细胞早衰对放射性皮肤损伤中伤口愈合的影响及机制研究

医疗照射、职业性暴露、应急照射均可引起放射性皮肤损伤, 相关的研究大多集中在放射性皮肤损伤的预防与治疗措施方面, 但相关分子尚未完全阐明。研究表明, 辐射诱导的细胞早衰在放射性皮肤损伤发生发展中发挥重要作用。本文从放射性皮肤损伤机制及细胞早衰促进放射性皮肤损伤的发生发展, 电离辐射诱导的细胞早衰信号通路, 及细胞早衰在伤口愈合中的作用3个方面进行综述, 探讨辐射诱导的细胞早衰与放射性皮肤损伤的关系。     

PERK参与调控砷化物诱导细胞自噬反应的信号转导机制研究

目的 探索PERK是否参与调控砷化物诱导的细胞自噬反应.方法 体外培养人肝癌细胞HepG2,以砷化物为刺激源,采用Western印迹方法检测自噬反应标志性蛋白的表达水平、PERK诱导活化状态以及敲低PERK表达水平后砷化物诱导细胞自噬反应和p53诱导活化水平变化情况;采用双荧光素酶报告基因技术检测敲低PERK表达水平后p53的转录激活活性变化.结果 砷化物刺激HepG2细胞后自噬反应相关蛋白Beclin-1诱导表达、LC3发生剪切、p62发生降解反应,同时PERK活化水平显著增强;敲低PERK表达水平后,砷化物刺激作用下Beclin-1的诱导表达、LC3的剪切以及p62的降解均被显著抑制;同样条件下p53在Ser15和Ser392位的磷酸化修饰反应水平和转录激活活性显著下降、p53下游靶基因DAPK1的诱导表达水平被显著抑制.结论 PERK能通过调节p53活化及其下游靶基因DAPK1表达从而介导砷化物诱导的细胞自噬反应.     

褪黑素通过调控p53介导的细胞早衰抑制紫外线诱导的HaCaT细胞中黑色素合成

目的 探索褪黑素对中波紫外线（UVB）引起人永生化表皮角质形成（HaCaT）细胞黑色素合成的影响及机制,为褪黑素的皮肤保护机制提供理论基础。方法 80 mJ/cm² UVB照射10^-5 mol/L褪黑素预处理的HaCaT细胞,照后48和72 h,利用NaOH法检测细胞黑色素水平。照后72 h,利用β-半乳糖苷酶染色试剂盒检测早衰阳性细胞并分析其比例,蛋白免疫印迹法检测衰老相关蛋白p53和酪氨酸酶（TYR）的表达变化。80 mJ/cm² UVB照射分别经毛细血管扩张性共济失调突变激酶/毛细血管扩张性共济失调Rad3相关激酶（ATM/ATR）抑制剂、p53抑制剂和褪黑素预处理的HaCaT细胞,照后72 h检测细胞早衰阳性比例和黑色素水平变化。结果 褪黑素抑制UVB诱导的黑色素水平增加（t=56.65、13.39,P<0.05）,同时抑制UVB诱导的TYR表达增加（t=16.46,P<0.05）;并可缓解UVB诱导的HaCaT细胞早衰（t=6.37,P<0.05）,抑制UVB诱导的p53表达增加（t=19.08,P<0.05）;ATM/ATR抑制剂、p53抑制剂和褪黑素预处理均可抑制UVB诱导的HaCaT细胞黑色素水平升高（t=13.88、7.86、8.96,P<0.05）。结论 褪黑素通过调控p53介导的细胞早衰,抑制UVB诱导的HaCaT细胞黑色素水平增加。     

DAPK1调控砷化物诱导的细胞自噬反应机制研究

目的 探究死亡相关蛋白激酶1(DAPK1)在砷化物As203诱导细胞自噬反应中的作用及分子机制.方法 应用As203刺激HepG2细胞后,蛋白质免疫印迹和逆转录PCR实验检测DAPK1、自噬标志性信号分子和DNA损伤调节自噬相关蛋白1(DRAM1)的表达水平及p53活化能力;采用双荧光素酶报告分析实验检测敲低DAPK1后p53的转录激活能力变化情况,流式细胞术检测敲低DAPK1对As203诱导细胞自噬反应的影响.结果 As203刺激能诱导HepG2细胞中DAPK1表达;敲低DAPK1表达显著抑制了细胞自噬反应水平;同时p53的诱导活化反应强度显著下调,但自噬反应特异性p53靶基因DRAM1的表达水平无明显变化.此外,敲低p53表达可显著抑制砷化物刺激作用下DAPK1的诱导表达水平.结论 DAPK1在As203诱导细胞自噬反应中可通过与p53形成正反馈通路实现对自噬信号的放大效应.     

