The Mechanism Of Rat Bone Marrow-derived Mesenchymal Stem Cells Migrating Toward C6 Glioma

ObjectiveGliomas are the most common malignant tumors in central nervous system,and because of their highly invasive and infiltrative growth pattern,gliomas are easy to be recurrent even after extensive surgical treatment,and radio- and chemotherapy usually are ineffective.The mortality of patients suffering from gliomas is high.Thus,there is a great need to look for new therapy to improve therapeutic effect on gliomas.Recently, with the advances in the pathogenetic mechanism of gliomas,gene transfer targeted on specific cell receptors,critical genes and regulatory molecules has increasingly called reseachers' attention.And it is undoubted that there is great value for researchers to look for the delivery vehicles for targeted gene therapy which can migrate toward gliomas,and release active factors against gliomas.Bone marrow-derived mesenchymal stem cells originating from mesoderm have showed the plasticity for differentiation and the capacity of migration and it is easy to be isolated and cultured in vitro.The expression of exogenous genes is stable after they are transferred into BMSCs.And the immunological rejection and ethnic problems can be avoided by drawing bone marrow from the patient himself and isolating BMSCs from it.The action of some cytokines and chemotactic factors on BMSCs can cause them to migrate directionally,and BMSCs have the tropism for intracranial lesions such as ischemia, inflammation and tumors.Stem cell factor is the ligand of tyrosine kinase coding by c-kit proto-oncogene.SCF can not only act as an chemotactic factor for stem cells migrating toward damaged brain tissue,but also play an important role in angiopoiesis of gliomas by interacting with its receptor,c-kit.Intercellular adhesion molecule-1 is a member of immunoglobulin superfamily.As an adhesion molecule,ICAM-1 is one of cell surface antigens used for the identification of BMSCs,and expressed on the surface of BMSCs,mediating the response of intercellular contact and conjuction.As a member of MAPK family,p38MAPK signaling pathway acts as an important intracellular signaling pathway,and p38MAPK extensively participates a series of physiological and pathological reaction including cell migration.The research focusing on exploring the effect of p38MAPK on SCF-induced migrating capacity and ICAM-1 expression change of BMSCs can help us to understand the mechanism of BMSCs migrating toward gliomas.In this study,at first we established the in vitro culture system of rat BMSCs.And based on this we explored the tropism of rat BMSCs for C6 gliomas by in vitro model, evaluated the capacity of BMSCs passing through blood-brain barrier and migrating toward C6 cells by C6 rat intracerebral glioma model,analyzed the influence of SCF on the migrating capacity change of BMSCs toward C6 glioma and the expression of ICAM-1,and investigated the relation between p38MAPK signaling pathway and the migration of BMSCs and the expression of ICAM-1 induced by SCF.The results obtained by this study will provide experimental evidence and theoretical basis for targeted gene therapy against gliomas using BMSCs as delivery vehicles,and offer new ideas for improving the migrating efficiency of BMSCs toward gliomas.Materials and MethodsPartâ… :The isolation and culture of BMSCs,and the tropism of BMSCs migrating toward C6 glioma1.Materials(1)Animals:Wistar rats of 4-6weeks old and weighing 60-80 grams were used for the preparation of BMSCs.And Wistar rats of weighing 200-250grams were applied for the establishment of C6 rat intracerebral glioma model. (2)Experimental reagents:Ficoll-paque centrifugate;rabbit anti-rat CD34,CD44, CD54,CD105,GFAP and NSE multicolonal antibody;mouse anti-rat CD45 multicolonal antibody;BrdU and mouse anti-BrdU monoclonal antibody;SABC kit and DAB kit;β-mercaptoethanol and propidium iodide.(3)Experiment intruments:superclean bench;CO₂ incubator;invert microscope, flow cytometry,Transwell inserts,micro-perfusion pump,and microtome.2.Methods(1)Rat BMSCs were isolated and cultured by density gradient centrifugation and adherent culture method.(2)The surface markers of BMSCs were identified by immunohistochemistry method.