首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organismTetrahymena.We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agree- ment with a previous study [17], indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn2+and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion inTetrahymena,suggesting a requirement here for an endonuclease which is Ca2+-independent and Zn2+-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration.  相似文献   

2.
Programmed cell death (PCD) and phagocytotic activity of immune cells play a pivotal role in insect development. We examined the influence of Zn2+, an important element to fundamental biological processes, on phagocytosis and apoptosis of hemocytes in two fly species: Musca domestica and Drosophila melanogaster. Hemocytes were isolated from the third instar larvae of both species and treated for 3 h with zinc chloride solutions, containing 0.35 mM or 1.7 mM of Zn2+, and untreated as control. Phagocytotic activity of hemocytes was examined by flow cytometry after adding latex fluorescent beads to the medium, while apoptosis was evaluated by application of annexinV-FITC and pan-caspase-FITC inhibitor. Mitochondrial viability was determined by measuring resazurin absorbancy in the cell medium. The obtained results showed that Zn2+ increases phagocytosis and affects PCD of both species hemocytes but each in a different way. Zinc decreases fraction of annexin-positive hemocytes in M. domestica but increases it in D. melanogaster. The pan-caspase analysis revealed low and high activity of caspases in hemocytes of M. domestica and D. melanogaster, respectively. Zn2+ also decreased the viability of hemocyte mitochondria but only in D. melanogaster. It suggests that flies use different pathways of PCD, or that Zn plays a different role in this process in M. domestica than in D. melanogaster.  相似文献   

3.
4.
Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.  相似文献   

5.

Key message

An increase in Ca 2+ concentration in the nucleus may activate the PCD of secretory cavity cells, and further Ca 2+ accumulation contributes to the regulation of nuclear DNA degradation.

Abstract

Calcium plays an important role in plant programmed cell death (PCD). Previously, we confirmed that PCD was involved in the degradation of secretory cavity cells in Citrus sinensis (L.) Osbeck fruits. To further explore the function of calcium in the PCD of secretory cavity cells, we used potassium pyroantimonate precipitation to detect and locate calcium dynamics. At the precursor cell stage of the secretory cavity, Ca2+ was only distributed in the cell walls. At the early stage of secretory cavity initial cells, Ca2+ in the cell walls was gradually transported into the cytoplasm via pinocytotic vesicles. Although a small amount of Ca2+ was present in the nucleus, the TUNEL signal was scarcely observed. At the middle stage of initial cells, a large number of pinocytotic vesicles were transferred to the nucleus, where the vesicle membrane fused with the nuclear membrane to release calcium into the nucleoplasm. In addition, abundant Ca2+ aggregated in the condensed chromatin and nucleolus, where the TUNEL signal appeared the strongest. At the late stage of initial cells, the chromatin and nucleolus gradually degraded and disappeared, and the nucleus appeared broken-like, as Ca2+ in the cell wall had nearly completely disappeared, and Ca2+ in the nucleus was also rapidly reduced. Furthermore, the TUNEL signal also disappeared. These phenomena indicated that an increase in Ca2+ concentration in the nucleus might activate the PCD of secretory cavity cells, and further Ca2+ accumulation contributed to the regulation of nuclear DNA degradation.  相似文献   

6.

Key message

Kinetin-induced programmed cell death, manifested by condensation, degradation and methylation of DNA and fluctuation of kinase activities and ATP levels, is an autolytic and root cortex cell-specific process.

Abstract

The last step of programmed cell death (PCD) induced by kinetin in the root cortex of V. faba ssp. minor seedlings was explained using morphologic (nuclear chromatin/aggregation) and metabolic (DNA degradation, DNA methylation and kinases activity) analyses. This step involves: (1) decrease in nuclear DNA content, (2) increase in the number of 4′,6-diamidino-2-phenylindole (DAPI)-stained chromocenters, and decrease in chromomycin A3 (CMA3)-stained chromocenters, (3) increase in fluorescence intensity of CMA3-stained chromocenters, (4) condensation of DAPI-stained and loosening of CMA3-stained chromatin, (5) fluctuation of the level of DNA methylation, (6) fluctuation of activities of exo-/endonucleolytic Zn2+ and Ca2+/Mg2+-dependent nucleases, (7) changes in H1 and core histone kinase activities and (8) decrease in cellular ATP amount. These results confirmed that kinetin-induced PCD was a specific process. Additionally, based on data presented in this paper (DNA condensation and ATP depletion) and previous studies [increase in vacuole, increase in amount of cytosolic calcium ions, ROS production and cytosol acidification “in Byczkowska et al. (Protoplasma 250:121–128, 2013)”], we propose that the process resembles autolytic type of cell death, the most common type of death during development of plants. Lastly, the observations also suggested that regulation of these processes might be under control of epigenetic (methylation/phosphorylation) mechanisms.  相似文献   

