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对海绵中呋喃萜类化合物的细胞毒和抗氧化活性的研究

作　者: 蒋雅红
导　师: 游松；金东奎
学　校: 沈阳药科大学
专　业: 微生物与生化药学
关键词: furanosesterterpenes cytotoxicity apoptosis DNA replication antioxidant
分类号: R96
类　型: 博士论文
年　份: 2005年
下　载: 143次
引　用: 0次
阅　读: 论文下载

内容摘要

During the course of our search for bioactive metabolites from the marine sponges collected from Korean waters, 10 linear furanosesterterpenes characterized by a furan group and a terminal tetronic acid moiety were identified from Psammocinia sp. (order Dictyoceratida). An inseparable 1:1 mixture of (8E,13Z,20Z)-strobilinin and (7E,13Z,20Z)-felixinin (abbreviated as EZZ) was found to deliver significant cytotoxicity against some human solid tumor cell lines. Therefore, we carried out a study mainly focused on the mechanism of EZZ-induced cytotoxicity. In addition, as a part of our growing interest to screen dual anticancer and antioxidant drugs and inspired by the multi-double bonds in the structure of EZZ, the antioxidant activity of it was also evaluated.In my study, human cervical HeLa cells treated with EZZ showed significant decreases in its proliferation rate and viability (IC50 about 50 μM), which was associated with evidence of apoptosis. Nuclear changes observed under fluorescence microscope confirmed apoptosis occurrence and showed a typical pattern of chromatin condensation, which was further proved by DNA fragmentation by gel analysis. Furthermore, Annexin V-FITC/PI dual staining demonstrated that early apoptosis has occurred mainly at 24 h, which then progressed to late apoptosis after further treatment. In addition, the cell cycle was characterized by an arrest at S phase indicating EZZ might affect DNA replication or interfere in certain signal transduction pathway.It is well known that DNA replication in S phase is a strictly regulated event. Any stress exerting on this progress would lead to cell cycle arrest or apoptosis. The inhibitory effect of EZZ on DNA replication was proved both in [~3H]dTTP incorporation assay and in SV40 DNA in vitro replication system. In order to find the molecule target (or targets) of EZZ, we further checked the effects of EZZ on three important replication proteins: topo I, DNA pol α-primase and RPA. It was found that EZZ could significantly inhibit the DNA relaxation activity of topo I and this inhibition reached to 80% at 40 μM of EZZ. In addition, more than 70% of DNA pol α-primase activity was reduced by 20 μM of EZZ, while the ssDNA binding activity of RPA was slightly affected by 50 μM of EZZ. Herein, we suggest that EZZ-induced

全文目录

Abbreviations  7-9
Abstract  9-11
Introduction  11-16
Chapter 1.EZZ-Induced HeLa Cell Apoptosis and Cell Cycle Arrest  16-30
  Abstract  16-17
  1.1 Background  17-18
  1.2 Materials and Methods  18-21
    1.2.1 Chemicals and reagents  19
    1.2.2 Determination of cell viability and proliferation  19
    1.2.3 Cell morphology  19-20
    1.2.4 Annexin V-FITC/PI apoptosis analysis  20
    1.2.5 Detection of DNA fragmentation  20-21
    1.2.6 DNA content analysis  21
  1.3 Results  21-28
    1.3.1 Effects of EZZ on the viability and proliferation of HeLa cells  21-22
    1.3.2 EZZ-induced morphological changes of HeLa cells  22-24
    1.3.3 Annexin V-FITC/PI double staining assay  24-25
    1.3.4 DNA fragmentation  25-26
    1.3.5 Cell cycle arrest  26-28
  1.4 Discussion  28-30
Chapter 2.Inhibitory effects of EZZ on DNA replication and some replication proteins  30-53
  Abstract  30-31
  2.1 Background  31-35
    2.1.1 Eukaryotic DNA replication  31-32
    2.1.2 SV40 DNA replication  32-35
  2.2 Materials and Methods  35-39
    2.2.1 Chemicals and reagents  35-36
    2.2.2 [~3H]Thymidine incorporation  36
    2.2.3 In vitro SV40 DNA replication reaction  36-37
    2.2.4 Analysis of replication products  37
    2.2.5 Initiation stage assay of SV40 DNA replication  37-38
    2.2.6 Plasmid DNA relaxation by human DNA topoisomerase I  38
    2.2.7 DNA Pol α-primase assay  38
    2.2.8 ssDNA-binding activity of RPA  38-39
    2.2.9 Statistical analysis  39
  2.3 Results  39-49
    2.3.1 Inhibition of [~3H]thymidine incorporation in HeLa cells by EZZ  39-40
    2.3.2 In vitro SV40 DNA replication  40-41
    2.3.3 Replication products analysis  41-43
    2.3.4 Initiation stage of SV40 DNA replication  43-44
    2.3.5 Topo I-mediated DNA relaxation inhibited by EZZ  44-45
    2.3.6 Effect of EZZ on the activity of mammalian DNA pol α-primase  45-47
    2.3.7 Effect of EZZ on the ssDNA-binding activity of RPA  47-49
  2.4 Discussion  49-53
Chapter 3.Study on the Antioxidant Activity of EZZ  53-62
  Abstract  53-54
  3.1 Background  54
  3.2 Materials and methods  54-56
    3.2.1 Chemicals and reagents  54-55
    3.2.2 DPPH free radical scavenging activity  55
    3.2.3 Measurement of TEAC  55
    3.2.4 NBT(Superoxide scavenging) assay  55-56
    3.2.5 Inhibitory effect on hydroxyl radical-induced DNA damage  56
  3.3 Results  56-60
    3.3.1 DPPH free radical scavenging activity  56-57
    3.3.2.Measurement of TEAC  57-58
    3.3.3 NBT(Superoxide scavenging)assay  58-59
    3.3.4 Inhibitory effect on hydroxyl radical-induced DNA damage  59-60
  3.4 Discussion  60-62
References  62-73
Acknowledgements  73-74
Publication list  74

对海绵中呋喃萜类化合物的细胞毒和抗氧化活性的研究

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