UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotese-mediated shedding

PURPOSE: L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITC-labelled annexin-V in the presence ofpropidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. RESULTS: PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. CONCLUSION: UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte trafficking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.     

The effect of the ratio of CD4+ to CD8+ T-cells on radiation-induced apoptosis in human lymphocyte subpopulations

PURPOSE: Apoptosis occurs spontaneously in cultured human peripheral blood lymphocytes but is enhanced by exposure to ionizing radiation. Subpopulations of lymphocytes are known to have varying radiosensitivities to radiation-induced apoptosis. The purpose of this study was to examine the radiation-induced apoptotic response of CD4(+) and CD8(+) T-cells incubated as a complete lymphocyte population. MATERIALS AND METHODS: Using a four-colour flow-cytometry method, which measures annexin-V binding to phosphatidyl serine and propidium iodide, spontaneous and radiation-induced apoptosis was measured in the total lymphocyte fraction and in CD4(+) and CD8(+) T-cell subpopulations. RESULTS: It was found that CD8(+) T-cells were more sensitive to radiation-induced apoptosis than CD4(+) T-cells at doses up to 2 Gy. The yield of radiation-induced apoptosis in the total lymphocyte fraction decreased with increasing ratios of CD4(+) to CD8(+) T-cells (CD4/CD8 ratio). By manipulating the CD4/CD8 ratio within lymphocyte cultures, it was found that the CD4/CD8 ratio had a dramatic effect on the yield of spontaneous apoptosis of total lymphocytes fraction and CD4(+) T-cells but not CD8(+) T-cells. CONCLUSION: The CD4/CD8 ratio affects the apoptotic response of human lymphocytes and CD4(+) T-cells.     

The effect of the ratio of CD4 + to CD8 + T-cells on radiation-induced apoptosis in human lymphocyte subpopulations

Purpose: Apoptosis occurs spontaneously in cultured human peripheral blood lymphocytes but is enhanced by exposure to ionizing radiation. Subpopulations of lymphocytes are known to have varying radiosensitivities to radiation-induced apoptosis. The purpose of this study was to examine the radiation-induced apoptotic response of CD4 + and CD8 + T-cells incubated as a complete lymphocyte population. Materials and Methods: Using a four-colour flow-cytometry method, which measures annexin-V binding to phosphatidyl serine and propidium iodide, spontaneous and radiation-induced apoptosis was measured in the total lymphocyte fraction and in CD4 + and CD8 + T-cell subpopulations. Results: It was found that CD8 + T-cells were more sensitive to radiation-induced apoptosis than CD4 + T-cells at doses up to 2 Gy. The yield of radiation-induced apoptosis in the total lymphocyte fraction decreased with increasing ratios of CD4 + to CD8 + T-cells (CD4/CD8 ratio). By manipulating the CD4/CD8 ratio within lymphocyte cultures, it was found that the CD4/CD8 ratio had a dramatic effect on the yield of spontaneous apoptosis of total lymphocytes fraction and CD4 + T-cells but not CD8 + T-cells. Conclusion: The CD4/CD8 ratio affects the apoptotic response of human lymphocytes and CD4 + T-cells.     

脑缺血再灌注后神经细胞凋亡的研究

 目的通过大鼠脑缺血再灌注模型,观察缺血1.5 h后,再灌注12、24、48和72h皮层、尾壳核和海马细胞凋亡的变化.方法采用线栓法制成大鼠左侧大脑中动脉缺血再灌注模型(n=21),应用末端转移酶介导的dUTP-DIG缺口末端标记法(TUNEL),标记凋亡细胞断裂的DNA.结果脑缺血再灌注后12 h,左侧大脑中动脉缺血区皮层和尾壳核出现大量凋亡细胞,于再灌注后24～48 h达高峰.结论缺血再灌注早期可诱发细胞凋亡.     

神经酰胺与辐射诱导细胞凋亡

神经酰胺作为神经鞘脂类的主要成员之一,是一个主要调节细胞活动的第二信使,以转导细胞凋亡等细胞生长抑制活动信号为主。研究表明,神经酰胺通路不同水平的功能性变化直接影响细胞的药敏感性和辐射敏感性,肿瘤细胞神经酰胺缺陷可导致肿瘤细胞对药物和电射的敏感性下降。     

DNA repair foci and late apoptosis/necrosis in peripheral blood lymphocytes of breast cancer patients undergoing radiotherapy

Abstract

Purpose: Double-strand breaks (DSB) repair and apoptosis are assumed to be key factors in the determination of individual variability in response to radiation treatment. In this study we investigated tumor protein p53 (TP53) binding protein 1 (53BP1) and phosphorylated histone 2A family member X (γH2AX) foci, γH2AX pan-staining and late apoptosis/necrosis (LAN) in lymphocytes from breast cancer (BC) patients undergoing radiotherapy.