NADH对X射线诱导的细胞凋亡信号传递的影响

目的　研究NADH抗辐射诱导的细胞凋亡及其作用机制。方法　通过MTT法和流式细胞仪检测L 0 2细胞受照后加和不加NADH(4 0 0 μg·ml-1)条件下细胞活性、细胞凋亡率和凋亡相关蛋白p5 3、p2 1、p16、bcl 2阳性细胞表达率。结果　细胞活性随X射线辐射剂量增大而减少 ,细胞凋亡率增加 ,NADH能增加肝细胞受照后细胞活性和减少细胞凋亡率。与假照射组相比 ,L 0 2细胞离子辐射后p5 3、p2 1和p16阳性表达率上升 ,bcl 2下降 ;加入NADH后 ,明显上调受照后肝细胞bcl 2蛋白表达和下调p5 3、p2 1和p16表达 ,差异非常显著 (P <0 .0 5 )。结论　X射线诱导正常肝细胞L 0 2凋亡及NADH抗辐射作用可能与p5 3信号传递途径有关     

p38 MAPK对PDGF诱导的细胞迁移的调控作用

目的 研究p38丝裂原活化蛋白激酶(MAPK)在血小板源性生长因子(PDGF)诱导的细胞迁移中的作用.方法 PDGF刺激小鼠NIH 3T3细胞后,利用免疫印迹法检测p38磷酸化程度的改变情况.利用Transwell细胞迁移系统来观察PDGF对NIH 3T3细胞迁移的诱导作用,以及p38基因敲除对PDGF诱导的细胞迁移的影响.结果 PDGF能显著诱导NIH 3T3细胞迁移(P＜0.001),且在此过程中p38能被磷酸化激活,p38基因敲除能阻断PDGF诱导的细胞迁移(P＜0.001).结论 p38 MAPK参与了PDGF诱导的细胞迁移的调控.     

p53 K370位乙酰化调控紫外线诱导的p53活化及细胞凋亡反应

目的探讨p53 K370位乙酰化修饰反应在紫外线B（UVB）诱导p53活化及细胞凋亡反应中的作用。方法体外培养野生型和p53基因缺陷型小鼠胚胎成纤维细胞（wt-MEFs和p53-/-MEFs）。以UVB为刺激源,双荧光素酶报告基因法分析wt-MEFs细胞中p53的转录诱导活化情况;Western印迹检测UVB诱导p53在K370位发生乙酰化修饰反应及p53总蛋白表达情况;构建p53点突变体K370R并在p53-/-MEFs细胞中比较UVB诱导wt-p53和p53 K370R的转录活化情况及野生型和突变体蛋白介导细胞凋亡反应的差异。结果 UVB刺激wt-MEFs细胞后p53转录激活活性呈剂量和时间依赖性明显上调;同样条件下UVB能够有效诱导p53在K370位发生乙酰化修饰反应;p53 K370位突变后并不影响UVB刺激作用下p53的蛋白质稳定性,但其转录激活活性显著下降;同时K370位突变后能够有效抑制UVB诱导的细胞凋亡反应。结论 p53 K370位乙酰化修饰反应在UVB诱导的p53活化及细胞凋亡反应中具有重要作用。     

体外诱导表皮细胞去分化的初步实验研究

目的 建立表皮细胞去分化体外模型,探索与去分化过程有关的信号通路.方法 分离培养人表皮细胞,分别用双氧水、ERK阻断剂PD98059加双氧水处理细胞,采用免疫细胞化学染色法和Western blot法检测不同方式处理后表皮细胞表型的变化情况,Western blot法检测双氧水处理后ERK信号通路相关蛋白的表达情况.结果 双氧水处理后,表皮细胞干细胞标志物(CK19、β1整合素、Oct4和p63)表达增加,分化细胞标记物(CK10)表达减少,ERK信号通路中相关的磷酸化蛋白表达增强.以PD98059阻断ERK通路的活化后,双氧水诱导的表皮细胞表型逆转受到了抑制.结论 ERK通路参与了双氧水诱导的表皮细胞去分化过程.     

