Target Delivery Of SOD And Env In Vitro And In Vivo

Cancer has become one of most harmful disease.Thereare almost 1.4 million citizens die from cancer every year in China.In the worldwide,AIDS is developing into another disaster.In past twenty years,governments and scientists cost much to develop AIDS vaccine,but failed. Human being has overcome measles,smallpox,and flu,but been discouraged in overcoming AIDS. Cancer and AIDS will continue existing with us for a long time.On one hand,human keep studying on their pathologys;on the other hand,human try to design drugs aiming directly at these two diseases,for example,superoxide dismutase(SOD) which can eliminate cellular superoxide anions in cancer,and human immune deficiency virus envelop glycoprotein(HIV Env),which can elicit HIV neutralizing antibodies in vivo.Current studies focus on these macromolecules, mainly on SOD.At present most researches are about Cu/Zn-SOD,while few are about Fe-SOD.There are few reports about Fe-SOD structure,function,or clinically anti-oxidant effect.Fe-SOD industrialization and clinic application are still remote from us.However,half-life of Cu/Zn-SOD in vivo is only 7 min,while that of Fe-SOD is up to several hours,it implies the potential of Fe-SOD in clinic application.We hence focus our study on Fe-SOD.There are still two disadvantages for SOD in tumor-clinic application:nonspecific tumor cell targeting, inability to permeate cell membrane(as its large molecular weight).What is more,anti-tumor mechanism of SOD is still being argued,as its substrate(Reactive oxygen species,ROS) plays multiplex and complicated roles in tumor cells.Current studies are based on above 2 disadvantages and 1 argument.In chapter 2,we modified Fe-SOD on protein level,to target-deliver Fe-SOD in vitro and in vivo;in chapter 3,we fused fe-sod with single chain variable fragment(scfv) gene,to target SOD to tumor cells,in which two critical disadvantages of Fe-SOD were overcome simultaneously.SOD-induced pathways were further studied.In chapter 4,we explored what roles ROS play in tumor cells,and how they play. This part was based on an individual cell,so that an interesting micro-world was presented to reader.In chapter 5,we purified Fe-SOD in industrial scale and applied it on mice and human bodies,to confirm its anti-oxidant effect in clinic.Elicitation of potent and broadly neutralizing antibodies against HIV-1 infection(AIDS vaccine) is still one of the top priorities in HIV field,even in scientific field.Reasons for failure in eliciting neutralizing antibodies are complicated:firstly,there are lots of non-functional Env on HIV virus surface,which can elicit non- neutralizing antibodies of high titer;Secondly,HIV can mutate easily and develop into other HIV strains,there are few antibodies can broadly neutralize HIV strains.In last chapter,we achieved functional Env trimer with high homology,and then applied target-liposome to deliver it to immune organ in vivo,in order to elicit potential HIV-1 neutralizing antibodies.Contents,conclusions and novelties for current studies are as followed:1,Clone,expression,and target delivery of Fe-SOD in vitro and in vivo(protein modification) Intact Nostoc commune fe-sod(Accession number:AY830114) was cloned,and used to construct a high-express pET-SOD vector in E.coli(accounts approximately 78%of total proteins).We further developed a dual-fluorescent-labeled magnetic nano-liposome,by which expressed Fe-SOD was delivered in vitro and in vivo.In vitro,this nano-liposome can deliver Fe-SOD into cells by endocytosis;Fe-SOD was subsequently released into cytosol,thereby decreased cell oxidative stress.In vivo,nano-liposome with small size was easier to move into circulation,while co-encapsulated magnetic particles efficiently targeted Fe-SOD to object tissue.2,Molecular Cloning,target delivery and anti-tumor characterization of Fe-SOD targeted to lung adenocarcinoma tumor(gene modification)Evolution and 3rd structure predictions of above Fe-SOD imply that its C end is fit to be modified.Single chain variable fragment(ScFv) not only keeps high immune binding to antigen, but also has a low molecular weight.We extracted total mRNA from LC-1 cell line secreting anti-lung adenocarcinoma antibody,and