Ever since 2002,when tumor-targeting Salmonella VNP entered its Phase Ⅰ clinical trails,tumor-targeting bacteria has become a hot topic for tumor biotherapy.The study of tumor-targeting bacteria is mainly focused on the following fields:tumor-targeting bacteria as a vector for tumor gene therapy,the mechanism and application of bacteria’s tumor-targeting charactor,the effect on anti-tumor ability of certain bacteria genes as well as the mechanism mediating bacteria’s anti-tumor effect.This thesis is mainly about the following studies:the application of tumor-targeting attenuated Salmonella typhimurium VNP as a vector carrying tumor-specific promoter to conduct gene therapy,the usage of Triptolide to enhance VNP’s tumor-targeting ability,the decrease of bacteria’s anti-tumor ability after knocking out flagella,as well as the mechanism of bacteria’s anti-tumor effect.In the first chapter,we discover that even though Salmonella VNP has relatively high tumor-targeting ability(tumor:liver and spleen>1:1000-10000),there is still a small amount of VNP growing within other organs during a short period after infection.So it will cause extra toxin to other organs by unspecific gene expression while using VNP as a vector for tumor therapy.Here,we use a E.Coli hypoxia-induced promotor PnirB to target the anoxia micro-environment within tumor,confining the expressing of drug in the tumor.We construct plasmids expressing traditional anti-tumor drugs TRAIL and Endostatin using hypoxia-induced promotor PnirB,and then confirm PnirB’s hypoxia-specificity as well as the expression of therapeutic genes using luciferase reporter essay and GFP expressing analysis in vitro.We also prove PnirB’s tumor-specificity as well as it stability via luciferase reporter essay in vivo.furthermore,we build mice model carrying subcutaneous melanoma to conduct anti-tumor effect analysis using VNP to carry hypoxia-induced promotor PnirB and therapeutic genes TRAIL and Endostatin,then we find this tumor-targeting gene therapy system achieve significant effect on repressing tumor growth.At the same time,we conduct a study about the usage of therapeutic gene TRAIL to induce tumor cells’ apoptosis and to enhance the apoptosis caused by chemo-therapy;we also study about the mechanism how Endostatin repress the CD31+vascular endothelial cell as well as the production of VEGF(Vascuoar endothelial growth factor)within tumor.We conclude that it is effective to express therapeutic genes via PnirB,a hypoxia-induced promotor,using Salmonella VNP as a vector,finally to achieve the goal of tumor-targeting gene therapy.In the second chapter,we find that exogenous promotor might alter VNP’s metabolism as well as the expression of exogenous genes while using VNP to conduct gene therapy.So we conduct a two dimensional electrophoresis(2D)as well as a mass spectrum analysis(MS)to screen the endogenous hypoxia-induced promotor of salmonella VNP.We discover some endogenous proteins which are highly induced in anoxia environment after analysing the 2D and MS results of anoxia sample.Among those proteins,we choose alcohol dehydrogenase(AdhE)with the highest score,then we clone 500,1000 or 1200bp of AdhE’s promotor.After analysing their ability of inducing expression in anoxia environment,we finally determine PadhE,a 1200bp long promotor of AdhE,as a highly specific hypoxia-induced promotor.First,we confirm that PadhE is only activated in an anoxia environment using GFP expressing analysis in vitro.At the same time,we also use luciferase reporter essay to prove that PadhE is specificly activated within tumor and has no activity in liver or spleen.Secondly,we conduct a chick chorioallantoic membrane experiment to prove the repression of angiogenesis after using PadhE to expression Endostatin.We find that the lysis supernatant of VNP expressing Endostatin induced by PadhE in anoxia environment can significantly repress the angiogenesis on chorioallantoid membrane.To better explain the feasibility of VNP-PadhE system,we use the subcutaneous melanoma mice model and lewis tumor model to evaluate the anti-tumor ability of using VNP-PadhE to target tumor and to express Endostatin with tumor.We find that the gene therapy groups achieve significant anti-tumor effect.We also conduct immunohistochemistry and ELISA to confirm its anti-angiogenesis effect.What’s more,we discover that the sepression of angiogenesis can increase the titer of VNP within tumor,which induces a greater extent of tumor cell apoptosis.In the third chapter,we mainly study about the tumor-targeting ability of bacteria.The Phase Ⅰ clinical trail results of VNP showed that it failed to produce a significant anti-tumor effect in human patients,and only a small amount of bacteria could colonise within tumor.So enhancing the tumor colonization of VNP is necessery for its further application in clinical trails.Here we discover that Triptolide,a monomer chemotherapy drug,can inhance the tumor colonization of VNP at certain dosage,without causing any increase of bacteria titer in other organs,which means that Triptolide can significantly improve the tumor-targeting ability of VNP.Furthermore,we discover that Triptolide can inhibit granulocytes’ infiltration into tumor,and granulocytes’ functional depletion expreriment result in a greater titer and wider colonization of VNP within tumor.Then we confirm that Triptolide inhance the colonization of VNP via inhibiting the granulocytes infiltration into tumor.Besides,we prove that Triptolide can repress the angiogenesin tumor,which is illustrated by the repression of CD31+ vascular endothelial cell as well as the production of VEGF after Triptolide treatment.Furthermore,we discover that neutralizing VEGF within tumor can achieve a great expansion of