Anti-inflammatory effect of low-dose X-irradiation and the involvement of a TGF-beta1-induced down-regulation of leukocyte/endothelial cell adhesion

PURPOSE: Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the underlying radiobiological and immunological mechanisms remain elusive. In recent studies, we observed a reduced adhesion of peripheral blood mononuclear cells (PBMC) to endothelial cells (EC) after LD-RT (0.3-0.7 Gy). This shows that this treatment affects the initial steps of the inflammatory response. To explore the role of inflammatory mediators in this process, we investigated the expression of Transforming growth factor beta(1) (TGF-beta(1)) and Interleukin 6 (IL-6) after LD-RT. MATERIALS AND METHODS: EC were grown to subconfluence and irradiated with single-dose LD-RT. Twenty-hours after irradiation, EC were treated with IL-1beta for 4 h and then incubated with peripheral blood mononuclear cells (PBMC). Adherent PBMC were counted when using light microscopy. Expression of the cytokines TGF-beta(1) and IL-6 was measured by ELISA, and mRNA levels were analysed by the RNAse-protection assay (RPA). Surface expression of E-selectin was quantified by flow cytometry. RESULTS: A relative minimum of adhesion was observed after LD-RT between 0.3 and 0.7 Gy. This was paralleled by an expression maximum of TGF-beta(1) and IL-6, as shown by protein and mRNA levels, respectively. Neutralization of TGF-beta(1) by monoclonal antibodies, but not of IL-6, increased PBMC adhesion to EC nearly to control levels. In addition, fluorescence activated cell sorter (FACS) analysis of irradiated EC demonstrated a down-regulation of E-selectin in the same dose range. CONCLUSION: Low-dose X-irradiation between 0.3 and 0.7 Gy induced a relative maximum of TGF-beta(1) production by stimulated EC. This results in a down-regulation of leukocyte/PBMC adhesion and may contribute to the anti-inflammatory effect of LD-RT.     

Anti-inflammatory effect of low-dose X-irradiation and the involvement of a TGF-β 1 -induced down-regulation of leukocyte/endothelial cell adhesion

Purpose : Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the underlying radiobiological and immunological mechanisms remain elusive. In recent studies, we observed a reduced adhesion of peripheral blood mononuclear cells (PBMC) to endothelial cells (EC) after LD-RT (0.3-0.7 Gy). This shows that this treatment affects the initial steps of the inflammatory response. To explore the role of inflammatory mediators in this process, we investigated the expression of Transforming growth factor β 1 (TGF- β 1) and Interleukin 6 (IL-6) after LD-RT. Materials and Methods : EC were grown to subconfluence and irradiated with single-dose LD-RT. Twenty-hours after irradiation, EC were treated with IL-1 β for 4 h and then incubated with peripheral blood mononuclear cells (PBMC). Adherent PBMC were counted when using light microscopy. Expression of the cytokines TGF- β 1 and IL-6 was measured by ELISA, and mRNA levels were analysed by the RNAse-protection assay (RPA). Surface expression of E-selectin was quantified by flow cytometry. Results : A relative minimum of adhesion was observed after LD-RT between 0.3 and 0.7 Gy. This was paralleled by an expression maximum of TGF- β 1 and IL-6, as shown by protein and mRNA levels, respectively. Neutralization of TGF- β 1 by monoclonal antibodies, but not of IL-6, increased PBMC adhesion to EC nearly to control levels. In addition, fluorescence activated cell sorter (FACS) analysis of irradiated EC demonstrated a down-regulation of E-selectin in the same dose range. Conclusion : Low-dose X-irradiation between 0.3 and 0.7 Gy induced a relative maximum of TGF- β 1 production by stimulated EC. This results in a down-regulation of leukocyte/PBMC adhesion and may contribute to the anti-inflammatory effect of LD-RT.     