Materials and methods: BC patients were subjected to local radiotherapy with fractionated doses using linear accelerator. Adverse reactions of patients were classified according to the Radiation Therapy Oncology Group (RTOG)/European Organization for Research and Treatment of Cancer (EORTC) criteria. Blood samples were collected before treatment, at various time-points during and after radiotherapy. Residual 53BP1 and γH2AX foci, γH2AX pan-staining were analyzed in peripheral blood lymphocytes (PBL) using the Metafer system and confocal laser scanning microscopy. LAN cells were counted by the trypan blue (TB) exclusion assay. Statistical analysis was performed using Mann–Whitney test, Spearman rank correlation test and analysis of covariance (ANCOVA).

Results: No statistically significant changes were observed in the levels of γH2AX foci during radiotherapy. In contrast, radiation-induced residual 53BP1 were detected already after the first fraction. Increased individual variability in the 53BP1 focus formation was observed during treatment. The background level of DNA repair foci and its individual variability in response to radiotherapy decreased after the end of radiotherapy indicating successful removal of DNA-damaging effects. A correlation between stage of cancer and 53BP1 focus formation was established which suggests the prognostic value of this test. We show that the fraction of LAN cells negatively correlates with the level of 53BP1 and positively correlates with individual radiosensitivity. Only weak correlation was observed between γH2AX pan-staining and LAN cells. Due to large interindividual variability, both in vivo assays, LAN and focus formation, have shown relatively low predictive power at the individual level.

Conclusions: It is likely that radiosensitive patients have less efficient mechanisms of elimination of apoptotic cells with DNA damage resulting in accumulation of LAN cells and facilitating adverse reactions. Our data suggested that the grade of adverse reaction may positively correlate with LAN cells in PBL before and during radiotherapy.     

类风湿关节炎患者蛋白聚糖aggrecan G1区特异性T细胞诱导SCID小鼠产生膝关节炎的实验研究

目的探讨针对蛋白聚糖的自身免疫反应在类风湿关节炎（RA）发病中的作用。方法从人软骨细胞中克隆获得aggrecan G1区的cDNA片段,应用杆状病毒表达系统获取重组人aggrecan G1区蛋白（rAG1）。用rAG1刺激来自RA患者关节液的单个核细胞,采用β-液闪计数仪对培养细胞进行放射性检测并计算刺激增长指数（SI）,取SI超过3者为rAG1特异性T细胞。将取自3个人的rAG1特异性T细胞克隆腹腔注射至8只SCID Beige小鼠,同时在小鼠左膝注射rAG1。于注射后第8天处死小鼠,取双膝关节行组织病理学检查,观察炎细胞浸润及软骨和骨糜烂情况。结果从RA患者关节液单个核细胞分离得到rAG1特异性T细胞,主要为CD4^＋CD8^-T细胞,分泌Th1型细胞因子（γ-干扰素）。部分腹腔注射人rAG1特异性T细胞的小鼠左膝出现了关节滑膜单个核细胞浸润和早期软骨侵蚀改变。结论人特异性rAG1反应性T细胞在rAG1刺激下可向关节局部归巢而导致关节炎症反应,提示针对rAG1的自身免疫反应可能参与了RA的发病。     

Involvement of MAPK activation and ROS generation in human leukemia U937 cells undergoing apoptosis in response to sonodynamic therapy

Abstract

Purpose: To elucidate the underlying events in Chlorin e6 (Ce6)-mediated sonodynamic therapy (SDT) (Ce6-SDT)-induced apoptosis of human leukemia cell line U937.

Materials and methods: The viability of cells was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) test. Apoptosis was analyzed using a ?ow cytometer as well as ?uorescence microscopy with 4′-6-Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, poly ADP- ribose polymerase (PARP) and mitogen-activated protein kinase (MAPK).

