| Based on the above background, on the basis of predecessors, we had synthesized a varietyof ruthenium complexes, empolyed the different research methods to study the inhibition ofthese complexes on telomerase activity, as well as the interaction with bFGF inhibition of tumorangiogenesis with Ru complexes. The paper is divided into four parts.In chapter1, it elaborated the structural characteristics and biological functions of theG-quadruplex, in particular, the structural characteristics and biological functions of humantelomeric G-quadruplex DNA and the relationship between the telomere, telomerase and tumor,then the characteristics of the G-quadruplex structure and biological function of G-quadruplexstructure of bcl-2promoter region. The second part summarized the mechanism of apoptosis;Then it introduced a number of biological functions of bFGF, and the research of promotion ofangiogenesis. This chapter also described the significance of the topic of ruthenium complexesin cell imaging and as a Fluorescence Probe of progress and the subject.In chapter2, We could find that RPD and RBD had cytotoxic effect on HUVECs, theycould effectively inhibit cell proliferation and there was a dose-dependent after incubating withbFGF by MTT. We could see that the RPD can inhibit bFGF-mediated HUVECs migration andthe role of RBD was poor from Cell Scratch and Transwell experiments. Results ofangiogenesis in vitro experiment showed that the RPD and RBD could inhibit thebFGF-mediated angiogenesis and the RPD had stronger effect on HUVEC. At the last, CAM invivo experiment showed that the RPD strongly inhibited the bFGF-mediated newborn ofmicrovascular. The role of bFGF in promoting angiogenesis is regulated by a variety of signaltransduction pathways in cells, so we reacherded the effcet of the ruthenium complex on signaltransduction pathways that activated by bFGF/bFGFR, the results showed that the RPD caninhibit the expression of p-Akt and had no effect on the expression of AktIn chapter3, We further studied the mechanism of action of the RuPOP inducing apoptosisin HepG2cells by Westernblot and found that the complex in addition to the induction of apoptosis through the mitochondrial signaling pathway, but also through the death receptorsignaling pathway, as well as the P53-mediated signaling pathways promoted the apoptosis ofHepG2cells, these results suggested that RuPOP induced mechanism of cell apoptosis throughmultiple signaling pathways crosstalk with each other and the interaction of co-exerts itsbiological effects; then the Pu39or MutPu39binding properties of complexes have beenstudied by emission spectra, CD spectra, FRET, PCR-stop assay. These results showed thatRuPOP and RuBOP could selectively induce Pu39not MutPu39to a hybrid G-quadruplex.Among them, the bioactivity of RuPOP was the strongest, and areneRuMOP basicly had nobioactivity.In chapter4, we used the circular dichroism spectra experiment, FRET melting experiment,gel mobility shift assay, PCR-stop assay and other experiments systematically studied theinteraction of the chiral ruthenium complexes with telomeric G-quadruplex. The differentquadruplex binding properties of these compounds were evaluated by circular dichroismspectroscopy, fluorescence resonance energy transfer melting assay. The results show that fourcomplexes can bind to the telomere DNA and the order of their binding affinity (A) is: A (NPH)> A (NBH)> A (NP3)> A (NB3). Among them, NPH has the biggest binding constant withtelomere G4-DNA, and can induce the hybrid quadruplex, while NBH can induce the antiparallelquadruplex. Moreover, polymerase chain reaction-stop assay, electrophoretic mobility shift assay,telomerase repeat amplification protocol demonstrate that NPH can also stable G-quadruplex andshown better inhibition of telomerase activity. It is worth noting that the properties betweennickel complexes and telomere G4-DNA are weaker than the ruthenium complexes, whichindicate that the metal center pays an important role in binding and stabilizing Human telomericQuadruplexes. |
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