Funktionelle und molekulare Aspekte der antiinflammatorischen Wirkung niedrig dosierter Radiotherapie

PURPOSE: Low-dose radiotherapy (LD-RT) with single fractions between 0.1 and 1.0 Gy is known to exert an antiinflammatory effect. Although different mechanisms for the clinical efficiency were proposed, only few experimental data are still available. This paper focuses on functional and molecular aspects of LD-RT. METHODS AND RESULTS: The antiinflammatory efficiency of LD-RT in clinical studies could be confirmed in experimental models of osteoarthritis and rheumatoid arthritis. In a model of adjuvants arthritis, 5 x 1.0 Gy as well as 5 x 0.5 Gy, given at the maximum of the acute inflammation, could prevent clinically and histologically progression of the disease without affecting existing signs of inflammation. The effect of LD-RT on the adhesion of peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) was analyzed in in-vitro assays. In the dose range between 0.3 and 0.7 Gy almost 4 hours after irradiation adherent cells reached a relative minimum of adhesion compared to unirradiated controls. In PBMC an discontinuous increase of apoptosis with a maximum between 0.3 and 0.5 Gy, the proteolytic shedding of L-selectin and an increased expression of the antiinflammatory cytokine interleukin 10 as well as downregulation of TNF alpha could be identified as potential mechanisms for the observed reduced adhesion. Conversely, reduced expression of E-selectin and an increased induction of transforming growth factor beta (TGF beta 1) with a maximum at 0.5 Gy could be observed in endothelial cells. Macrophages immigrating the site of inflammation are known to express inducible nitrix-oxide synthase (iNOS), which in turn mediates cytotoxic and immunmodulatory effects by producing nitric oxide (NO). LD-RT of stimulated macrophages within the dose range between 0.6 and 1.25 Gy reduced NO production and iNOS-protein expression without affecting iNOS-mRNA expression. CONCLUSION: Our experimental data have confirmed the antiinflammatory efficiency of LD-RT in vitro and in vivo, indicating effects on different cellular components and mechanisms of inflammation. The regulation of the adhesion between PBMC and endothelial cells and the effects on activated macrophages may mediate the antiinflammatory properties of LD-RT. Ongoing experiments will help to clarify the molecular mechanism.     

Funktionelle und molekulare Aspekte der antiinflammatorischen Wirkung niedrig dosierter Radiotherapie

