海马神经元凋亡的影响。结果:加药后2 h除模型MCM组海马神经元凋亡数高于正常对照组和正常MCM组(P<0.05)外,其余各组之间海马神经元凋亡数无明显差异。加药后24 h,模型MCM组海马神经元凋亡数较正常对照组和正常MCM组显著增多(P<0.01);低剂量用药MCM组和高剂量用药MCM组海马神经元凋亡数与模型MCM组比较显著降低(P<0.05,P<0.01);高剂量用药MCM组海马神经元凋亡数较低剂量用药MCM组明显降低(P<0.01)。结论:雷公藤内酯醇对AD细胞模型海马神经元的凋亡具有抑制作用。     

已烯雌酚诱导T细胞肿瘤Jurkat细胞系凋亡中转录因子Oct-1的表达

研究己烯雌酚对Ｔ细胞肿瘤的细胞凋亡诱导作用,以及凋亡过程中转录因子Ｏｃｔ－１的表达,方法采用ＤＮＡ梯形片段化,荧光染色流式细胞技术分析细胞凋亡,以电泳泳 动度迁移率法检测Ｏｃｔ－１的表达。结果己烯雌酚可诱导Ｔ细胞出现细菌凋亡,并有典型的ＤＮＡ梯形片段化,１０μｇ己烯雌酚诱导１２ｈ后,凋亡过程中细胞数达３０％以上。己烯雌酚诱导Ｔ细胞凋亡过程与Ｏｃｔ－１转录因子的表达有关。结论己烯雌酚可诱导Ｔ细胞肿瘤     

雷公藤甲素对嘌呤霉素所致足细胞凋亡的抑制作用

目的 为了探讨雷公藤治疗足细胞病变的机制,利用嘌呤霉素诱导足细胞凋亡的体外损伤模型,观察雷公藤甲素对足细胞损伤的保护作用及其机制.方法 用CCK法观察雷公藤甲素对足细胞增殖活性的影响;应用TUNEL染色法定性观察雷公藤甲素对嘌呤霉素诱导的足细胞凋亡的保护作用;采用PI染色后再经流式细胞仪来定量分析雷公藤甲素对足细胞凋亡的抑制效应.结果 雷公藤甲素质量浓度小于1 μg/mL时对足细胞生存率没有明显影响;100 μg/mL嘌呤霉索诱导足细胞凋亡,但是此凋亡效应可以被1 μg/mL雷公藤甲素抑制;而且这种抑制作用具有时间依赖性.结论 雷公藤甲素通过抑制细胞凋亡来保护足细胞.雷公藤甲素对足细胞的保护作用可能是临床上雷公藤有效治疗足细胞病变的机制之一.     

雷公藤红素通过ROS/JNK途径诱导Saos-2细胞发生caspase依赖的凋亡

目的:探讨雷公藤红素对人骨肉瘤细胞Saos-2的杀伤作用及相关机制。方法:将人骨肉瘤细胞Saos-2用各种不同的浓度的雷公藤红素治疗后,采用MTT法检测雷公藤红素对Saos-2细胞活力的抑制作用;采用流式细胞术检测用雷公藤红素处理后Saos-2细胞的凋亡及活性氧簇(ROS)的产生;Western blot检测雷公藤红素处理后Saos-2细胞cleaved caspase-9、cleaved caspase-3和磷酸化JNK的表达水平。结果:雷公藤红素有显著的体外抗骨肉瘤作用,能显著抑制Saos-2细胞的活力。雷公藤红素可显著诱导Saos-2细胞发生凋亡,产生ROS。雷公藤红素处理后Saos-2细胞cleaved caspase-9、cleaved caspase-3和磷酸化JNK的表达水平均显著上升。结论:雷公藤红素通过ROS/JNK途径诱导人骨肉瘤Saos-2细胞发生caspase依赖的凋亡。     

Analysis of susceptibility of mature human T lymphocytes to dexamethasone-induced apoptosis

We present evidence that dexamethasone (Dex), a synthetic glucocorticosteroid, causes apoptosis in mature human T cells, similarly to what has been reported for murine T lymphocytes. Human T cell clones and short-term activated T lymphocytes treated with Dex show the characteristic pattern of apoptotic cells, such as hypodiploid nuclei, chromatin condensation and DNA fragmentation into oligonucleosomal fragments. However, Dex susceptibility of T cells to apoptosis is cell cycle-dependent. The progression in the proliferative cell cycle (G1 versus S) rescues Dex-treated T cells from apoptosis. Moreover, occupancy of the T cell receptor reverses Dex-induced apoptotic phenomena. These observations suggest that glucocorticoids contribute to the regulation of the proliferative or the suicidal response of antigen-activated human T cells.     