7.
20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD is mediated via a nongenomic pathway that includes Ca2+ as a second messenger and the activation of protein kinase C/caspase-3-like protease; however, the steps leading to a concomitant buildup of intracellular Ca2+ are unknown. We employed pharmacological tools to identify the components of this pathway. ASGs were cultured in the presence of 1 μM 20E and one of the following inhibitors: a G-protein-coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, and an L- or T-type Ca2+ channel blocker. The T-type Ca2+ channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast, PCD was induced by 20E in Ca2+-free medium, indicating that the source of Ca2+ is an intracellular reservoir. The IP3R antagonist inhibited nuclear and DNA fragmentation, suggesting that the endoplasmic reticulum may be the Ca2+ source. Finally, the GPCR and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results indicate that 20E increases the intracellular level of Ca2+ by activating IP3R, and that this effect may be brought about by the serial activation of GPCR, PLC, and IP3.  相似文献   

8.
Endonucleases   总被引:4,自引:0,他引:4  
Programmed cell death (PCD) involves hydrolysis of genomic DNA, which must be catalyzed by endonuclease(s) capable of digesting dsDNA. Plants have two major classes of endonucleases active towards dsDNA, Zn2+-dependent endonuclease and Ca2+-dependent endonuclease. Both classes are found among endonucleases nominated for machineries of PCD in plants. Survey of plant endonucleases in relation to PCD leads to a possibility that a different class of endonuclease reflects a different phase of PCD-associated DNA hydrolysis.  相似文献   

9.
Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn2+ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H2O2 induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn2+]i, and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H2O2 increased concentration-dependently the [Zn2+]i and caused lysosomal dysfunction and Zn2+ loss and, furthermore, mitochondrial Zn2+ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn2+ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn2+ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn2+ uptake in isolated mitochondria, which was prevented by TPEN. H2O2-induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H2O2-induced ROS generation, lysosomal dysfunction and Zn2+ release, and mitochondrial Zn2+ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn2+ release, and mitochondrial Zn2+ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.  相似文献   

10.
This review considers modern data about the set, nature, specificity of action, and other properties of plant endonucleases involved in various forms of programmed cell death (PCD) in various plant tissues (organs). Apoptosis is an obligatory component of plant development; plant development is impossible without apoptosis. In dependence on the conditions of plant growth, this process can be induced by various biotic and abiotic factors, including stressors. Endonucleases accomplishing apoptotic degradation of nuclear material in the plant cell play one of the main roles in PCD. Plant endonucleases belong to at least two classes: (1) Ca2+- and Mg2+-dependent and (2) Zn2+-dependent nucleases. The set and activities of endonucleases change with plant age and during apoptosis in a tissue-specific manner. Apoptosis is accompanied by the induction of specific endonucleases hydrolyzing DNA in chromatin with the formation firstly of large domains and then internucleosomal DNA fragments; the products produced are of about 140 nucleotides in length with their subsequent degradation to low-molecular-weight oligonucleotides and mononucleotides. About 30 enzymes are involved in apoptotic DNA degradation. Histone H1 modulates endonuclease activity; separate (sub)fractions of this nuclear protein can stimulate or inhibit corresponding plant endonucleases. In the nucleus and cytoplasm of the plant cells, Ca2+/Mg2+-dependent endonucleases recognizing substrate DNA methylation status were revealed and described for the first time; their action resembles that of bacterial restrictases, which activity is modulated by the donor of methyl groups, S-adenosylmethionine. This indicates that higher eukaryotes (higher plants) might possess the system of restriction-modification to some degree analogous to that of prokaryotes.  相似文献   