Hintergrund: Niedrig dosierte Radiotherapie (LD-RT) mit Dosen zwischen 0,1 und 1,0 Gy hat eine empirisch gut belegte antiinflammatorische Wirksamkeit. Obwohl verschiedene Hypothesen über die Mechanismen für die klinische Wirkung vorgeschlagen wurden, liegen bislang nur wenige experimentelle Untersuchungen vor. Aktuelle Daten über funktionelle und molekulare Aspekte der LD-RT im Hinblick auf ihre adhäsionsbiologischen und antiinflammatorischen Effekte werden vorgestellt. Methoden und Ergebnisse: In experimentellen Modellen der Osteoarthritis und rheumatoiden Arthritis konnten klinisch nachweisbare antiinflammatorische Effekte der LD-RT objektiviert werden. Im Modell der Adjuvansarthritis führten sowohl 5 2 1,0 als auch 5 2 0,5 Gy, wenn zum Zeitpunkt des akuten Entzündungsmaximums appliziert, klinisch und histologisch zu einer Prävention der weiteren Arthritisprogression, ohne bereits vorhandene Zeichen zu vermindern. In einem In-vitro-Assay wurde der Einfluss von LD-RT auf die Adhäsion von mononukleären Zellen des peripheren Blutes PBMC) an Endothelzellen (EC) untersucht. Dabei zeigte die Adhäsion bereits 4 Stunden nach Bestrahlung mit Einzeldosen von 0,3-0,7 Gy ein relatives Minimum im Vergleich zur Kontrolle. Als mögliche Mechanismen der verminderten Adhäsion wurden auf Seite der PBMC ein diskontinuierlicher Anstieg der Apoptoserate mit einem lokalen Maximum bei 0,3-0,5 Gy, die proteolytische Abspaltung des L-Selektins von der Oberfläche und eine gesteigerte Expression des antiinflammatorischen Zytokins Interleukin 10 bzw. die Herunterregulation von TNF! bestimmt. Auf Seiten der Endothelzellen konnte neben einer Reduktion von E-Selektin die Induktion des antiinflammatorisch wirksamen Zytokins Transforming Growth Factor beta (TGFş) mit einem Maximum bei 0,5 Gy festgestellt werden. In den Entzündungsherd migrierende Makrophagen exprimieren induzierbare Stickoxidsynthase (iNOS), welche über die Bildung von Stickoxid (NO) sowohl zytotoxische als auch immunmodulatorische Effekte vermittelt. LD-RT aktivierter Makrophagen mit 0,6-1,25 Gy reduzierte die NO-Produktion und iNOS-Protein-Expression ohne nachweisbaren Effekt auf die iNOS-mRNA-Expression in vitro. Schlussfolgerung: Es wird deutlich, dass LD-RT wahrscheinlich mit unterschiedlichen zellulären Komponenten und Mechanismen des Entzündungsprozesses interferiert. Die Untersuchungen bestätigen antiinflammatorische Effekte der LD-RT in vitro und in vivo. Dabei steht derzeit die funktionelle Regulation der adhäsiven Interaktion von PBMC und Endothelzellen sowie von aktivierten Makrophagen im Mittelpunkt. Weiterführende experimentelle Ansätze könnten zur weiteren Klärung der molekularen Mechanismen führen. Purpose: Low-dose radiotherapy (LD-RT) with single fractions between 0.1 and 1.0 Gy is known to exert an antiinflammatory effect. Although different mechanisms for the clinical efficiency were proposed, only few experimental data are still available. This paper focuses on functional and molecular aspects of LD-RT. Methods and Results: The antiinflammatory efficiency of LD-RT in clinical studies could be confirmed in experimental models of osteoarthritis and rheumatoid arthritis. In a model of adjuvans arthritis, 5 2 1.0 Gy as well as 5 2 0.5 Gy, given at the maximum of the acute inflammation, could prevent clinically and histologically progression of the disease without affecting existing signs of inflammation. The effect of LD-RT on the adhesion of peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) was analyzed in in-vitro assays. In the dose range between 0.3 and 0.7 Gy almost 4 hours after irradiation adherent cells reached a relative minimum of adhesion compared to unirradiated controls. In PBMC an discontinuous increase of apoptosis with a maximum between 0.3 and 0.5 Gy, the proteolytic shedding of L-selectin and an increased expression of the antiinflammatory cytokine interleukin 10 as well as downregulation of TNF! could be identified as potential mechanisms for the observed reduced adhesion. Conversely, reduced expression of E-selectin and an increased induction of transforming growth factor beta (TGFş) with a maximum at 0.5 Gy could be observed in endothelial cells. Macrophages immigrating the site of inflammation are known to express inducible nitric-oxidase synthase (iNOS), which in turn mediates cytotoxic and immunmodulatory effects by producing nitric oxide (NO). LD-RT of stimulated macrophages within the dose range between 0.6 and 1.25 Gy reduced NO production and iNOS-protein expression without affecting iNOS-mRNA expression. Conclusion: Our experimental data have confirmed the antiinflammatory efficiency of LD-RT in vitro and in vivo, indicating effects on different cellular components and mechanisms of inflammation. The regulation of the adhesion between PBMC and endothelial cells and the effects on activated macrophages may mediate the antiinflammatory properties of LD-RT. Ongoing experiments will help to clarify the molecular mechanism.     

Feasibility and limits of bone marrow mononuclear cell expansion following irradiation

PURPOSE: To define the ability of bone marrow mononuclear cells (BMMNC) to expand after irradiation and to determine the amount of apoptosis in irradiated expanded cells. MATERIALS AND METHODS: Non-human primate BMMNC were irradiated in vitro at doses ranging from 0 to 4 Gy and were cultured during 1 week in the presence of interleukin 3, interleukin 6, stem cell factor, thrombopoietin and fms-like tyrosine kinase-3 ligand. The expansion yield of BMMNC, colony-forming cells and CD34(+) cells were compared with non-irradiated control cultures. Apoptosis in expanded cells was also defined by annexin V/propidium iodine staining. RESULTS: Irradiation of BMMNC up to 1 Gy did not modify the ability of haematopoietic cells to expand. At higher doses, expansion of haematopoietic cells is reduced as compared with non-irradiated cultures but it remains significant. This reduction in expansion of BMMNC was related to radiation-induced apoptosis. CONCLUSION: The results suggest that it is possible to expand haematopoietic cells after irradiation doses at least up to 2 Gy. This suggests a possible use of cell therapy for the treatment of radiation accident victims.     

