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.     

Beta-irradiation used for systemic radioimmunotherapy induces apoptosis and activates apoptosis pathways in leukaemia cells

Beta-irradiation used for systemic radioimmunotherapy (RIT) is a promising treatment approach for high-risk leukaemia and lymphoma. In bone marrow-selective radioimmunotherapy, beta-irradiation is applied using iodine-131, yttrium-90 or rhenium-188 labelled radioimmunoconjugates. However, the mechanisms by which beta-irradiation induces cell death are not understood at the molecular level. Here, we report that beta-irradiation induced apoptosis and activated apoptosis pathways in leukaemia cells depending on doses, time points and dose rates. After beta-irradiation, upregulation of CD95 ligand and CD95 receptor was detected and activation of caspases resulting in apoptosis was found. These effects were completely blocked by the broad-range caspase inhibitor zVAD-fmk. In addition, irradiation-mediated mitochondrial damage resulted in perturbation of mitochondrial membrane potential, caspase-9 activation and cytochrome c release. Bax, a death-promoting protein, was upregulated and Bcl-x_L, a death-inhibiting protein, was downregulated. We also found higher apoptosis rates and earlier activation of apoptosis pathways after gamma-irradiation in comparison to beta-irradiation at the same dose rate. Furthermore, irradiation-resistant cells were cross-resistant to CD95 and CD95-resistant cells were cross-resistant to irradiation, indicating that CD95 and irradiation used, at least in part, identical effector pathways. These findings demonstrate that beta-irradiation induces apoptosis and activates apoptosis pathways in leukaemia cells using both mitochondrial and death receptor pathways. Understanding the timing, sequence and molecular pathways of beta-irradiation-mediated apoptosis may allow rational adjustment of chemo- and radiotherapeutic strategies.     

In vitro apoptosis in peripheral blood mononuclear cells induced by low-dose radiotherapy displays a discontinuous dose-dependence

Purpose: Cells undergoing apoptosis contribute to the regulation of activated mononuclear cells (Voll et al. 1997). Low-dose radiotherapy (LD-RT) is known to improve inflammatory symptoms, but the mechanism of action is still unclear. The aim of this study was to investigate the rate of apoptosis of peripheral blood mononuclear cells (PBMC) induced by LD-RT within the therapeutic dose range of anti-inflammatory RT. Materials and methods: PBMC were isolated from venous blood of ten healthy volunteers and were irradiated with single doses between 0.1 and 3.0Gy. Apoptotic nuclei were detected by flow cytometry after propidium iodide (PI) triton staining, and apoptotic cells were detected by annexin V/PI staining and cell scatter analysis. Since apoptotic cells display increased cytoplasmatic granularity and concomitant reduced cell size, they can be distinguished from viable cells in forward/side scatter (FSC/SSC) histograms. Apoptotic PBMC were further subtyped by double staining with annexin V and directly labelled monoclonal antibodies recognizing the lineage-specific surface markers CD4, CD8, and CD19, respectively. The apoptosis rate of irradiated cells was analysed in a time and dose dependent fashion and was compared to a sham-irradiated control. Results: After irradiation, a dose-dependent increase in apoptosis was observed, with a discontinuity (plateau or peak) between 0.3Gy and 0.7Gy in 9/10 donors (90%) and 59/80 samples (74%). 8/10 donors (80%) and 38/80 samples (47%) showed not only a discontinuous increase with a plateau but a relative maximum of apoptosis peaking within the dose range of 0.3Gy and up to 0.7Gy. Conclusion: LD-RT induces a relative maximum of apoptosis in PBMC in the does range between 0.3Gy and 0.7Gy. This may contribute to its anti-inflammatory effect observed clinically.     

UV or X-Irradiation Increases the Cytoplasmic Accumulation of Rhodamine 123 in Various Cancer Cell Lines

PURPOSE: Previous studies indicated that ATP-binding cassette (ABC) membrane transporters protect against UV-induced apoptosis. We investigated the effect of UVB and X-ray irradiation on the export function of these ABC transporters in primary lymphocytes and various cancer cell lines. MATERIAL AND METHODS: We used rhodamine accumulation assays in various human malignant cell lines and peripheral blood lymphocytes (PBL). Cells were irradiated with up to 960 mJ/cm2 and up to 50 Gy of UVB and X-ray, respectively. RESULTS: We demonstrated that UVB as well as X-ray irradiation inhibit the export function of the ABC transporters in a dose-dependent fashion. For PBL, this effect did not correlate with an apoptotic phenotype. In the case of the tumor cell lines, even though the irradiation-induced inhibition of membrane transporters was accompanied by phosphatidylserine exposure, only a minority of cells had lost their mitochondrial membrane potential during the observation period. Furthermore, we demonstrated that the inhibition of membrane transporters is not a general feature of apoptosis. CONCLUSION: Irradiation inhibits the export function of ABC transporters. Although some of the irradiated cells undergo apoptosis following irradiation, the inhibition is an unique feature accompanying irradiation and not a general hallmark of apoptotic cell death. The inhibition of drug export by irradiation may offer new potential for reverting multidrug resistance of cancer cells.     