Results: Several distinct sonochemical effects were found after SDT treatment. The participation of MAPK signals in SDT which caused U937 cell damage was specifically examined and the inhibition of p38 MAPK and Jun-N-terminal kinase (JNK) both apparently exerted a negative effect on SDT-induced cell death, while extracellular signal-regulating kinase (ERK1/2) inhibition enhanced SDT-induced cell death. The intracellular reactive oxygen species (ROS) was significantly enhanced by SDT, and pre-treatment with ROS scavenger N-acetylcysteine (NAC) partially alleviated SDT-induced cell viability loss, DNA fragmentation, mitochondria membrane potential (MMP) dissipation, caspase-3 activation, but interestingly MAPK activation was not affected much by NAC.

Conclusions: In the present paper, cell apoptosis of U937 cells was markedly enhanced after Ce6-SDT. Meanwhile, p38 MAPK, JNK and ERK were all differently activated in this process. One possible explanation for the induced cell apoptosis could be the increased ROS generation in Ce6-SDT.     

等离子刀切除儿童肿大扁桃体的护理

儿童腭扁桃体及腺样体肥大是儿童阻塞性睡眠呼吸暂停低通气综合征(OSAHS)最常见的病因[1].OSAHS是指由于睡眠时上呼吸道梗阻导致的反复呼吸暂停及因此而来的低氧血症、高碳酸血症等一系列并发症[2],若患有此症的患儿未得到及时、准确的治疗,可能会造成儿童的生长发育落后及心肺功能改变、传导性耳聋及颜面部发育畸形等,严重者还可导致儿童猝死.等离子刀具有微创、切除彻底、岀血少、止血方便、创伤轻的优点.笔者自2005年8月～2008年4月对39例伴有OSAHS的扁桃体肿大患儿进行等离子刀扁桃体手术切除治疗,通过实施具有针对性护理措施,取得满意疗效,现将护理体会总结如下.     

辐射诱发淋巴细胞凋亡生成与抑制作用研究

研究了辐射诱发的人外周血淋巴细胞凋亡生成，以及水溶性维生素Ｅ类似物－Ｔｒｏｌｏｘ对辐射诱导人外周血淋巴细胞凋亡的抑制作用。照后３０分钟内Ｔｒｏｌｏｘ能有效地阻抑ＤＮＡ片段形成，而在照前或受照中加入Ｔｒｏｌｏｘ均不能抑制ＤＮＡ片段形成，揭示Ｔｒｏｌｏｘ并不是通过清除照射过程中产生的自由基而起作用。照后３０分钟内加Ｔｒｏｌｏｘ，２小时后撤去，同样能抑制ＤＮＡ片段形成，表明Ｔｒｏｌｏｘ能不可逆地阻抑细胞凋亡早期的＂关键＂事件。     

AIF促进缺氧/复氧诱导的肥大心肌细胞凋亡的研究

目的 探讨凋亡诱导因子(AIF)对缺氧/复氧致肥大心肌细胞凋亡的影响及其意义.方法 分离培养新生昆明系小鼠心肌细胞,应用血管紧张素Ⅱ(0.1μmol/L培养12h)诱导心肌细胞肥大并建立缺氧/复氧模型以模拟缺血/再灌注损伤.实验共分设7组:肥大对照组(HC组)、缺氧8h组(H8h组)、缺氧12h组(H12h组)、缺氧8h/复氧4h组(H8h/R组)、缺氧12h/复氧4h组(H12h/R组)、缺氧12h+siRNA基凶转染组(H12h+siRNA组)、缺氧12h/复氧4h+siRNA基因转染组(H12h/R+siRNA组).AIF小干扰RNA(siRNA)转染心肌细胞后分别采用RT-PCR、Western blotting、Hoechst 33258染色法检测AIF mRNA、蛋白及细胞凋亡率.结果 H8h组、H12h组AIFmRNA(分别为0.52±0.04、0.85±0.10)均较HC组(0.29±0.08)显著升高(P<0.05);H8h组、H12h组蛋白表达水平(分别为2.07±0.15、3.12±0.19)较HC组(1.00±0.04)显著升高(P<0.05),且H12组AIF mRNA、蛋白表达水平均高于H8h组(P<0.05).与单纯缺氧组比较,缺氧后给予复氧刺激,H8h/R组和H12h/R组AIF mRNA(分别为1.09±0.07、1.41±0.12)及蛋白表达水平(分别为4.57±0.25、5.71±0.27)均较单纯缺氧时对应时间组显著升高(P<0.05).H12h+siRNA组可显著抑制肥大心肌细胞AIF的表达,其细胞凋亡率(13.40%±1.53%)与H12h组(12.90%±1.55%)比较无显著差异(P>0.05);H12h/R+siR-NA组肥大细胞凋亡率(24.90%±3.90%)显著高于H12h/R组(14.50%±1.32%,P<0.05).结论 AIF siRNA转染显著减轻缺氧/复氧时肥大心肌细胞凋亡程度,提示AIF促进缺氧/复氧诱导的肥大心肌细胞凋亡.     

