Research On The Effect Of Robo4 Gene Expression To EPCs

Along with the rapid economic development,high-speed transpotation has been widely popular,resulting in the trauma incidence increased year by year.Thus the possibility of sepsis,infected because of the incidence,improved greatly.Then the incidence and mortality by multiple organ dysfuction syndromes（MODS）increased.The basis of the incidence of sepsis is a systemic inflammatory response syndrome（SIRS）,and subsequently severe sepsis and septic shock.Therefore how to prevent sepsis becomes a challenge to clinical medicine.Stepping into the 21^stt century,with the development of stem/progenitor cell technology and theory,it’s a new opportunity for the research of sepsis and MODs mechanism.Studies have approved that progenitor of mutilpotent differentiation in the blood could mobile,proliferate and migrate into the target damaged organ by the effect of inflammatory cytokines,and then differentiate into the endothelial progenitor cells（EPCs）to repair.On the other hand,EPSs are not only the participants in the inflammatory reaction,but alo the target damaged cells.The damage of EPCs could result in the damage of microcirculation,which is considered as the origination of organ dysfuction.In 1997,Asahara firstly isoloated and approved the existence of EPCs able to differentiate into endothelial cells in the human peripheral blood,and with the capability of proliferation,migration and differention into the mature enthothelial cells.Since then,more studies showed that EPSs were settled in the bone marrow,able to participate into the peripheral circulation after trauma or irritation under the inflammatory cytokines,and involved to repair and maintain the stability of cascular endothelial.Recent studies have found that autologous transplant EPCs can decrease the incidence of MODS and mortality,but exogenous EPCs transplantation under pathological environment not effectively differentiate into endothelial cells.it is weakening the vital organs of repair.in the process of sepsis,the lack of oxygen is not conducive EPCs directed differentiation and promote endothelial repair.How to make the maximum extent in the EPCs morbid environment directed to differentiate into endothelial cells,the mechanism of regulation of EPC directed differentiation is currently a hot research.The study found,that Slit2 is an important protein located in the lung,liver and kidney vascular endothelial cells.Robo4’s expression of vascular endothelial directed differentiation into mature endothelial and maintaining stability is closely related.After the process of sepsis in mice,administration of exogenous Slit2,Robo4 can be activated by calcium-dependent endothelial adhesion proteins（VE-cadherin）,preventing its uptake by cells,thereby enhancing endothelial stability,enhanced adhesion between endothelial cells,significantly increased the survival rate of sepsis in mice.My research will mainly focus on how the Robo4 gene expression could affect the number and function of EPCs,and induce its directed differentiation.It’ll be the theoretical and experimental basis of the prevention of sepsis and new therapeutic targrtes treatment.This study mainly contains three parts.The first part is to establish the model of Robo4^-/+、Robo4^-/-mice with sepsis after trauma knockout,by the gene knockout technology,cecal perforation and intraperitioneal injuction of endotoxin（LPS）.The second part is to dynamicly observe the Robo4 expression level and the number change of EPCs of the process of the post-traumatic sepisis development.The last part is in vitro and functional charactorerization of EPSs with three kinds of gene expression of the mice bone marrow.Part Ⅰ Establishment of mice with multiple organ dysfuction modelsObjective:the establishment of sepsis mice models by gene knockout is the experimental basis for the study of how Robo4 could affect the proliferation and diffentation of EPCs.Method:Divide 100 mice,weighing 20-25g and with three genes types(Robo4^-/+,Robo4^-/-and WT)into 2 groups:One is Model group（Group M）,made of 90 mice and operated the intrapertioneal injection of endotoxin and ccal perforation.The other one is Control group（Group C）,made of 10 mice and disguised opretation.Then observe the symptoms,signs,survival ratio,organ functional indicator（ALT,AST,Cr）,inflammatory cytokines（TNF-α,IL-1β,GF）,and the organ pathological changes under the naked eye and the microscope of the mice in each group.Result:The symptoms,physical signs and functional indicators of Group M changed