内毒素直接作用于血管内皮细胞激活信号分子的研究

目的：对内毒素（LPS）直接作用于血管内皮细胞（VECs)激活的多种信号通路的主要激酶及转录因子进行筛选研究。方法：10ng.ml^-1浓度的LPS刺激细胞至预定时相点后,用免疫细胞化学观察信号分子的磷酸化和／或核转位,结果：LPS直接作用于VECs在5－15min内即可观察到多种信号分子的激活,随刺激时间延长,信号分子激活程度逐步增,至一定时相点后活性又消失,这些信号分子的活性在持续时间上存在一定的差异。这些激活的信号分子包括p38、ERK1／2MAPK、CREB、STAT5、STAT6、NF－kB家庭成员p65、p50、p52、c-Rel、Rel-B等。结论：LPS直接作用于VECs可以迅速激活多种信号分子,诱导信号分子的磷酸化和／或核转位。LPS诱导的CREB磷酸化、不同NF－kB家族成员核转位持续时间的差异是第一次报道。     

黄芩苷诱导人肝癌HepG2细胞凋亡的p38MAPK途径研究

目的研究黄芩苷对人肝癌HepG2细胞增殖的抑制作用及其对p38MAPK信号通路的影响。方法以不同浓度的黄芩苷与人肝癌HepG2细胞共同培养24、48、72 h后,采用MTT法测定细胞增殖抑制率;通过活细胞计数,检测不同浓度黄芩苷处理后细胞活力的变化;采用western blotting方法检测黄芩苷处理72 h后HepG2细胞p38和p-p38蛋白的表达变化。结果黄芩苷对HepG2细胞生长有抑制作用,并随药物作用时间的延长和药物浓度的增加,其抑制效果越明显;通过对活细胞计数发现黄芩苷对HepG2细胞生长有抑制作用,随黄芩苷浓度的增加,细胞存活率下降,细胞增殖速度明显减缓;在黄芩苷处理72 h后p38蛋白表达兀明显变化,而p-p38蛋白表达上调且随黄芩苷浓度升高蛋白表达升高。结论黄芩苷可能通过激活p38MAPK信号通路诱导人肝癌HepG2细胞凋亡。     

Transcriptomic profiling suggests a role for IGFBP5 in premature senescence of endothelial cells after chronic low dose rate irradiation

Abstract

Purpose: Ionizing radiation has been recognized to increase the risk of cardiovascular diseases (CVD). However, there is no consensus concerning the dose-risk relationship for low radiation doses and a mechanistic understanding of low dose effects is needed.

Material and methods: Previously, human umbilical vein endothelial cells (HUVEC) were exposed to chronic low dose rate radiation (1.4 and 4.1 mGy/h) during one, three and six weeks which resulted in premature senescence in cells exposed to 4.1 mGy/h. To gain more insight into the underlying signaling pathways, we analyzed gene expression changes in these cells using microarray technology. The obtained data were analyzed in a dual approach, combining single gene expression analysis and Gene Set Enrichment Analysis.

Results: An early stress response was observed after one week of exposure to 4.1 mGy/h which was replaced by a more inflammation-related expression profile after three weeks and onwards. This early stress response may trigger the radiation-induced premature senescence previously observed in HUVEC irradiated with 4.1 mGy/h. A dedicated analysis pointed to the involvement of insulin-like growth factor binding protein 5 (IGFBP5) signaling in radiation-induced premature senescence.

Conclusion: Our findings motivate further research on the shape of the dose-response and the dose rate effect for radiation- induced vascular senescence.     

In vitro response of human dermal fibroblasts to X-irradiation: relationship between radiation-induced clonogenic cell death,chromosome aberrations and markers of proliferative senescence or differentiation

Purpose : To analyse the relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation. Materials and methods : Plateau-phase human dermal fibroblasts from 18 donors were irradiated with graded doses of 1-6 Gy 200kV X-rays. Cell survival was determined by a colony-forming assay. Markers of differentiation or senescence were: spontaneous and radiation-induced clonal differentiation, which was determined morphologically and by the cellular potential to proliferate in clonal culture, also single-cell g -galactosidase (g -gal) staining at pH 6.0; and the secretion of transforming growth factor- g (TGF- g 1) into the culture medium. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and the excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations for chromosomes 4, 7 and 9 using the FISH method. Results : A broad spread was found in the shapes of the survival curves, with SF2 ranging from 0.041 - 0.015 to 0.63 - 0.05. Radiation-induced clonal differentiation as well as the secretion of TGF- g 1 was elevated in radiosensitive samples. With respect to chromosome aberrations, a significant correlation was found between clonogenic survival and radiation-induced excess acentric fragments. Conclusions : In the fibroblast cell system, in vitro radiosensitivity is determined not only by processes directly involved in DNA-damage recognition and repair, but also by intracellular signalling cascades, which will lead to differentiation processes.     