硫芥诱导大鼠脾脏淋巴细胞凋亡的研究

目的 探讨硫芥诱导脾脏组织淋巴细胞凋亡以及caspase-3在其中的作用.方法 大鼠腹腔注射硫芥,各组动物分别于中毒后1、3、5d后麻醉,开胸取脾.HE染色法观察脾脏组织的病理改变,RT-PCR法检测大鼠脾脏组织caspase-3基因表达,West-em blot法检测caspase-3的蛋白表达.分离脾脏淋巴细胞,制备硫芥染毒细胞模型.DNA琼脂糖凝胶电泳观察caspase-3抑制剂Ac-DEVD-CHO对细胞DNA降解的影响;流式细胞仪检测Ac-DEVD-CHO对染毒细胞凋亡峰(即G1亚G1峰)及线粒体跨膜电位(△Ψm)的影响.结果 硫芥中毒大鼠脾脏组织形态发生病理改变,部分淋巴细胞出现了凋亡的特征;脾脏组织caspase-3的mRNA与蛋白表达均增加:mRNA在中毒后1d的表达量与对照组比较具有统计学意义(P＜0.05);蛋白表达在中毒后3d和5d与对照组比较具有统计学意义(P＜0.05,P＜0.01).caspase-3的特异性抑制剂Ac-DEVD-CHO对体外培养的硫芥染毒淋巴细胞的DNA降解和细胞(G1峰左侧凋亡峰的m现有抑制作用.硫芥可导致细胞线粒体跨膜电位降低,随着中毒时间延长,线粒体膜电位的降低更明显.结论 硫芥中毒可导致大鼠脾脏组织损伤,细胞凋亡是其作用机制之一,caspase-3可能参与了这一过程并在其中发挥重要的作用.     

碳离子射线诱导细胞凋亡的研究进展

碳离子射线治疗肿瘤是利用其剂量分布优势,将能量集中于肿瘤组织释放,同时尽量避免损伤周围正常组织。相较于常规光子射线,碳离子不仅表现出上述物理学优势,还具有优越的生物学特性。笔者概述了碳离子射线在物理、生物学方面的特点,并着重综述了其在诱导细胞凋亡方面的进展。     

Reduction of arteriohepatovenous shunting by temporary balloon occlusion in patients undergoing radioembolization

Radioembolization with yttrium-90 resin microspheres is a treatment option that selectively targets hepatic tumors. One of the primary limiting factors for this therapy is the degree of arteriohepatovenous shunting, as excessive radiation to the lungs may cause radiation pneumonitis. To safeguard patients against this, a technetium Tc 99m macroaggregated albumin scan is performed before treatment to assess the degree of arteriohepatovenous shunting. As lung shunt fraction increases, activity reductions are mandated, with a 20% shunt sufficient to prohibit treatment. Temporary occlusion of shunts may be achieved by placement of balloon catheters in the hepatic veins. This endovascular technique used to reduce arteriohepatovenous shunting allows otherwise untreatable patients to undergo radioembolization.     

鞘氨醇激酶调节细胞凋亡的研究进展

鞘磷脂衍生物神经酰胺(Cer)、鞘氨醇(Sp)及1-磷酸鞘氨醇(SIP)在调控细胞增殖、存活及凋亡中发挥着重要作用。鞘氨醇激酶(SPK1)是调控细胞内Cer、Sp、SIP代谢平衡的关键酶。SPK1磷酸化Sp生成SIP，SIP通过细胞内和细胞外作用机制调节细胞生长和凋亡。SPK1参与细胞因子的信号传递。高表达SPK1抑制半胱天冬酶的裂解并上调Bcl-2基因的表达。本文综述了SPK的结构、调控及对细胞凋亡的调节作用。     