then modified it to be anti-lung adenocarcinoma scfv. The scfv was fused to C end of fe-sod,followed by SOD-ScFv fusion protein expression.Both fluorescence labeling assay and anti-Fe-SOD antiserum(prepared by mice immunization using native Fe-SOD) assays indicated fusion SOD could specifically recognize SPC-A-1 lung tumor cell membrane and permeated it.After permeating,fusion SOD can eliminate intracellular superoxide anions,decrease cell oxidative stress(exhibiting decreased ROS level and increased GSH level),thereby activated Akt/P27^kipl pathway,and inhibited SPC-A-1 cell proliferation.Current study overcame two critical disadvantages of SOD in clinical application(can not target tumor cells,hard to entry tumor cells).It was for the first time that the ScFv was used to target macromolecule;also,Akt/P27^kipl pathway triggered by SOD was firstly reported here.3,Dual-role of ROS in SPC-A-1 tumor cells(single cell level)ROS play dual roles as deleterious and beneficial species.They play positive roles in gene activation,and cellular growth,while excessive ROS will result in oxidative damage to cellular components and alter cellular functions.As a secondary message,Ca²⁺ signal plays potential roles in transferring oxidative signal,and triggerring growth-related pathways. Here we explored the roles of ROS in a single tumor cell,in which mitochondria were placed at the center of oxidative model:1) Ca^{+ signaling is indispensable for cellular ROS burst under exogenous oxidative stress: release of C2+ from endoplasmic reticulum and influx of extracellular Ca2+,leading to overload of cytosolic Ca2+ and mitochondrial Ca2+.2} Endogenous burst of ROS:exogenous H₂O₂ can not permeate cell membrane directly;burst of intracellular oxidative signal is original from mitochondria mediated by Ca²⁺ signal.On other words,increase intracellular ROS is endogenous.It answers the extensive argument "as an important message molecule,is H₂O₂ membrane-permeatable".3) ROS regulate growth-related P-Akt and Bcl-2/cyt c pathways:Here ROS were considered to be the "third message" regulated by Ca²⁺ message(second message).Change of ROS levels can co-activate proliferation-related pathways(P-Akt pathway and Bcl-2/cyt c pathway).This finding reveals the mechanism how ROS play dual role in tumor cells.Current study was based on an individual tumor cell,in which ROS dual-roles were explored more precisely.According to this theory,two anti-tumor strategies were put forward.4,Industrial preparation and clinical studies of Fe-SOD(industrial level,clinical level) Here we industrially prepared Fe-SOD from native algae,and its anti-oxidant effect in clinic was confirmed.Using improved purification procedure,7 500 U/mg Fe-SOD can be achieved.In addition,there is a considerable breakthrough in saving purification time and purification cost.Fe-SOD feeding experiments from 107 patients indicated Fe-SOD possessed of anti-oxidant effect in clinic.Fe-SOD pre-industrialization and clinic feeding experiments directly lay the groundwork for further Fe-SOD social application.5,Target delivery of EnvWe further targeted HIV immunogen to immune organ,in order to elicit potent and broadly neutralizing antibodies against HIV-1 infection.Here we modified immunogen(HIV envelop glycoprotein,Env) using different size of liposome,to target it to immune organ in vivo. After subcutaneous administration,the Env-liposome complex elicited HIV-1 antibody with higher neutralizing activity;in addition,it can at least neutralize 4 HIV-1 strains. Elicitation of potent and broadly neutralizing antibodies against HIV-1 infection discourages scientists during past twenty years.Current studied implies the feasibility of applying target strategy in this filed.In all,current studies overcame two critical disadvantages of SOD in clinic application. Also,Fe-SOD pre-industrialization directly laid the groundwork for its further social application,it makes considerable practical sense.According to ROS dual-roles theory, anti-tumor mechanism of SOD is illuminated here,and two anti-tumor strategies were thereafter put forward.As to studies on Env target delivery,we elicited broadly neutralizing antibodies against HIV-1 with some improvements.It moves us forward in this field.