In vitro response of human dermal fibroblasts to X-irradiation: relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation.

PURPOSE: To analyse the relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation. MATERIALS AND METHODS: Plateau-phase human dermal fibroblasts from 18 donors were irradiated with graded doses of 1-6 Gy 200kV X-rays. Cell survival was determined by a colony-forming assay. Markers of differentiation or senescence were: spontaneous and radiation-induced clonal differentiation, which was determined morphologically and by the cellular potential to proliferate in clonal culture, also single-cell beta-galactosidase (beta-gal) staining at pH 6.0; and the secretion of transforming growth factor-beta (TGF-beta1) into the culture medium. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and the excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations for chromosomes 4, 7 and 9 using the FISH method. RESULTS: A broad spread was found in the shapes of the survival curves, with SF2 ranging from 0.041+/-0.015 to 0.63+/-0.05. Radiation-induced clonal differentiation as well as the secretion of TGF-beta1 was elevated in radiosensitive samples. With respect to chromosome aberrations, a significant correlation was found between clonogenic survival and radiation-induced excess acentric fragments. CONCLUSIONS: In the fibroblast cell system, in vitro radiosensitivity is determined not only by processes directly involved in DNA-damage recognition and repair, but also by intracellular signalling cascades, which will lead to differentiation processes.     

Gamma radiation induces senescence in human adult mesenchymal stem cells from bone marrow and periodontal ligaments

Purpose: Mesenchymal stem cells isolated from bone marrow (BM-MSC) and periodontal ligament (PLSC) are cells with high proliferative potential and ability to self-renewal. Characterization of these cells under genotoxic stress conditions contributes to the assessment of their prospective usage. The aim of our study was to evaluate changes in BM-MSC and PLSC caused by ionizing radiation. Methods: Human BM-MSC and PLSC were irradiated with the doses up to 20 Gy by Co⁶⁰ and observed 13 days; viability, proliferation, apoptosis and senescence induction, and changes in expression and phosphorylation status of related proteins were studied. Results: Irradiation with the doses up to 20 Gy significantly reduces proliferation, but has no significant effect on cell viability. The activation of tumor suppressor protein 53 (p53) and its phosphorylations on serines 15 and 392 were detected from the first day after irradiation by 20 Gy and remained elevated to day 13. Expression of cyclin-dependent kinases inhibitor 1A (p21^Cip1/Waf1) increased. The cell cycle was arrested in G2 phase. Instead of apoptosis we have detected hallmarks of stress-induced premature senescence: increase in cyclin-dependent kinases inhibitor 2A (p16^INK4a) and increased activity of senescence-associated β-galactosidase. Conclusion: Mesenchymal stem cells isolated from bone marrow and periodontal ligament respond to ionizing radiation by induction of stress-induced premature senescence without apparent differences in their radiation response.     

Involvement of Wnt signaling in the injury of murine mesenchymal stem cells exposed to X-radiation

Abstract Purpose: To investigate the injury of murine mesenchymal stem cells (mMSC) exposed to 4 Gy X-radiation and the role of canonical and non-canonical wingless-type (Wnt) signaling in the radiation injury. Materials and methods: C3H10T1/2 cells were submitted to 4 Gy X-radiation. At different time points after radiation, Hoechst33258 staining and Annexin V-fluorescein isothiocyanate (FITC) flow cytometry analysis were performed to assess cellular apoptosis. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to analyze cellular senescence. Cell cycle was measured by flow cytometry. P53, p21, Wnt3a, Wnt5a, sonic hedgehog (Shh) mRNA was detected by Real time polymerase chain reaction (PCR) and Wnt5a protein was determined by Western blot. Results: A time-dependent cellular apoptosis was observed with a peak level 12 hours after radiation. Cellular senescence was detected 72 h after radiation. A remarkable up-regulation of Wnt5a mRNA expression (～ 269-fold) and protein expression was seen 72 h after radiation. Conclusions: The effect of 4 Gy X-radiation to mMSC was time-dependent in the form of cellular apoptosis in the early period and cellular senescence in the late period. Non-canonical Wnt signaling may be involved in mMSC senescence induced by 4 Gy X-radiation.     