In-vivo visualization of radiation-induced apoptosis using (125)I-annexin V

BACKGROUND: As apoptosis occurs in tumors within a short time after irradiation, the detection of the frequency of apoptosis may be useful as an indicator of the effect of treatment. For the evaluation of apoptosis under these conditions, tissue extraction from patients is indispensable. AIM: To develop a noninvasive imaging technique to measure and monitor apoptosis in tumor cells caused by X-irradiation using (125)I-radiolabeled annexin V. METHODS: The tumors used were human ependymoblastomas, which were transplanted into nude mice. The tumors were irradiated at 2, 5 or 10 Gy. (125)I-annexin V was administered intravenously 6 h after irradiation. In the 5 Gy irradiation group, the isotope was injected at various time intervals (3, 6 and 12 h) after irradiation. Three hours after the injection, the mice were sacrificed, the tumors were quickly removed and frozen sections were prepared at 6 and 40 microm thickness using a cryomicrotome. In autoradiographic imaging, the tumor-to-muscle ratios were compared in the respective irradiated groups. In addition, apoptosis detection by the in-situ end-labeling (Klenow) assay was conducted on the same sections. The number of Klenow-positive cells was counted in 100 x fields for each section. RESULTS: Both autoradiography and immunohistochemical staining showed a significantly higher frequency of apoptosis in the neoplasms in all irradiated groups than in the control group (P<0.05). Although immunohistochemical staining revealed a peak apoptosis frequency in the 5 Gy irradiated group, autoradiography revealed a peak in the group receiving a lower dose than 5 Gy. When the time from irradiation to annexin injection was varied, both imaging methods showed a peak apoptosis frequency in the group receiving the injection 6 h after irradiation. CONCLUSION: It is possible to predict the effect of treatment in cancer in a noninvasive manner by apoptosis imaging in vivo after radiotherapy.     

不同剂量γ射线照射后胎儿骨髓CD34~ 造血干细胞死亡规律

目的　探讨不同剂量γ射线照射后胎儿骨髓CD34 造血干细胞的死亡规律。方法　以流式细胞术PE -CD34 FITC AnnexinV 7AAD三色荧光法 ,多参数分析了受不同剂量γ射线照射后 ,不同时间点的胎儿骨髓CD34 造血干细胞凋亡、坏死和存活的时效和量效变化特点。结果　受大剂量率γ射线 1次 5Gy和 8Gy照射后 ,胎儿骨髓CD34 造血干细胞的死亡是在照射后为期 1周的连续性死亡 ,既有凋亡又有坏死 ,但是以凋亡为主 ;受大剂量率γ射线 1次 10Gy和 12Gy照射后 ,胎儿骨髓CD34 造血干细胞则在受照后当天即大量坏死 ,在持续 1周内全部死亡。结论　受大剂量率γ射线 1次照射后 ,造血干细胞在 1周内发生了增殖期死亡而连续性空出所占据的龛位     

Combined Effect of Tumor Necrosis Factor-alpha and Ionizing Radiation on the Induction of Apoptosis in 5637 Bladder Carcinoma Cells

BACKGROUND AND PURPOSE: Apoptosis can be induced by distinct but overlapping pathways. Ionizing radiation induces apoptosis by an "intrinsic", mitochondria-dependent pathway. Ligation of tumor necrosis factor-(TNF-)alpha, FAS (CD95) or TRAIL receptors are typical representatives of an extrinsic, death-receptor-mediated pathway. In this study the effect of irradiation, treatment with the cytokine TNF-alpha, or a combination of both on the induction of apoptosis and clonogenic survival of bladder carcinoma cells was investigated. MATERIAL AND METHODS: 5637 bladder carcinoma cells were treated with different concentrations of recombinant TNF-alpha (0-10 ng/ml), irradiated with single doses ranging from 0.5 to 10 Gy, or a combination of both modalities. Apoptotic cells were quantified by the TUNEL assay up to 96 h following treatment, clonogenic cell survival by a clonogenic assay. Synergistic effects of both modalities were evaluated using isobolographic analysis. RESULTS: Irradiation of 5637 carcinoma cells resulted in a discontinuous dose dependence of the apoptotic fraction with a pronounced increase in the range of 0-2 Gy and a slighter increase at 2-10 Gy. The percentage of apoptotic carcinoma cells also increased continuously after treatment with lower concentrations of TNF-alpha reaching a plateau at concentrations of 5.0-10.0 ng/ml. Isobolographic analysis revealed a supraadditive interrelationship between irradiation and TNF-alpha in the range between 0.005 and 0.5 ng/ml, and an additive effect for TNF-alpha concentrations>0.5 ng/ml. The additive effects were confirmed in clonogenic survival assays with reduced survival fractions following combined TNF-alpha administration and irradiation. CONCLUSION: The combination of two apoptosis-inducing modalities resulted in a synergistic effect on the induction of apoptosis in 5637 bladder carcinoma cells. Although a radiosensitizing effect still has to be proven in animal models, combined-modality treatment may increase the therapeutic effectiveness of irradiation in bladder cancer.     