Let-7和miR-24在紫外线B诱导的细胞凋亡中的作用

目的 研究let-7和miR-24在紫外线B(UVB)诱导的细胞凋亡中的可能作用。方法 NIH3T3细胞受50 J/m²UVB照射后,用Hoechest 33342/PI染色观察其凋亡情况;RT-PCR检测let-7和miR-24的表达水平。利用在线数据库PicTar等预测它们的靶基因,并用GOstat软件对这些靶基因进行功能分类。结果 荧光显微镜下,NIH3T3细胞经UVB照射后可见典型的凋亡和坏死细胞;let-7和miR-24在UVB照射组的表达水平较对照组明显增加。用GOstat进行功能分类后发现,casp3、bcl2l2、map3k1和cdk5等基因同时也是UVB诱导的细胞周期调节的靶基因。结论 let-7和miR-24可能参与UVB诱导的细胞凋亡。     

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.     

Alpha-tocopherol succinate-mobilized progenitors improve intestinal integrity after whole body irradiation

Abstract

Purpose: The objective of this study was to elucidate the action of α-tocopherol succinate (TS)- and AMD3100-mobilized progenitors in mitigating radiation-induced injuries.

Material and methods: CD2F1 mice were exposed to a high dose of radiation and then transfused intravenously with 5 million peripheral blood mononuclear cells (PBMC) from TS- and AMD3100-injected mice after irradiation. Intestinal and splenic tissues were harvested after irradiation and cells of those tissues were analyzed for markers of apoptosis and mitosis. Bacterial translocation from gut to heart, spleen, and liver in TS-treated and irradiated mice was evaluated by bacterial culture.

Results: We observed that the infusion of PBMC from TS- and AMD3100-injected mice significantly inhibited apoptosis, increased cell proliferation in the analyzed tissues of recipient mice, and inhibited bacterial translocation to various organs compared to mice receiving cells from vehicle-mobilized cells. This study further supports our contention that the infusion of TS-mobilized progenitor-containing PBMC acts as a bridging therapy by inhibiting radiation-induced apoptosis, enhancing cell proliferation, and inhibiting bacterial translocation in irradiated mice.

Conclusions: We suggest that this novel bridging therapeutic approach that involves the infusion of TS-mobilized hematopoietic progenitors following acute radiation injury might be applicable to humans as well.     

电离辐射对T淋巴细胞IL-2分泌和细胞毒活性的影响

正常人外周血单个核细胞（ＰＢＭＣ）体外经１～６Ｇｙγ射线照射后，间接免疫荧光法分析受照Ｔ细胞Ｔ细胞抗原受体（ＴＣＲ）、ＣＤ＿３及ＣＤ＿（２３）阳性细胞百分率，Ｈ￣３－ＴｄＲ掺入及释放法测定白细胞介素－２（ＩＬ－２）分泌及Ｔ细胞细胞毒活性，免疫细胞化学法分析Ｔ细胞内ＴＣＲ及ＣＤ＿３表达．结果表明，Ｔ细胞ＴＣＲ、ＣＤ．及ＣＤ＿（２５）表达及ＩＬ－２分泌和细胞毒活性皆呈照射剂量依赖性降低．受照Ｔ细胞ＩＬ－２分泌及细胞毒活性抑制在一定程度上与细胞表面ＴＣＲ、ＣＤ＿３及ＣＤ＿（２５）表达减少、活性损伤有关，而细胞表面ＴＣＲ和ＣＤ＿３表达降低可能与大量ＴＣＲ及ＣＤ＿３在受照细胞胞浆内堆积有关．     

Naive and memory T cell subsets are differentially mobilized during physical stress