Activation-induced T cell death occurs at G1A phase of the cell cycle

Peripheral negative selection of cycling T cells after TCR engagement and deletion of activated T cells after an immune response occur by an apoptotic process termed activation-induced cell death (AICD). The cross-linking of TCR-CD3 complex with anti-CD3 monoclonal antibody led to significant apoptotic cell death in peripheral blood T cells. To further define cell cycle restriction points for triggering AICD in T cells, we evaluated the association between cell cycle progression and death signal transduction. Simultaneous DNA / RNA quantification analysis revealed that T cells entering G1A phase of the cell cycle may acquire sensitivity to AICD. The activation of caspase-3 was induced when T cells entered G1A phase. Up-regulation of cyclin-dependent kinases (Cdk4 and Cdk6) and cyclin D3 was initiated in TCR-stimulated T cells entering G1A phase and expression of these markers steadily increased as T cells progressed from G1A into G1B phase. Interestingly, caspase-3 inhibitors could inhibit the up-regulation of these G1 cell cycle regulators and induce G0 / G1A arrest as well as the inhibition of AICD. On the basis of these results, AICD signals are most likely transduced into TCR-stimulated T cells entering G1A phase. T cells that fail to progress from G1A into G1B phase undergo AICD.     

Activation of caspase-3-like enzymes in non-apoptotic T cells

The activation of the caspase family of cysteine proteases is a key step in the implementation of apoptotic cell death leading to further downstream effects such as DNA fragmentation. In cultured tumor cells, caspase activity appears only when cells are undergoing apoptosis. Here we show that human and murine T lymphocytes acquire high intracellular activities of cell death-specific caspases upon activation by mitogens and IL-2 without evidence that apoptosis is proceeding. The highest activity is seen when cells are mitogen activated for 3 days. On a per cell basis, caspase activity in activated T cells is much higher than in tumor cells induced to undergo apoptosis. In the presence of exogenously added IL-2 cells stay alive and maintain a high level of caspase activity while IL-2 withdrawal results in cell death and decline of caspase activity. Caspase activity can also be measured in extracts from spleen and lymph nodes from mice injected with superantigen. While in tumor cell lines caspase activity correlates with cleavage of poly(ADP)-ribose polymerase (PARP) and DNA fragmentation, in activated T cells cleavage products of cellular PARP can be detected whereas DNA fragmenting activity appears only upon IL-2 withdrawal which coincides with cell death. These data show that caspase activation in intact cells does not necessarily lead to cell death and argue for a checkpoint in the apoptotic pathway downstream of caspases. Furthermore, they provide a molecular correlate for the high susceptibility of activated T cells for apoptosis.     

Propriocidal apoptosis of mature T lymphocytes occurs at S phase of the cell cycle

We found that mature nontransformed CD4⁺ and CD8⁺ T lymphocytes could be made susceptible to T cell receptor(TcR)-mediated apoptosis by pretreatment with interleukin-4 (IL-4) or interleukin-2 (IL-2). The degree of susceptibility to death could be correlated with the level of cell cycling as measured by thymidine incorporation, cell doubling times, or the number of cells incorporating bromodeoxyuridine during S phase. However, using pharmacologic cell cycle blocking agents, we found that progression through the cell cycle was not required for cell death. Rather, we found that cells must be in a certain phase of the cell cycle to be susceptible to TcR-mediated death. Cells blocked in G1 phase were resistant to T cell receptor-induced apoptosis, whereas cells blocked in S phase were susceptible. These observations suggest that an important feature of growth lymphokines is their ability to drive T cells into portions of the cell cycle where they are sensitive to antigen receptor-induced apoptosis. Furthermore, these results provide additional evidence that the T cell growth lymphokines IL-2 and IL-4 may participate in the down-regulation of T cell responses by apoptosis a pathway we have termed “propriocidal regulation”.     