人肺腺癌A549细胞低剂量辐射超敏感性及其机制的研究

目的 观察A549细胞的低剂量辐射超敏感性现象,探讨其发生的机制。方法 A549细胞接受0~2 Gy的⁶⁰Coγ射线照射后,流式细胞仪对其分选计数,克隆形成法检测细胞存活分数,Western blot法检测ATM1981Ser-P蛋白表达,Hoechst 33258荧光染色法、AnnexinⅤ-FITC/PI双染流式细胞仪检测细胞凋亡,PI单染流式细胞仪检测细胞周期。结果 细胞在0~0.3 Gy表现出单位剂量杀伤增强,在0.3~0.5 Gy表现出一定的辐射抗性,0.5 Gy后的区域存活分数随辐射剂量的增加而降低。照射后1 h,ATM激酶在0.2 Gy时开始活化,0.5 Gy时活化达高峰(t=7.96,P<0.05);与0.5 Gy相比1.0和2.0 Gy的活化水平无明显变化(t=0.69、0.55,P>0.05)。照射后24 h,部分细胞发生凋亡,其凋亡曲线与存活曲线相吻合。与未照射组相比,0.1和0.2 Gy组在各时间点(照射后6、12和24 h)的细胞周期无明显变化,而0.3、0.4和0.5 Gy 组,照射后6和12 h细胞发生G₂/M期阻滞(t＝2.87、2.88、4.92和3.70、3.12、8.11,P<0.05),照射后24 h G₂/M期细胞比例下降(t＝3.87、4.77、3.01,P<0.05)。结论 A549细胞存在HRS/IRR现象,其发生可能与ATM激酶、细胞周期变化有关,凋亡是细胞死亡的主要方式。     

Low-dose radiotherapy (LD-RT) and the modulation of iNOS expression in adjuvant-induced arthritis in rats

PURPOSE: Low-dose radiotherapy (LD-RT) of arthritic joints applied during the peak of the acute inflammatory response improves the clinical and histomorphological development of adjuvant arthritis. The study was undertaken to investigate the cellular composition of the inflammatory infiltrate and the expression of the pro-inflammatory and anti-inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclo-oxygenase 2 (COX-2) and haem-oxygenase 1 (HO-1), in response to LD-RT. MATERIALS AND METHODS: Adjuvant arthritis in female Lewis rats was induced by intradermal injection of heat-inactivated mycobacterium tuberculosis on day 0. Both arthritic hind paws were sham irradiated (group 1) or X-irradiated with either 5 x 1.0 Gy (group 2) or 5 x 0.5 Gy (group 3) from days 15 to 19 after induction (15 animals/group). On days 21 (n=12 joints/group) and 30 (n=18 joints/group), cryostat sections were analysed histologically and immunohistologically after specific staining for macrophages, iNOS, COX-2 and HO-1. RESULTS: A total of 5 x 1.0 Gy or 5 x 0.5 Gy led to a significant reduction of clinical symptoms from days 21 to 29, and a highly significant reduction of cartilage and bone destruction on day 30. Macrophage-positive areas could be detected continuously throughout the periarticular infiltrate, and were slightly reduced after LD-RT on days 21 and 30. This reduction was more pronounced after 5 x 1.0 Gy. Following LD-RT, the iNOS score was reduced by about 45-50% on days 21 (p<0.05) and 30 (p<0.001). In contrast, the HO-1 score was increased by about 50% on days 21 (p=0.08) and 30 (p=0.03). CONCLUSIONS: The clinically and histologically observed prevention of the progression of adjuvant arthritis after LD-RT given during the peak of the acute inflammatory response and the reduction of cartilage and bone destruction in the chronic phase appears to be related to the modulation of iNOS activity by low X-ray doses.     