This study examined the naive and memory phenotypic profiles of CD4+ and CD8hi T cells that were mobilized to the peripheral circulation during a combination of aerobic exercise and heat stress, determining expression of the adhesion molecules CD62L and CD11a on the recruited cells. Twelve recreationally active males (age 27.1 +/- 5.3 yr, height 1.77 +/- 0.08 m, mass 76.9 +/- 12.0 kg, VO2peak 43.9 +/- 6.7 mL x kg(-1) x min(-1)) completed a 40 min bout of cycle ergometry at 65 % of VO2peak while immersed to mid-chest in a water bath at 39 degrees C. Venous blood samples were collected before (T0), during (T40) and 30 min after (T70) exposure to combined exercise and heat stress. Specimens were analyzed by three-colour flow cytometry for CD4+ and CD8hi T cell expression of CD45RO, CD11a and CD62L. Some 80 % of the CD4+ T cells that were mobilized were of the CD45RO memory phenotype, with the numbers of CD11alo and CD62L+ cells increasing more than those of CD11ahi and CD62L- cells. For the CD8hi cells, there was a more equal recruitment of CD45RO- naive (43 %) and CD45RO+ memory (57 %) cells. The majority (84 %) of recruited CD8+ cells were CD11ahi; there was a trend to predominance of CD62L- cells (57 %) for the memory subset, but with almost equal recruitment of CD62L+/- for the naive subset. We conclude that the exercise + heat stress induced trend to an increase in CD4+ T cells is linked in some way to memory phenotype; it cannot be explained simply by a high density expression of CD11a and lack of the lymph node homing receptor (CD62L). Furthermore, although mobilization of CD8hi T cells is not linked to memory phenotype, a high density expression of CD11a and a lack of the lymph node homing receptor are important determinants of CD8hi T cell mobilization.     

HLA-G~+调节性T细胞在肾移植受者外周血中的比例及其功能研究

目的 确定人类白细胞抗原-G(HLA-G)区别于CD4~+ CD25~+ FoxP3~+ 调节性T细胞的细胞表型特征,观察HLA-G~+ T细胞在混合淋巴细胞培养中的免疫调节作用及其在移植免疫调节中的活性和意义.方法采用流式细胞术检测肾移植受者外周血CD4~+ HLA-G~+ GD8~+ HLA-G~+ T淋巴细胞的含量,分选HLA-G~+ T淋巴细胞,分析细胞表型,采用RT-PCR检测调节性T细胞的特异性标志FoxP3在HLA-G~+ T淋巴细胞的表达情况,以淋巴细胞分离液分离的PBMC、CD4~+ CD25~(high)、CD4~+ CD25~-细胞作为对照.取5对活体肾移植供受者的淋巴细胞进行混合培养,分别加入流式细胞术分选纯化后的HLA-G~+ 、HLA-G~- T淋巴细胞,对照组只加入供、受者淋巴细胞,MTT法观察细胞增殖和抑制情况.结果 肾移植受者外周血CD4~+ HLA-G~+ 、CD8~+HLA-G~+ T淋巴细胞表达率分别为1.95%±0.34%、3.13%±0.56%.流式细胞术分选HLA-G~+ T淋巴细胞后纯度可达55.0%～75.1%.RT-PCR 结果证明上述细胞CD25、FoxP3均为阴性表达.与HLA-G~- 组、对照组比较,混合淋巴培养3d后HLA-G~+ 组细胞增殖率明显受到抑制(P<0.05).结论 在肾移植受者外周血中存在CD4~+ HLA-G~+ 、CD8~+HLA-G~+ T淋巴细胞,这类细胞不同于CD4~+ CD25~+ FoxP3~+调节性T细胞,它们不表达CD25和FoxP3而表达免疫耐受分子HLA-G,在混合淋巴培养体系中,能显著抑制反应性细胞增殖,具有免疫调节功能.     

Increased and decreased expression of CD69 and CD23, respectively, in gravity-stressed lymphocytes

BACKGROUND: Recent studies have shown that gravity-changing stress modulates expression levels of cell surface molecules on human lymphocytes. However, previous in vitro microgravity studies have been performed with lymphocytes treated with mitogenic agents. HYPOTHESIS: The aim of the study was to test if exposure of cells to gravity-changing stress alone alters the expression levels of cell surface molecules. Specifically, we examined whether the expression of activation markers is altered after exposure of lymphocytes to combinations of microgravity and hypergravity. METHODS: We used free-fall in parabolic flight for human subjects and a drop-shaft to expose peripheral blood mononuclear cells (PBMC) to gravity-changing stress. After such exposure, PBMC were isolated, and expression levels of CD69, CD23 and CD38 were estimated using three-color flow cytometry. RESULTS: Increased percentages of CD69-positive cells were observed with PBMC from 3 of 4 volunteers who undertook 10 parabolic flights. Exposure of blood to gravity-changing stress in the drop-shaft increased both ratios of CD69-positive cells and levels of CD69 expression on T and B cells. In contrast, the percentages of CD23-positive B cells was decreased. However, gravity-changing stress was not always followed by significant alteration in CD38 expression. CONCLUSIONS: Our findings suggest that CD69 and CD23 might be useful markers that are up- and down-regulated, respectively, after exposure of lymphocytes to gravity-changing stress.     