significally comparing with Group C,especially for the organ pathology change of lung,liver,heart,kidney and gastrointestinal tract.Conclusion:Sepsis mice models could establish successfully by cecal perforation and LPS technology,with a higher mortality and able to further development into MODS.This technology is also with reproducible stability.Part Ⅱ Number of EPCs and Robo4 expression level changes in the process of SepsisObjective:By monitoring the number change of the EPCs in the normal mice bone marrow,and inspecting the mRNA and protein of Robo4 changes in all stages of sepsis,discover the correlation of the change of Robo4 expression level and EPCs number.Method:with the models introducted in Part I,extract the mice bone marrow at T1（Under normal condition）,T2（2h）,T3（4h）,T4（12h）,T5（24h）T6（48h）,T7（72h）,T8（92h）after injection of endotoxin under normal condition,get the peripheral mononuclear cells（PMC）according to the density gradient centrifugation,cultivate on the culture plates with the density of 5×10⁵PMC/species in the hole covered with fibronectin（FN）,collect the non-adherent cells after 48 hours and re-cultivate on the culture plates with the density of 11×10⁵PMC/species in the hole covered with FN,and then count the number of adherent cells after 72 hours.By Lysing the 1×10⁵cells and RealTime-PCR,discover the Robo4 mRNA and protein changes at all stages of EPC.Result:In the process of sepsis development,Robo4 expression level gradually increases reaches peak at T4,and then decreased.The number of EPCs increased gradually,reaches peak at T5,and then decreased.Correlation analysis showed that Robo4 expression level and the number of EPCs are in positive correlation in the progression of sepsis.Conclusion:in the LPS-mediated sepsis mice models,Robo4 expression level and the number of EPCs in the bone morrow are in positive correction,therefor Robo4 is with high possibility of participating in the regulation of proliferation of EPCs.Part Ⅲ Indentification of culture and comparision of functional characteristics of bone marrow-derived EPCs in three different phenotypesObjecive:Isolate,culture,identify and compare the feature of bone marrow-derived EPCs in three different phenotypes.Method:Isolate mononuclear cells from the mice bone marrow,induce differentiation culture,indentify and analysize the cell morphology,flow cytometry,phagocytosis and dual function in vitro angiogenesis of cultured EPC of P6 generation.Result:the result of EPC indentification in three different phenotypes is as follows:adherent cells uptakes Dil-Ac-LDLand FITC-UEA-1 with a rate of 80%.The result of immunohistochemistry:Group Robo4^-/-:CD133（+）,CD34（+）,CD31（++）,KDR（++）;Group Robo4^+/-:CD133（+）,CD34（+）,CD31（+++）,KDR（+++）;Group WT:CD133（+）,CD34（+）,CD31（+++）,KDR（+++）.The result of Flow cytometry indentification:Group Robo4^-/-:the positive rate of CD133 is 17.25±6.43%,that of CD34 is 48.75±6.83%,that of CD31 is 72.53±12.38%,and that of KDR is 87.25±10.23%.Group Robo4^+/-:the positive rate of CD133 is 16.55±7.29%,that of CD34 is 46.65±7.23%,that of CD31 is75.93±11.28%,and that of KDR is 89.25±11.33%.Group WT:the positive rate of CD133 is 18.25±8.57%,that of CD34 is 47.15±5.72%,that of CD31 is 74.61±10.69%and that of KDR is 88.25±11.35%.The result of in vitro angiogenesis function:Group Robo4^-/+:28.49±4.38/HP;Group Robo4^-/-:25.82±3.92/HP;GroupWT:42.32±3.62/HP（P<0.05）.The result of adhesion function（adhesion rate）:Group Robo4^-/+:42.7±3.1%,Group Robo4^-/-:39.5±1.7%;Group WT:64.5±2.6%.The result of migration（migration rate）:Group Robo4^-/+:18.5±1.7%;Group Robo4^-/-:13.3±2.6%;Group WT:27.5±1.8%（P<0.05）.The result of Proliferation:Group Robo4^-/+:23.06±2.63×10⁴;Group Robo4^-/-:19.24±2.65×10⁴;Group WT:40.33±3.65×10⁴.Conclusion:there’s no significant difference of the cell morphology and indentification of EPC in three different phenotypes.The angiogenic,migration,adhesion and Proliferation capacity of mice of gene knockout(Robo4^+/-and Robo4^-/-)are worse than WT group.Summary:The establishment of sepsis mice models by CLP and LPS is similar to the clinical way.In the progression of sepsis development,Robo4 expression level and the number of EPCs increased gradually,but decreased along with the increasing of death.Comparing with and analysizing the bone morrow-derived EPCs in three different phenotypes,the result showed that the angiogenic,migration,adhesion and Proliferation capacity of mice of gene knockout(Robo4^+/-and Robo4^-/-)are worse than WT group.In conclusion,Robo4 phenotype could affect some function of EPCs.