In vitro evaluation of 213Bi-rituximab versus external gamma irradiation for the treatment of B-CLL patients: relative biological efficacy with respect to apoptosis induction and chromosomal damage

External source radiotherapy and beta radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha emitters is attractive because of the high linear energy transfer (LET) and short path length, allowing higher tumour cell kill and lower toxicity to healthy tissues. We assessed the relative biological efficacy (RBE) of alpha RIT (in vitro) compared to external gamma irradiation with respect to induction of apoptosis in B chronic lymphocytic leukaemia (B-CLL) and induction of chromosomal damage in healthy donor B and T lymphocytes. The latter was measured by a micronucleus assay. ²¹³Bi was eluted from a ²²⁵Ac generator and conjugated to CD20 antibody (rituximab) with CHX-A'-DTPA as a chelator. B-CLL cells from five patients were cultured for 24 h in RPMI/10% FCS while exposed to ²¹³Bi conjugated to CD20 antibody or after external ⁶⁰Co gamma irradiation. Binding assays were performed in samples of all patients to calculate the total absorbed dose. Apoptosis was scored by flow cytometric analyses of the cells stained with annexin V-FITC and 7-AAD. Apoptosis was expressed as % excess over spontaneous apoptosis in control. Full dose range experiments demonstrated ²¹³Bi-conjugated CD20 antibody to be more effective than equivalent doses of external gamma irradiation, but showed that similar plateau values were reached at 10 Gy. The RBE for induction of apoptosis in B-CLL was 2 between 1.5 and 7 Gy. The micronucleus yield in lymphocytes of healthy volunteers was measured to assess the late toxicity caused by induction of chromosomal instability. While gamma radiation induced a steady increase in micronucleus yields in B and T cells, the damage induced by ²¹³Bi was more dramatic, with RBE ranging from 5 to 2 between 0.1 Gy and 2 Gy respectively. In contrast to gamma irradiation, ²¹³Bi inhibited mitogen-stimulated mitosis almost completely at 2 Gy. In conclusion, high-LET targeted alpha particle exposure killed B-CLL cells more effectively than did external gamma irradiation at a low dose (RBE=2), while a plateau was reached at a high dose. Long-term toxicity on healthy B and T lymphocytes was systematically higher for the alpha emitter (RBE=5 to 2).     

低剂量率裂变中子照射对大鼠外周血淋巴细胞亚群的影响

目的 探讨低剂量率中子长期照射对大鼠外周血细胞亚群的影响。方法 96只雄性大鼠分为对照组和照射组,照射组每天用低剂量率中子²⁵²Cf(吸收剂量率为0.35 mGy/h)照射20.5h,在照射的第14、28、42、56和70天(累积剂量分别为0.1、0.2、0.3、0.4和0.5 Gy)及停止照射后第35天各取8只大鼠,用血细胞计数仪检测大鼠外周血WBC、用流式细胞仪检测外周血CD4⁺CD3⁺、 CD8⁺CD3⁺、CD45RA⁺/CD161α⁺亚群的变化。结果 累积剂量为0.3、0.4及0.5 Gy时WBC明显低于对照组(P<0.05),停止照射后35 d,WBC显著低于对照组(P<0.01);累积剂量为0.1、0.3、0.4、0.5 Gy及停止照射后35 d,外周血CD4⁺CD3^-细胞比例显著高于对照组(P<0.01或<0.05);累积剂量为0.2和0.3 Gy时CD8⁺CD3^-细胞比例显著高于对照组(P<0.05或P<0.01)。而累积剂量为0.1 Gy时的CD4⁺CD3⁺细胞比例及0.1和0.2 Gy时的CD8⁺CD3⁺细胞比例明显高于同一天对照组(P<0.01或<0.05)。另外,低剂量率中子长期照射可使累积剂量为0.2~0.3 Gy的外周血NK细胞(CD161α⁺ CD45RA^-)显著升高,累积剂量为0.1~0.5 Gy及停止照射后35 d照射组的外周血B细胞(CD161α^- CD45RA⁺)比例明显下降。结论 低剂量率裂变中子长期照射可使外周血淋巴细胞TCR基因突变,使大鼠外周血WBC减少,淋巴细胞中B细胞减少,NK细胞细胞比例升高,这种变化在停止照射后一段时间仍可能难以恢复。     

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.     

