Chemotherapy Sensitization Of Ovarian Cancer Resistant Cells(SKOV3-DDP)by The Co-delivery System Of Curcumin And P53

Ovarian cancer is a gynecological malignant tumor with no obvious initial symptoms,and most patients can only be diagnosed when they enter the middle and late stage,so the mortality rate is high.In recent years,with the combination of paclitaxel and platinum,the efficacy of ovarian cancer has been improved,but the overall five-year survival rate is still low.The main limitation is the emergence of chemotherapy resistance.The process of chemotherapy resistance is complex,in which tumor suppressor genes play an important role.Studies have shown that nearly 80%of ovarian cancer patients have p53 gene mutation,and the expression of p53 is closely related to their chemotherapy resistance.Therefore,how to successfully deliver p53 gene to ovarian cancer drug-resistant cells is the key to effectively reverse their chemotherapy resistance.On the other hand,drug resistance is usually associated with drug efflux.P-gp has attracted much attention in recent years because of its drug efflux function.It relies on intracellular adenosine triphosphate(ATP)to expel therapeutic drugs out of the cell,increasing the drug resistance of tumor cells.Therefore,down-regulation of P-gp expression and inhibition of drug efflux can effectively reduce chemotherapy resistance.Curcumin(CUR)can significantly inhibit the efflux of P-gp on cytotoxic drugs.Based on the above analysis,the purpose of this study is to prepare a non-viral transgenic vector with high efficiency and low toxicity,and to prepare a co-delivery system for simultaneous delivery of curcumin and p53 by enhancing the transfection efficiency of p53 in vivo and using CUR to inhibit drug efflux,so as to effectively improve the chemosensitivity of ovarian cancer drug-resistant cells to cisplatin and improve the chemotherapeutic effect of ovarian cancer.The first chapter summarys the mechanism of drug resistance,the defects of polyethyleneimine(PEI)as a transgenic carrier and the construction of co-drug loading system based on PEI.The second chapter describes the synthesis and characterization of CUR-PEI-K14.Firstly,bifunctional peptide K14(C-t Lyp-1-NLS-C)and CPLGLAG peptide were synthesized by solid phase method,and the sequence was identified by electrospray ionization mass spectrometry(ESI-MS).The purity was determined by HPLC.Then,a new non-viral gene vector PEI-K14,was obtained by cross-linking low molecular weight PEI with K14.Curcumin intermediate CUR-CPLGLAG,was prepared by acid-amine reaction and curcumin was grafted onto the surface of PEI-K14 by CPLGLAG peptide to obtain CUR-PEI-K14 polymer.The structure of curcumin polymer was analyzed by ~1H-NMR.The results showed that bifunctional peptides K14 and CPLGLAG were successfully synthesized,and the purity of bifunctional peptides reached 97%.The structural characterization of ~1H-NMR showed that CUR-PEI-K14 has been successfully synthesized,indicating that the synthesis method is stable and controllable.The third chapter evaluates the physical and chemical properties of CUR-PEI-K14.The morphology of CUR-PEI-K14 and CUR-PEI-K14/DNA was observed by scanning electron microscope(SEM),and the particle size and surface potential were measured by DLS.The DNA condensation ability of CUR-PEI-K14,the dissociation ability of antiserum and antiheparin sodium were evaluated by 1%agarose gel electrophoresis,and its buffering ability was evaluated by acid-base titration.The results showed that the CUR-PEI-K14/DNA complex system was uniformly dispersed and spherical,with moderate particle size and potential,which was suitable for transport in vivo between 20-150 nm and 18-37 m V.When the w/w ratio was 0.8,the charge of DNA was completely neutralized by the polymer,indicating that CUR-PEI-K14 had a strong ability of DNA condensation.Through the evaluation of its humoral stability,it was found that CUR-PEI-K14/DNA could stably exist in the solution containing 25%serum and 1200?g/m L heparin sodium,indicating that the CUR-PEI-K14/DNA system had good antibody solution dissociation ability.At the same time,CUR-PEI-K14 had good p H buffering capacity,indicating that CUR-PEI-K14/DNA system had a good proton sponge effect.These physical and chemical properties showed that CUR-PEI-K14 has the potential to efficiently deliver p53 and CUR in vivo.In chapter 4,the effect of CUR-PEI-K14/p53 on chemosensitivity of SKOV3-DDP cells was discussed.Firstly,PEI 2 k Da and PEI 25 k Da were selected as controls,and p EGFP-N2 green fluorescent protein plasmid and p GL3-Control luciferase plasmid were used as reporter genes to qualitatively and quantitatively investigate the transfection ability of CUR-PEI-K14 to SKOV3-DDP cells.The results showed that the transfection efficiency of PEI 25 k Da was lower than that of PEI 2 k Da in SKOV3-DDP cells because of its excessive toxicity.In addition,compared with PEI 2 k Da,the transfection efficiency of CUR-PEI-K14/DNA showed obvious advantages,and the modification of K14 significantly improved the transfection efficiency of the complex.Among them,the transfection effect of CUR-PEI-K14-3 was the best,which was close to 1×10~8 RLU/mg protein,which provided the feasibility for efficient delivery of p53 gene.The results of cytotoxicity evaluation showed that compared with PEI 25 k Da,at the same concentration,the toxicity of CUR-PEI-K14 was much lower than that of PEI25KDa,at low concentration,which showed good biological safety and was suitable for gene delivery in vivo.However,it showed another result when combined with cisplatin.The cell inhibition rate of SKOV3-DDP cells by CUR-PEI-K14/p53 combined with cisplatin showed that CUR-PEI-K14/p53 significantly increased the sensitivity of SKOV3-DDP cells to cisplatin.Furthermore,cisplatin,curcumin,p53 and their combination were used in SKOV3-DDP cells to explore the synergistic effect between p53 and curcumin.The results showed that curcumin and p53 alone had a certain inhibitory effect on the growth of SKOV3-DDP cells,but the effect was limited,which was not as good as 10?g/m L cisplatin alone(the inhibition rate was 49.76%).In the combined groups,the inhibition rate of SKOV3-DDP cells treated with curcumin,p53 and cisplatin(CUR-PEI-K14/p53+DDP)was the highest,about79.76%,which significantly increased the inhibitory effect of cisplatin on drug-resistant cells,indicating that CUR and p53 could synergistically enhance the sensitivity of SKOV3-DDP cells to cisplatin.Finally,through the QPCR experiment,it was further verified that CUR-PEI-K14/p53 enhanced p53 gene transfection,inhibited the function of P-gp protein,upregulated and promoted the expression of apoptosis gene bax,and enhanced the sensitivity of ovarian cancer cells to chemotherapeutic drugs by regulating p53 gene,drug pumping pathway and inducing apoptosis pathway.Aiming at the molecular target of chemotherapy resistance of ovarian cancer,a new co-loading system CUR-PEI-K14/p53,containing CUR and p53 was constructed,which organically combines gene drugs with natural drugs,combined with a variety of sensitization methods to play a synergistic effect,so as to explore an effective way to solve the current problem of chemotherapy resistance of ovarian cancer and its practical application.