Radiation sensitivity and apoptosis in human lymphoma cells

PURPOSE: The impact ofapoptosis on radiation-induced eradication of clonogenic tumour cells is uncertain. The aim was to analyse the relationship of different functional stages during the apoptotic process to cell death and clonogenic cell eradication. MATERIALS AND METHODS: Apoptosis in Jurkat T-cells was studied by morphology, light scatter and caspase activation. Mitochondrial integrity was determined by the mitochondrial membrane potential (delta(phi)m). Cell death was quantified using propidium iodide exclusion. Clonogenic cell death was determined using a dilution survival assay. The influence of Bcl-2 was tested using a Bcl-2 transfected Jurkat clone. RESULTS: Irradiation induced profound apoptosis within 48 h associated with caspase activation and breakdown of delta(phi)m. Inhibition of caspases abrogated the apoptotic morphology with no influence on breakdown of delta(phi)m and survival. Over-expression of Bcl-2 abrogated all hallmarks of apoptosis; delayed cell death, however, had no influence on clonogenic survival after irradiation. CONCLUSION: Based on Bcl-2 as a positional marker, radiation-induced apoptosis can be divided into two stages: the initiation/decision phase, characterized by a breakdown of the mitochondrial membrane potential, and the execution phase, characterized by caspase activation. The execution phase had no influence on survival, whereas the initiation/decision phase controls immediate survival. However, abrogation of both phases did not influence radiation sensitivity.     

细胞因子治疗4.5Gyγ射线照射比格犬造血损伤的机制初探

目的 探讨多种细胞因子配伍应用治疗4.5 Gy γ射线照射比格犬造血系统损伤的作用及可能机制。方法 16只比格犬均给予4.5 Gy ⁶⁰Co γ射线全身照射,随机分为照射对照、综合对症和细胞因子3组。细胞因子组在综合对症支持治疗的基础上应用rhG-CSF、rhIL-11和rhIL-2联合治疗,采用流式细胞术检测外周血中CD34+细胞含量、有核细胞周期及凋亡比例。结果 4.5 Gy γ射线照射犬外周血中CD34+细胞含量在照射后1 d明显下降(照射对照组和综合对症组分别为照前值的61.3%和52.1%),G₀/G₁期有核细胞比例增加(分别为99.27%和99.49%),且凋亡率(分别为26.93%和21.29%)和坏死率(分别为3.27%和4.14%)明显升高(与照前值比较, P<0.05);而经过细胞因子治疗后,外周血中CD34+细胞含量在照射后1 d即明显升高(为照前值的135.6%),G₀/G₁期有核细胞比例(99.71%)进一步增加,其凋亡率(5.66%)和坏死率(1.60%)明显低于照射对照和综合对症组。结论 本研究的细胞因子组合可能通过动员骨髓中CD34+细胞到外周血,使细胞周期阻滞在G₀/G₁期,减少细胞凋亡,从而促进极重度骨髓型急性放射病犬造血功能的恢复。     

Ultraviolet exposure of thymocytes: selective inhibition of apoptosis

PURPOSE: To evaluate selective effects of ultraviolet (UV) irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes. MATERIALS AND METHODS: Cells were exposed to UV radiation with emission peaks of 365 nm (UVA) exposures of 1620-10200 J m(-2), of 312 nm (UVB) exposures of 34-1620 J m(-2) or of 254 nm (UVC) exposures of 1.5-1620 J m(-2), and incubated for 5.5 h with or without hydrocortisone, phorbol-12-myristate-13-acetate or anti-Fas antibody. Additionally, cells were irradiated with gamma-rays (5 Gy) before UVB exposure (408 J m(-2)) at different times. Apoptosis was quantified by DNA fragmentation. RESULTS: Up to an irradiation of 5000 J m(-2), UVA exposure did not show any effect on thymocyte apoptosis, while at 10200 J m(-2) irradiation, considerable DNA fragmentation was observed. In contrast, UVB and UVC irradiation clearly inhibited natural and cortisone-induced apoptosis. Moreover, UVB inhibited apoptosis triggered by phorbol-12-myristate-13-acetate and gamma-irradiation, but not by anti-Fas antibody. CONCLUSIONS: The response of mouse thymocytes in culture to UV irradiation strongly depends on the wavelength used. It is suggested that either a survival or an apoptotic pathway occurs depending on the physiological state of the cell, spectral composition of the UV light and cell type. The possible involvement of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase in the apoptotic pathway is discussed.     