Dose-rate effects in normal and malignant cells of human origin

Human cell lines derived from normal tissue and malignant tumours were irradiated in plateau phase under acute (81 Gy/h) or protracted (0.1-0.7 Gy/h) exposure to determine initial survival curve slopes, assess sublethal damage repair (SLDR) capability, and establish differences in survival due to SLDR over a range of dose rates. No correlation was found between clinical resistance of the tumor types and initial slope or survival at 2 Gy. Sublethal damage repair was assessed by comparing survival curves for acute and continuous irradiation. All cell lines showed significant SLDR, with the more clinically resistant tumour types demonstrating the best recovery rates. The survival data also demonstrated little difference in cell killing over the 0.1-0.7 Gy/h range. Using a modified linear-quadratic model with provisions for SLDR, it was found that for these cell lines the repair half-time was less than 1 h, with no significant change in the amount of recovery from sublethal damage at dose rates below 1 Gy/h.     

低剂量辐射对恶性肿瘤患者自体CIK细胞免疫功能影响的实验研究

目的 观察低剂量辐射对恶性肿瘤患者自体CIK细胞增殖、表型和杀伤活性的影响,为临床应用CIK细胞过继免疫治疗提供依据。方法 取10例恶性肿瘤患者外周血,常规分离出单个核细胞,用不同细胞因子培养诱导CIK细胞。培养10 d后,以30、50、80、100和200 mGy X射线照射,24 h后采用³H-TdR掺入法检测对CIK细胞增殖的影响;流式细胞术检测对CD3+CD56+表型CIK细胞百分率的影响;³H-TdR释放法检测对其杀伤活性的影响。采用³H-TdR释放法检测80 mGy X射线照射对自体CIK细胞在12、24、48和72 h杀伤活性的影响,及80 mGy连续照射3 d对CIK细胞杀伤活性的影响。结果 CIK细胞增殖活性与对照组(0 mGy)相比均有明显增高(t =2.2、3.5、3.3和2.2, P ＜0.05)。50、80和100 mGy组的CD3+CD56+细胞的百分率均有显著性增高(t =2.3、4.2和2.4, P ＜0.05)。CIK细胞接受LDI,80 mGy组和100 mGy组与对照组(0 mGy)相比,CIK细胞杀伤活性均有显著性增高(t =3.3和2.3, P ＜0.05)。80 mGy照射后24 h,CIK细胞的杀伤活性出现高峰,为(54.2±5.0)%(t =3.2, P ＜0.01),48和72 h下降到正常水平。80 mGy连续照射3 d,24和48 h CIK细胞杀伤活性分别为(55.2±5.3)%和(61.9±4.4)%,72 h达到最高水平为(67.2±5.7)%(t =2.6、4.7和5.7, P ＜0.05)。结论 低剂量辐射对肿瘤患者CIK细胞显示兴奋效应,具有临床应用前景。     