bacteria within tumor,resulting more necrosis area.So we conclude that Triptolide can also increase the bacteria titer in tumor via producing wider necrosis area by repressing angiogenesin.Wide colonization of VNP in tumor can induce the infiltration of a great number of CD8+T cell,accompanied by an obvious increase of TNFa and IFN-y levle in serum of mice carrying tumor,which result in a anti-tumor efect.What’s more,we find that Triptolide can repress the activatin of NFκB signaling pathway by VNP in tumor cells,which will also cause cell apoptosis.So we conclude that the repression of angiogenesis and granulocytes’ infiltration can inhance the tumor-targeting ability of VNP,which offers theoretical foundations for the success of further clinical trails.Furthermore,based on previous study we explore the mechanism of bacteria’s anti-tumor effect by knocking out flagella related genes flhD(main regulator gene)and fliE(Hook-basal body,flagellar basal body structure gene).We find that the flagella related genes are crucial for VNP’S anti-tumor ability,since deletion of flhD or fliE will result in disappearance of VNP’s anti-tumor ability.Further more,we confirm that flagella defected strains ΔflhD and ΔfliE have significantly impaired infectivity to B16F10 in vitro,especially for ΔflhD.We also get a greatly reduced number of flagella defected bacteria in circulation after infection,finally resulting in a diminished bacteria titer in tumor.So that raises a question:whether does the deficiency of infectivity cause the significant impairment of anti-tumor ability?We address this problem by treating tumors with adjusted number of bacteria to achieve the same bacteria titer in tumor for different strains,and then we eliminate the difference of infectivity for different strains.In this situation,we still find a decreased anti-tumor effect for flagella defected strains,compared with wild type VNP,but we should notice that ΔfliE can still present a significant anti-tumor effect while ΔflhD have no effect at all.Even at an incredible high dosage of 1010 cfu,we can not get a satisfying bacteria titer in tumor.These phenomenons suggest that there are other factors deciding bacteria’s anti-tumor ability other than its infectivity mediated by flagella.We conduct further analysis using nude mice,and find that the anti-tumor ability of VNP is closely related with immune system,so we guess immune response also mediate the anti-tumor effect of bacteria.The immune response induced by VNP may break the immune-repression environment within tumor,and then stimulate the immune response against tumor,and flagella may play a role during this process.To prove our hypothysis,we use a method of bacteria subcutaneous immune combined with drug treatment in tumor to test the anti-tumor ability of wild type and flagella defecient VNP.After that,we challenge these mice again with B16F10 to confirm that this treatment can induce an immune response against B16F10 to protect these mice from getting more tumors.We find that flagella is involved in the immune response induced by bacteria and bacteria’s infectivity can largely determine their anti-tumor efficiency mediated by immune response,but at the same time,there are other factors which alse participant in breaking the immune repression environment with tumor.In the tumors treated with VNP,we find a great amount of white cells including T cells,granulocyte and macrophagocytes,but only a small number of these cells can be found in the tumors treated with flagella defected strains.Meanwhile,we find that the MAPK pathway is highly activated after VNP infection,which stimulate a significantly enhanced expression of the downflow inflammatory factor and chemokines.To figure out whether this MAPK stimulation is caused by infection or by other factors,we analysis the expression of proteins related to MAPK pathway in a condition that every tumor is infected with relatively equal number of bacteria(which means the same number of ΔfliE and VNP can be find in tumor).We find that flagella itself can induce the activation of MAPK pathway and the release of downflow chemokines,even though a high infectivity can improve the stimulation effect at a great extend.Since Flagellin is the only known specific ligand for Toll-Like-Receptor5(TLR5),we guess that TLR5 may be involved in VNP’s ability of stimulating MAPK pathway as well as suppressing the growth of tumor cells.We prove that tumor cells stably express TLR5 both in vitro and in vivo,and we also find that flagellin extracted from VNP can stimulate TLR5 pathway in B16F10 in vitro.To further confirm that TLR5 stimulation influences tumor growth,we overexpress TLR5 as well as TLR5 dominant negative construcs,and then conduct anti-tumor expreriments with or without bacteria treatment.We find that the growth of tumor overexppressing TRL5 is significantly suppressed after VNP treatment,while this anti-tumor effect of VNP is greatly reduced by overexppressing two dominant negative constructs LRR and TIR.From that,we conclude the anti-tumor effect of VNP is partially depended on TLR5 signaling pathway.Becides,we also discuss other factors that might facilitate the anti-tumor ability of VNP,for example,Foxp3+Treg cell and cytokines inhibiting DC maturating(VEGF and TGF-β1),and the existence or disappearance of fagella can influence these factors.We prove that the VNP invasion and the immune response against it are crucial for its anti-tumor efficiency by conducting this flagella knockout experiment.Meanwhile,the regulation of TLR signaling pathway in tumor,which is stimulated by bacteria,also participants in its anti-tumor process.It is also possible that the metabolism of bacteria itself can have considerable effect on its anti-tumor efficiency. |