11.
We applied the alkaline version of the single-cell gel electrophoresis (comet assay) to seedlings of heterozygous tobacco (Nicotiana tabacum L. var. xanthi) treated with zinc acetate dihydrate (20 to 80 mM Zn2+ for 2 h or 2 to 12 mM Zn2+ for 24 h). A dose dependent increase in DNA damage expressed by the tail moment values were observed in nuclei isolated from the roots after 2 and 24 h Zn2+ treatments. In contrast, Zn2+ did not induce significant DNA damage to leaf nuclei, with the exception of 10 or 12 mM Zn2+ for 24 h. Somatic mutations, identified as dark green, yellow, and dark green/yellow double sectors on the pale green tobacco leaves were not detected after any Zn2+ treatments. The accumulation of Zn in roots and shoots was determined by inductively coupled plasma optical emission spectrometry and the Zn content in roots was about three times higher than in shoots.  相似文献   

12.
The nectaries of Ipomoea purpurea wilt in the late flowering period. The senescence process of nectaries is frequently associated with cell lysis. In this paper, various techniques were used to investigate whether programmed cell death (PCD) was involved in the senescence process of nectaries in I. purpurea. Ultrastructural studies showed that nectary cells began to undergo structural distortion, chromatin condensation, mitochondrial membrane degradation, and vacuolar-membrane dissolution and rupture after bloom. 4′,6-Diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine-5′-triphosphate (dUTP) nick end-labeling (TUNEL) assay showed that nectary cell nuclear DNA began to degrade during the budding stage, and disappeared in the fruiting stage. DNA gel electrophoresis showed that degradation of DNA was random. Together, these results suggest that PCD participate in the senescence of the nectary in I. purpurea. PCD began during the budding period, followed by significant changes in nectary morphology and structure during the flowering period. During the fruiting stage, the PCD process is complete and the nectary degrades.  相似文献   

13.
Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca2+), nitric oxide (NO) and hydrogen peroxide (H2O2) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca2+ ([Ca2+]cyt) elevation, NO and H2O2 production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca2+ and NO played a negative role in this process by modulating cytochrome c release.  相似文献   

14.
PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed.  相似文献   

15.
Microalgae are rich resources for high-value nutrients and biodiesel production. However, extraction of these valuable compounds from them requires costly energy-consuming procedures due to their rigid cell walls. Application of cell-disruptive agents, the AES-Bt agents, extracted from an algicidal bacterium, Bacillus thuringiensis ITRI-G1, are a promising way to reduce the cost of cell disruption. Treatment with AES-Bt agents resulted in a rapid decline of photosynthesis ability and caused cell death in Chlorella vulgaris. Hallmarks of programmed cell death (PCD), including chromatin condensation, DNA fragmentation, and phosphatidylserine externalization, were detected in C. vulgaris cells treated with the AES-Bt agents. Therefore, the cell disruption effect caused by application of the AES-Bt agents can be due to the occurrence of PCD. Similar to other PCDs, the PCD caused by AES-Bt agents was also associated with increased reactive oxygen species (ROS). However, co-treatments with diphenyleneiodonium chloride (DPI), an NAD(P)H oxidase inhibitor, or N,N′-dimethylthiourea (DMTU), a hydrogen peroxide (H2O2) trap, with the AES-Bt agents successfully reduced ROS production, and more cells displayed a feature of PCD detected after the co-treatments. In conclusion, the AES-Bt agents can promote PCD of microalgae; however, the mechanism may not be through induction of ROS.  相似文献   

16.
Inoculation of wheat (Triticum aestivum L. cv. Huamai 8) leaves with wheat powderly mildew fungus (Blumeria graminis f. sp. tritici) induced cell death in wheat adventitious roots, where no fungal structures were observed. The cytological and molecular characterization of this cell death was shown as following: cell nuclei were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; chromatin condensed and formed peripheral conglomeration in nuclei; and perinuclear spaces partly dilated. These results suggested that, without pathogen spread, the infection could induce systemic PCD in adventitious roots. Comparison with a leaf-cutting experiment (LC)enabled us to speculate that lack of assimilates was not the only reason for the systemic PCD in wheat roots in powdery mildew experiment and that such systemic PCD might be mediated by long-distance signals. In addition, reactive oxygen species (ROS) and Ca2+ were related to the systemic PCD.  相似文献   