Th2 cells as effectors in postirradiation pulmonary damage preceding fibrosis in the rat.

PURPOSE: Radiation-induced pneumonitis and subsequent pulmonary fibrosis are important dose-limiting complications of radiotherapy. Their pathogenesis is known only in part. T-lymphocytes comprise a significant part of the infiltrating cells but little is known about their role. The aim of this study was to define the function of T-lymphocytes during development of postirradiation pneumonitis and pulmonary fibrosis. MATERIALS AND METHODS: Rats received a unilateral lung irradiation of 20 Gy. Kinetics of T-lymphocytes isolated from irradiated and non-irradiated lungs were analysed. Subsequent CD4 depletion experiments were performed to affirm the importance of CD4+ T-cells in the development of lung fibrosis. Finally, the T helper-cell subtype of the T-lymphocytes was analysed by determining the cytokine mRNA by RT-PCR. RESULTS: A selective increase of CD4+ T-cells was observed peaking 4 weeks after irradiation in the irradiated lungs. When rats were depleted of these cells, the postirradiation thickening of parenchyma was significantly reduced as determined by morphometric analysis of lung tissue sections. In addition, it was found that IL-4 mRNA was selectively increased in the CD4+ T-cells isolated from irradiated lungs, which indicates a lymphocyte reactivity dominated by Th2 cells. CONCLUSION: The results suggest a critical role for Th2 CD4+ T-lymphocytes in the pathogenesis of radiation-induced pneumonitis preceding lung fibrosis.     

低剂量照射对慢性髓细胞白血病干细胞p16基因mRNA表达水平的影响及其意义

目的 探讨低剂量辐射(LDR)对慢性髓细胞白血病干细胞(LSCs)中P16基因转录表达的影响及其临床意义。 方法 取20例健康产妇脐血100 ml,免疫磁株法分离纯化CD34+、CD38-的正常造血干细胞(HSCs)作为对照组;取20例初诊慢性髓细胞白血病(CML)慢性期患者骨髓液5~10 ml,免疫磁株法分离纯化CD34+、CD38-、CD123+的LSCs作为实验组。将HSCs及LSCs依据LDR剂量(0、12.5和50 cGy)辐照后收集细胞行RT-PCR和荧光实时定量PCR检测两种干细胞中p16基因的变化;另辐照后分别于24、48和72 h收集细胞,流式细胞仪检测两种干细胞的细胞周期和凋亡率。结果 CML-LSCs经12.5 cGy剂量照射后P16 mRNA表达略上调,50 cGy照射后明显增高(Z=-3.39,P＜0.01),而HSCs经各剂量点LDR处理后p16基因转录水平无明显变化。CML-LSCs经12.5 cGy剂量处理后48 h出现G₀/G₁期阻滞,50 cGy剂量点照射后72 h出现了G₀/G₁期阻滞现象。CML-LSCs经LDR处理后,细胞的早期凋亡率随时间推移逐渐增加,50 cGy剂量照射后72 h处达到(17.75±11.76) %,与未照射组 (6.13±4.71)%相比差异有统计学意义(Z=-2.37,P＜0.05)。 结论 LDR可诱导CML-LSCs中P16基因转录水平的表达上调,使得CML-LSCs阻滞在G₀/G₁期,促进LSCs的凋亡,为P16基因作为CML中治疗的靶点及LDR在白血病中的应用提供理论依据。     

辐射小鼠骨髓CD34~ 细胞的变化及其意义

目的　研究γ射线照射后C5 7小鼠骨髓中CD34 细胞的数量变化规律及其意义。方法　流式细胞仪测定CD34 细胞在骨髓有核细胞中的比例 ;AnnexinV FITC试剂盒检测骨髓细胞的凋亡 ;细胞固定后PI染色测定细胞周期。结果　①CD34 细胞在骨髓有核细胞中的比例随照射剂量的加大而降低 ,在 5 5Gy照射后 14d内小鼠CD34 细胞的减少表现为持续性 ;②小鼠照射后6h骨髓细胞凋亡率最高 ,以 5 5Gy照射组最为明显 ;③ 5 5Gy照射后小鼠骨髓细胞周期紊乱。 结论　γ射线损伤骨髓中的干祖细胞 ,造成骨髓中干祖细胞的数量减少 ,其途径之一是诱导骨髓细胞凋亡。     

Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells

Purpose : To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. Materials and methods : HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm -2) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. Results : The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. Conclusion : The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.     

Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells

PURPOSE: To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. MATERIALS AND METHODS: HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm(-2)) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. RESULTS: The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. CONCLUSION: The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.