褐藻糖胶对照射大鼠免疫功能和淋巴细胞凋亡影响

目的　研究不同剂量褐藻糖胶对受γ射线照射大鼠免疫功能影响的作用机理。方法不同剂量褐藻糖胶经口给予实验组大鼠 ,连续给药 10d后行一次性 6 0Coγ射线照射 ,18h后测定各组大鼠体液免疫、细胞免疫和非特异性免疫相关指标 ,并测定大鼠脾淋巴细胞凋亡率。结果　 10 0mg kg褐藻糖胶即能显著提高照射后大鼠T、B淋巴细胞增殖能力、巨噬细胞吞噬功能、血清溶血素含量 ,增强大鼠迟发性超敏反应 ,抑制照射后脾淋巴细胞凋亡 ,并呈现剂量 效应关系 ,与阳性对照组比较差异有非常显著性 (P <0 0 1)。结论　褐藻糖胶是一种抗照射免疫调节剂 ,对照射后大鼠的免疫系统具有恢复作用 ,其抗照射作用与抑制淋巴细胞凋亡有关     

裂变中子诱导小鼠胸腺细胞凋亡的剂量效应

目的 研究反应堆裂变中子诱导小鼠胸腺细胞凋亡的剂量效应,并与＾６０Ｃｏγ射线的效应相比较,探讨两种辐射诱导细胞凋亡的异同。方法 用光镜和电镜形态学观察与ＤＮＡ琼脂糖凝胶电泳方法对细胞凋亡进行定性研究;用流式细胞术（ＦＣＭ）和二苯胺（ＤＰＡ）法对其进行定量研究。     

Enhanced ultrasound-induced apoptosis and cell lysis by a hypotonic medium

PURPOSE: To test the hypothesis that non-lethal hypotonia will enhance ultrasound-induced cell killing in vitro and that the mechanism is mechanical in nature. MATERIALS AND METHODS: Hypotonic RPMI medium (146 mOsm) was used to induce non-lethal osmotic swelling of human myelomonocytic leukaemia U937 cells. Hypotonia for 10 min was started just before exposure to 1 MHz ultrasound at 0.5 or 1.0 Wcm(-2) for 10 min, or 5 min before exposure to 2.0 Wcm(-2) for 1 min. Surviving intact cells were then determined by the trypan blue dye exclusion test immediately after treatment. After 6-h incubation of the treated cells, early apoptosis and secondary necrosis were measured using a flow cytometer. Intracellular free calcium ion imaging by Fura-2 fluorescence and cellular ion scanning using a secondary ion mass spectrometer were also performed. RESULTS: Enhancement of ultrasound-induced cell lysis was observed at all intensities, and most prominently at 2.0 Wcm(-2), while apoptosis induction was significantly enhanced at intensities of 0.5 and 1.0 Wcm(-2), but not at 2.0 Wcm(-2). The enhanced cell lysis is attributed to the increased susceptibility of the cells to mechanical damage. This is consistent with previous reports describing the effects of mechanical stresses on cell membranes. Cellular ion scanning images also suggest that hypotonia has an effect on the membrane damage-and-repair mechanism of the cells. CONCLUSIONS: The results support the hypothesis that non-lethal hypotonia can enhance ultrasound-induced cell killing. These findings also suggest the 'sonomechanical' nature of the effects on the cells.     

DNA lesion complexity and induction of apoptosis by ionizing radiation

Purpose : To determine whether murine lymphoid cell lines can discriminate between high- and low-LET (linear energy transfer) radiation-induced DNA lesions. Materials and methods : Sensitivity to killing by DNA-incorporated 3 H and 125 I decays, accumulated during storage in the gas phase of a liquid nitrogen tank, was determined by clonogenic survival assay. Results : Induction of a lethal event in the STRij-4-2.2, WEHI-22.1, and L5178Y-R cell lines required approximately 30 times more 3 H than 125 I decays. Hence, the same ratio of 3 H to 125 I decays was found irrespective of whether the cell lines contained mutant or wild-type p53 and irrespective of whether they underwent rapid interphase or mitosis-related apoptosis after irradiation. The 18-81 cell line differed in showing a ratio of around 21 and it is argued that this may be a consequence of v-ABL over-expression. The assumption that DNA-incorporated 3 H and 125 I decays are low- and high-LET-like events respectively was confirmed by the similar sensitivity of L5178Y-R and -S cells to killing by 125 I decays in contrast with their difference in sensitivity to 3 H decays. Conclusions : The difference in lethal effectiveness between DNA-incorporated 3 H and 125 I decays can be explained by the hypothesis that simple DSB (double-strand breaks) are non-lethal and that cell killing is attributable to complex DSB. The low-LET radiation-specific sensitization of L5178Y-S cells may reflect defective repair of a DNA lesion class (presumably simple DSB) that is differentially induced by high- and low-LET radiation and is non-lethal to cells with normal repair capacity.