白藜芦醇对IEC-6细胞辐射损伤的防护作用及机制研究

目的 探讨白藜芦醇对大鼠小肠隐窝细胞（IEC-6）辐射损伤的防护作用及机制。方法 采用不同剂量（2、4、6、8、10 Gy）的6 MeV高能X射线对大鼠IEC-6细胞进行照射,应用克隆形成实验检测细胞增殖,建立IEC-6细胞辐射损伤模型。应用CCK-8检测不同浓度白藜芦醇（0.1、0.5、1、5、10、20、40 μmol/L）对IEC-6细胞活力的影响,以及不同浓度白藜芦醇（0.1、1、2、4、6、8 μmol/L）对照射后细胞活力的影响。将细胞分为对照组、模型组（10 Gy）和白藜芦醇组（10 Gy,1 μmol/L）,对照组接受伪照射并予等量载体孵育;模型组接受6 MeV高能X射线照射,予等量载体孵育;白藜芦醇组在照射前2 h予相应浓度的白藜芦醇预处理,接受与模型组等剂量的高能X射线照射。采用 Annexin V-PI染色检测细胞凋亡;DCFH-DA荧光探针检测活性氧;β-Gal染色检测细胞衰老;Real-time qPCR、Western Blot检测p16、p21、CAT和SOD2的mRNA水平及相应蛋白质表达情况。多组间数据比较采用单因素方差分析,应用Tukey’s HSD法进行事后两两比较。结果 4~10 Gy电离辐射明显抑制IEC-6细胞增殖,呈剂量依赖性（均P<0.05）;浓度为0.1、0.5、1、5 μmol/L的白藜芦醇对细胞活力无明显影响（均P>0.05）;浓度为0.1 μmol/L、1 μmol/L的白藜芦醇可增加电离辐射后细胞活力（均P<0.05）。白藜芦醇组细胞凋亡率、DCFH-DA荧光强度、β-Gal 阳性率均低于模型组（均P<0.05）;白藜芦醇组细胞中CAT、SOD2的mRNA水平及相应蛋白质表达较模型组升高（均P<0.05）,但p16、 p21的mRNA水平及相应蛋白质表达较模型组降低（均P<0.05）。结论 白藜芦醇可以降低活性氧水平,抑制细胞衰老和凋亡,从而减轻IEC-6细胞辐射损伤。     

Low dose ionizing irradiation suppresses cellular senescence in normal human fibroblasts

Purpose: Exposure to high dose ionizing radiation leads to premature cell senescence and suppression of cell proliferation. In contrast, low dose and low dose-rate gamma-irradiation can lead to stimulation of cell proliferation. We aimed to examine whether the low dose radiation-induced proliferation of normal human fibroblasts can lead to a progressive depletion of proliferation potential and to an early onset of senescence.

Materials and methods: Normal human embryonic lung fibroblasts (HELF-104) at passage 22–24 were gamma-irradiated with doses of 0 (sham-irradiation), 10, 30, 50, 90, 120, 150, 200, and 500 mGy as well as 1 and 2?Gy. After irradiation, the fraction of cells positively stained for senescence-associated β-galactosidase activity was measured weekly until the cell culture completely ceased to proliferate.

Results: We show that single irradiation of HELF-104 cells with 30 and 50 mGy resulted in deceleration of senescence. The suppression of senescence was observed during almost the entire length of the study up to a complete arrest of cell growth.

Conclusions: Our data, together with the previously published observation of delayed stimulation of proliferation in HELF-104 cells exposed to 30 mGy, suggest that low dose gamma-irradiation can increase the overall proliferative potential of normal human fibroblasts.     

Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways

Purpose: Epidemiological studies indicate that radiation doses as low as 0.5?Gy increase the risk of cardiovascular disease decades after the exposure. The aim of the present study was to investigate whether this radiation dose causes late molecular alterations in endothelial cells that could support the population-based data.

Materials and methods: Human coronary artery endothelial cells were irradiated at 0.5?Gy (X-ray) and radiation-induced changes in the proteome were investigated after different time intervals (1, 7 and 14 d) using ICPL technology. Key changes identified by proteomics and bioinformatics were validated by immunoblotting and ELISA.

Results: The radiation-induced alteration of the endothelial proteome was characterized by sustained perturbation of Rho GDP-dissociation inhibitor (RhoGDI) and nitric oxide (NO) signalling pathways. At later time-points, this was accompanied by reduced proteasome activity, enhanced protein carbonylation indicating augmented oxidative stress, and senescence.

Conclusions: These molecular changes are indicative of long-term premature endothelial dysfunction and provide a mechanistic framework to the epidemiological data showing increased risk of cardiovascular disease at 0.5?Gy.