脂肪干细胞治疗放射性肠炎的实验研究

目的：评价脂肪干细胞对放射性肠炎的治疗作用。方法：选用成年雄性SD大鼠,共52只。随机数字表法选取46只给予全腹15 Gy X射线照射制作放射性肠炎动物模型。造模结束后2 h,随机数字表法从受照大鼠中选取23只受照大鼠给予P6代青年女性脂肪干细胞腹腔注射治疗（Ad-MSC治疗组）;取另23只受照大鼠给予双磷酸盐缓冲液（PBS）腹腔注射治疗（PBS溶剂对照组）;剩余6只未照射大鼠作为健康对照组。造模后首个10 d内,分次抽取受照大鼠外周血,ELISA法检测血清中白介素-10（IL-10）的含量;分离受照大鼠胸腺细胞及外周血单个核细胞（PBMC）,流式细胞术检测CD4/CD25/Foxp（3）阳性调节性T细胞的百分含量;获取受照小肠组织,HE染色分析炎症细胞浸润程度,Masson三色染色分析胶原成分的沉积程度,髓系过氧化物酶（MPO）免疫组织化学染色分析浸润的炎症细胞类型。Kaplan-Meier方法对受照大鼠进行生存分析。结果：在照射后30 d内,与PBS腹腔注射治疗的大鼠相比,接受脂肪干细胞治疗的大鼠的生存时间显著延长（t=4.53,P<0.05）。另外,脂肪干细胞能够上调受照大鼠外周血中IL-10的含量（7 d： t=13.93,P<0.05）;上调外周血（3.5 d：t=7.72,7 d：t=11.11,10 d：t=6.99,P<0.05）和胸腺（7 d：t=16.17,10 d：t=12.12,P<0.05）CD4/CD25/Foxp（3）阳性调节性T细胞的百分比;进而减轻炎症细胞在受照小肠中的浸润及胶原的沉积。结论：脂肪干细胞对放射性肠炎具有治疗作用。     

波长510.6 nm铜蒸气激光诱导兔血管平滑肌细胞凋亡的照射剂量筛选

目的　筛选对离体培养的兔血管平滑肌细胞 (VSMC)能诱导出较高凋亡率的激光照射剂量参数。方法　取离体培养的第 4代兔VSMC ,予波长 5 10 6nm铜蒸气激光照射 ,照射剂量为功率密度 10 0、2 0 0、30 0mW cm2 ,能量密度10 0、15 0、180、2 0 0、2 10、30 0J cm2 。脱氧核苷酸末端转移酶介导的缺口末端标记法 (TUNEL)标记检测各照射剂量组细胞凋亡率。结果　与对照组比较 ,各照射组VSMC凋亡率明显增高 ,达对照组凋亡率的 2 9～ 14 7倍 (P <0 0 5 )。以功率密度 30 0mW cm2照射 6 6 7s照射组之VSMC凋亡率最高 ,达 30 %。结论　以波长 5 10 6nm、功率密度 30 0mW cm2 铜蒸气激光照射兔VSMC 6 6 7s可诱导发生较高的细胞凋亡率     

Radiobiological mechanisms in inflammatory diseases of low-dose radiation therapy

PURPOSE: Whereas X-irradiation with high doses is established to exert pro-inflammatory effects, low-dose radiotherapy (LD-RT) with single fractions below 1.0 Gy and a total dose below 12 Gy is clinically well known to exert anti-inflammatory and analgesic effects on several inflammatory diseases and painful degenerative disorders. Experimental studies to confirm the effectiveness, the empirical dose and fractionation schemes, and the underlying radiobiological mechanisms are still fragmentary. METHOD: The anti-inflammatory efficiency of LD-RT was confirmed in several experimental in vitro and in vivo models. RESULTS: In vitro studies revealed a variety of mechanisms related to the anti-inflammatory effect, in particular the modulation of cytokine and adhesion molecule expression on activated endothelial cells and leukocytes, and of nitric oxide (NO) production and oxidative burst in activated macrophages and native granulocytes. CONCLUSION: Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. It is, therefore, reasonable to assume that further molecular pathways and cellular components contribute to the anti-inflammatory effect of LD-RT. This review discusses data and models revealing aspects of the mechanisms underlying the anti-inflammation induced by low doses of X-irradiation and may serve as a basis for systematic analyses, necessary to optimize LD-RT in clinical practice.     

Lack of inverse dose-rate effect on fission neutron induced transformation of C3H/10T1/2 cells

Exponential and density-inhibited cultures of C3H/10T1/2 cells were exposed to a single dose of 0.3 Gy of fission neutrons delivered at rates ranging from 0.005 to 0.1 Gy/min. No discernible effect upon cell survival or transformation was observed by a lowering of the fission neutron dose rate in either exponential or plateau cultures. At the level of 2.3 x 10(-4) transformants per surviving cell, the RBE for neoplastic transformation was three at acute dose rates and ten at the lowest dose rate studied (0.005 Gy/min for neutrons and 0.01 Gy/min for X-rays).