17.
Zn2+, an element that is essential to all life forms, can play a catalytic or a solely structural role. Previous works have shown that Zn2+ binds preferentially to water molecules and His in catalytic sites, but to Cys instructural sites, but the molecular basis for the observed ligand preference is unclear. Here, we show that the different Zn2+ roles are also reflected in the different bond distances to Zn2+ in structural and catalytic sites. We reveal the physical basis for the observed differences between structural and catalytic Zn sites: In most catalytic sites, water is found bound to Zn2+ as it transfers the least charge to Zn2+ and is less bulky compared to the protein ligands, enabling Zn2+ to serve as a Lewis acid in catalysis. In most structural sites, however, ≥ 2 Cys are found bound to Zn2+, as Cys transfers the most charge to Zn2+ and reduces the Zn charge to such an extent that Zn2+ can no longer act as a Lewis acid; furthermore, steric repulsion among the bulky Cys(S) prevents Zn2+ from accommodating another ligand. Based on the observed ligand preference and Zn-ligand distance differences between structural and catalytic Zn sites, we present a simple method for distinguishing the two types of sites and for verifying the catalytic role of Zn2+. Finally, we discuss how the physical bases revealed aid in designing potential drug molecules that target Zn proteins.  相似文献   

18.
Alterations in intracellular Zn2+ concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn2+ deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn2+ for Ca2+ and Mg2+ within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential ( m). Therefore, cytoplasmic and mitochondrial events are critical to this process.  相似文献   

19.
Pear (Pyrus pyrifolia L.) possesses an S-RNase-based gametophytic self-incompatibility (GSI) system and S-RNase, the self-incompatibility (SI) determinant in the pistil, has also been implicated in the rejection of self-pollen and genetically identical pollen. We have demonstrated that S-RNase depolymerises actin cytoskeleton, triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube, which indicates programmed cell death (PCD) may occur in SI response of Pyrus pyrifolia. Recently, we have identified that S-RNase specifically disrupted tip-localized reactive oxygen species (ROS) of incompatible pollen tube via arrest of ROS formation in mitochondria and cell walls in Pyrus pyrifolia. Furthermore, tip-localized ROS disruption not only decreased the Ca2+ current and depolymerised the actin cytoskeleton, but it also induced nuclear DNA degradation in the pollen tube. The results mentioned above indicate that a cascade signal pathway may occur in SI of Pyrus pyrifolia and PCD is used to terminate the incompatible pollen tubes growth. In this addendum, we review the cascade signal pathway of Pyrus pyrifolia SI.Key words: S-RNase, programmed cell death, reactive oxygen species, actin cytoskeleton, Ca2+ current, nuclear DNA  相似文献   

20.
Zinc (Zn) is an essential element for plants but limited information is currently available on the molecular basis for Zn2+ transport in crop species. To expand the knowledge on Zn2+ transport in barley (Hordeum vulgare L.), a cDNA library prepared from barley roots was expressed in the yeast (Saccharomyces cerevisiae) mutant strain Δzrt1/Δzrt2, defective in Zn2+ uptake. This strategy resulted in isolation and identification of three new Zn2+ transporters from barley. All of the predicted proteins have a high similarity to the ZIP protein family, and are designated HvZIP3, HvZIP5 and HvZIP8, respectively. Complementation studies in Δzrt1/Δzrt2 showed restored growth of the yeast cells transformed with the different HvZIPs, although with different efficiency. Transformation into Fe2+ and Mn2+ uptake defective yeast mutants showed that the HvZIPs were unable to restore the growth on Fe2+ and Mn2+ limited media, respectively, indicating a specific role in Zn2+ transport. In intact barley roots, HvZIP8 was constitutively expressed whereas HvZIP3 and HvZIP5 were mainly expressed in ?Zn plants. These results suggest that HvZIP3, HvZIP5 and HvZIP8 are Zn2+ transporters involved in Zn2+ homeostasis in barley roots. The new transporters may facilitate breeding of barley genotypes with improved Zn efficiency and Zn content.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号