Differential effects of sex steroids on T and B cells: modulation of cell cycle phase distribution, apoptosis and bcl-2 protein levels.

Sex steroids have dramatic and differential effects on classic endocrine organ proliferation and apoptosis. In this investigation we sought to delineate similar effects of sex steroids on proliferation, cell cycle phase and apoptosis in lymphocyte cell lines as models for T and B cells. Estrogen and testosterone inhibited T cell line proliferation, induced accumulation of cells in S/G(2)M phases of the cell cycle, and increased apoptosis in a concentration- and time-dependent manner. There was a more modest effect of estrogen and testosterone on cell cycling and apoptosis in B lymphocyte cell lines, suggesting that estrogen and testosterone are inhibitory to T but not B cell lines. In comparison, progesterone induced cytostasis and modestly increased apoptosis in both T and B cell lines. Estrogen and testosterone were not antagonistic or synergistic to each other in their effects on cell cycle phase distribution, and only minimally synergistic for apoptosis. In contrast, progesterone antagonized cell cycle and apoptotic effects of estrogen in T cells. Estrogen-induced cell cycle and apoptotic effects in T cell lines were associated with suppression of bcl-2 protein levels, which were unaffected in Raji B cells. Progesterone also antagonized the estrogen-induced changes in T cell bcl-2 protein levels. These results suggest that there may be significant and differential sex steroid effects on T and B lymphocytes that may be important to sexual dichotomies in immune and autoimmune responses.     

IFN-beta induces caspase-mediated apoptosis by disrupting mitochondria in human advanced stage colon cancer cell lines.

Various human colon cancer cell lines tested in vitro differed significantly in susceptibility to growth inhibition of recombinant human interferon-beta (rHuIFN-beta). Two p53-mutant lines, COH and CC-M2, derived from high-grade colon adenocarcinoma, showed signs of apoptosis after treatment with 250 IU/ml of HuIFN- beta in the culture medium. The similarly p53-mutated HT-29 line from a grade I adenocarcinoma showed no apoptosis, however, and only cell cycle G1/G0 or S phase retardation with 1000 IU/ml HuIFN-beta. After HuIFN-beta exposure, COH and CC-M2 cells showed increased levels of Fas and FasL proteins, alteration of mitochondrial membrane potential, and activation of caspase-9, caspase-8, and caspase-3 in a time-dependent manner. Treatment of COH and CC-M2 cells with anti-FasL antibodies or rFas/Fc fusion protein, however, could not prevent the apoptosis induced by HuIFN-beta. In contrast, cell-permeable specific inhibitors of the three caspases could inhibit the DNA fragmentation and cell death but not the mitochondrial membrane potential changes. Treatment with mitochondria-stabilizing reagents could significantly abrogate the apoptosis and caspase activation induced by HuIFN-beta. These results suggest that in COH and CC-M2 colon cancer cell lines, HuIFN-beta induces apoptosis mainly through mitochondrial membrane alteration and subsequent activation of the caspase cascade pathway, but not by the Fas/FasL interaction or the p53-dependent apoptotic mechanism.     

浅蓝菌素诱发人结肠癌细胞凋亡的实验研究

目的　探讨脂肪酸合酶抑制剂———浅蓝菌素能否阻遏人结肠癌细胞增殖与诱发凋亡。方法　采用人结肠癌细胞系LoVo ,应用细胞形态学观察 ,噻唑蓝法 (MTT法 ) ,片断DNA琼脂糖凝胶电泳及流式细胞仪等方法进行检测和观察。结果　LoVo细胞在浅蓝菌素作用下 ,细胞增殖被阻遏 ,并呈现剂量效应关系 ,浅蓝菌素浓度 10 -9～ 10 -5mol/L增殖抑制率由 ( 2 1.0± 15 .9) %到 ( 96 .3± 2 7) %(P <0 .0 5或 0 .0 1)。同时诱发细胞发生凋亡。凋亡细胞表现为细胞固缩 ,核染色质凝聚、边集或断裂。细胞DNA裂解片段呈典型的“阶梯状”排列的条带 ,流式细胞仪显示“凋亡”峰 ,细胞周期分析浅蓝菌素能阻滞肿瘤细胞从S期进入G2 M期 ,并诱导其细胞凋亡。浅蓝菌素对人成纤维细胞增殖无明显影响。结论　脂肪酸合酶抑制剂可能通过抑制LoVo细胞内源性脂肪酸合成 ,诱发细胞凋亡来阻遏LoVo细胞的增殖     

Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division

Cells expressing indoleamine 2,3‐dioxygenase (IDO), an enzyme which catabolizes tryptophan, prevent T‐cell proliferation in vitro, suppress maternal antifetal immunity during pregnancy and inhibit T‐cell‐mediated responses to tumour‐associated antigens. To examine the mechanistic basis of these phenomena we activated naïve murine T cells in chemically defined tryptophan‐free media. Under these conditions T cells expressed CD25 and CD69 and progressed through the first 12 hr of G0/G1 phase but did not express CD71, cyclin D3, cdk4, begin DNA synthesis, or differentiate into cytotoxic effector cells. In addition, activated T cells with their growth arrested by tryptophan deprivation exhibited enhanced tendencies to die via apoptosis when exposed to anti‐Fas antibodies. Apoptosis was inhibited by caspase inhibitor and was not observed when T cells originated from Fas‐deficient mice. These findings suggest that T cells activated in the absence of free tryptophan entered the cell cycle but cell cycle progression ceased in mid‐G1 phase and T cells became susceptible to death via apoptosis, in part though Fas‐mediated signalling. Thus, mature antigen‐presenting cells expressing IDO and Fas‐ligand may induce antigen‐specific T‐cell tolerance by blocking T‐cell cycle progression and by rapid induction of T‐cell activation induced cell death in local tissue microenvironments.     

用反义寡核苷酸阻断热休克蛋白70表达诱导卵巢癌细胞凋亡

目的 观察特异性热体克蛋白７０（ｈｅａｔ－ｓｈｏｃｋ ｐｒｏｔｅｉｎ,ＨＳＰ）反义寡核苷酸阻断卵巢癌细胞的ＨＳＰ７０表达,及其对卵巢癌细胞生长和增殖的影响。方法 用胎盘蓝拒染法计算卵巢癌细胞的生长抑制率。Ｇｉｅｎｓａ染色法从形态上了解凋亡的发生,用琼脂糖凝胶电脉进一步检测发生凋亡的特征性ＤＮＡ降解,流式细胞仪定量分析凋亡发生率及周期性异性。结果 用ＨＳＰ７０反义寡核苷酸处理的卵巢细胞表现有显的生长     

T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma

BACKGROUND: Asthma is an inflammatory airway disease associated with an infiltration of T cells and eosinophils, increased levels of pro-inflammatory cytokines, and shedding of bronchial epithelial cells (ECs). OBJECTIVE: Shedding of bronchial ECs is characterized by loss of the normal bronchial pseudostratified epithelium and the maintenance of a few basal cells on a thickened basement membrane. The aim of this study was to investigate whether, and by which mechanism, T cells and eosinophils can cause damage to airway ECs. METHODS: Bronchial ECs, cultured and exposed to cytokines, eosinophil cationic protein, activated T cells, and eosinophils were studied for the expression of apoptosis receptors (flow cytometry, immunoblotting, and RNA expression) and for the susceptibility for undergoing apoptosis. In addition, bronchial biopsy specimens from patients with asthma were evaluated for EC apoptosis. RESULTS: We demonstrate herein that the respiratory epithelium is an essential target of the inflammatory attack by T cells and eosinophils. Bronchial ECs underwent cytokine-induced cell death with DNA fragmentation and morphologic characteristics of apoptosis mediated by activated T cells and eosinophils. T cell- and eosinophil-induced EC apoptosis was blocked by inhibition of IFN-alpha and TNF-alpha; the Fas ligand-Fas pathway appears to be less important. Recombinant eosinophil cationic protein induced mainly necrosis of ECs. Furthermore, we demonstrated in situ apoptotic features of ECs in bronchial biopsy specimens of asthmatic patients. CONCLUSION: T cell- and eosinophil-induced apoptosis represents a key pathogenic event leading to EC shedding in asthma.