(3)The growth curve and cell-cycle of BMSCs were measured.And the capacity of BMSCs differentiating into neuron-like cells was evaluated.(4)The chemokinetic activity of BMSCs in response to C6 glioma cells was evaluated by Transwell inserts.(5)The rat C6 glioma models were established by stereotactic procedure.After being marked with BrdU,BMSCs were injected into ipislateral internal carotid artery of glioma to investigate their tropism for C6 gliomas and assess the feasibility of BMSCs serving as delivery vehicles for gene therapy aganist gliomas.Partâ…¡:The preliminary study on the mechanism of BMSCs migrating toward C6 glioma1.Materials(1)Animals:Wistar rats of 4-6weeks old and weighing 60-80 grams.(2)Experimental reagents:Ficoll-paque centrifugate;rabbit anti-rat SCF and c-kit multicolonal antibody;mouse anti-rat ICAM-1 monoclonal antibody;recombinant rat stem cell factor and anti-rat SCF neutralizing antibody;p38MAPK inhibitor SB203580 and DMSO;Trizol Reagent;RNA PCR kit(AMV ver3.0). (3)Experiment intruments:superclean bench,CO₂ incubator,invert microscope, Transwell inserts,UV-VIS spectrophotometer,PCR thermal cycler,electrophoresis,gel imaging system,fluorescene micoscope.2.Methods(1)The expression of SCF on C6 glioma cells and c-kit on BMSCs were evaluated by RT-PCR and immunohistochemistry method.(2)Analyze the influence of SCF on the migratory and adhesion capacity of BMSCs by in vitro model.(3)Evaluate the impact of SCF on the expression of ICAM-1 by RT-PCR and immunofluorescence method.(4)Explore the regulatory role of SB203580,a p38MAPK inhibitor,on the SCFinduced migrating capacity change of BMSCs and expression alteration of ICAM-1 by RT-PCR and immunofluorescence method.ResultsPartâ… :The isolation and culture of BMSCs,and the tropism of BMSCs migrating toward C6 glioma1.BMSCs were successive subcultured and purified by density gradient centrifugation and adherent culture method.The surface phenotypic characterization of BMSCs demonstrated the expression of CD44⁺,CD45⁺,CD105⁺,CD34^- and CD45^-. Flow cytometry detection showed that there were more than 70%of cells at G0/G1 stages,and a few at S and G2/M stages,which was consistent with the characteristics of stem cells.And we confirmed the cells obtained possessing the capacity of differentiating into neuron-like cells.2.The results obtained from in vitro model showed that the supernatant of C6 glioma cells induced directional migration of BMSCs.3.After being marked by BrdU and injected into internal carotid artery,BMSCs showed the capactity of passing through BBB and extensive tropism for C6 gliomas, and they could survive in the brain of rats bearing glioma.BMSCs scattered in glioma and the junction area between glioma and normal brain,but were not found in normal brain tissue.Partâ…¡:The preliminary study on the mechanism of BMSCs migrating toward C6 glioma1.The results of RT-PCR and immunohistochemistry showed that C6 glioma cells expressed SCF and rat BMSCs expressed c-kit,the receptor of SCF.2.The data of in vitro migratory experiment demonstrated that SCF could induced the directional migration of BMSCs,and the number of cells passing through polycarbonate membrane decreased after adding neutralizing antibody of SCF into the supernatant of C6 glioma cells.SCF was one of cytokines which contribute to the migration of BMSCs toward C6 glioma cells.3.SCF could up-regulate the expression of ICAM-1 on BMSCs,and enhance the migratory and adhesion capacity of BMSCs.4.The migration of BMSCs and up-regulatory expression of ICAM-1 on BMSCs induced by SCF were inhibited after blocking p38MAPK.Conclusions1.The culture supernant of C6 glioma cells can induce BMSCs to migrate toward it.BMSCs have the capacity to penetrate blood tumor barrier and migrate toward C6 glioma,and they can serve as delivery vehicles for the gene therapy against glioma. Internal carotid artery is an effective way for the transplantation of BMSCs.2.Stem cell factor is one of cytokines which contribute to the migration of BMSCs toward C6 glioma cells,and SCF incubation can enhance the adhesion ability of BMSCs. SCF can up-regulate the expression of ICAM-1,and enhance the migration and adhesion capacity of BMSCs.And p38MAPK is one of important signaling molecules correlated with the signal transduction of migration and adhesion capacity change induced by SCF.