Study Of MSCs Differentiation Into Cardiomyocytes And Endothelial Cells

Baekgroud The incidence and the fatality of coronary artery disease are ascending with the development of the living standard of the people, acute myocardial infarction is one of the main causes leading to the death to the people who have the cornary artery disease.After myocardial infarcation,the necrosis and the apoptosis of the cardiomyocytes resulted in the decrease of the cells' number,the necrosis cardiomyocyte substitute by the scar tissue,and ventricular remodeling appeared step by step and at last,cardiac systolic function decreased and even congestive heart failure was happened.Now the thearpy of the cardial infarcion include drugs therapy,interventional therapy and surgical theatment.By decrease the consumption myocardium oxygen,the drugs can release the symptom of the patients to a certain degree.Interventional therapy and surgical treatment can improve the blood supply to the heart and retrieve the survival cells,deferred the ventricular remodeling and improved the prognosis,but they cannot regenerate the death cells,so there are some patients finally developed in the cardiac insufficiency and even resulted in cardiac death.Cell transplant therapy providing a new method to the cardial infraction and received wide public concern.By increase the cell's number,cell transplant therapy will be a effective therapy to cardial infraction.In addition,angiogenes therapy have been developed into a new method for the ischemic disease at the moment,but the key problem is the resouce of the seed cells.Mesenchymal stem cells(MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells such as adipocyte,muscle cells,cartilage cells,nerve cells, endothelial cells,osteoblasts and so on.Because of the multilineage potential of MSCs for differentiation and relatively easy isolation,there has been a continuing interest in therapeutic applications of MSCs from the bone.Makino first sussessfully induced MSCs differentiate into cardiomyocyte by 5-azacytidine(5-aza) in vitro.At the same time,MSCs have basic theory and experiment background to differentiate into endothelial cells.Oswald has discovered that VEGF can induced MSCs differentiate into endothlial cells in vitro.These studies provide some experiment bases for the cell transplant therapy for coronary cardial diease.The notch pathway is an evlutionary highly conserved signaling machinery with roles in invertebrates as well as in almost erery veterbrate organ and tissue.The notch pathway is a versatile regulator of cell fate specification,growh,differentiation,and patterning processes in metazoan organisms.It has a important role in the development of the cardiovascular,its dificient or overdose will result in death because of the abnormal in cardiovascular.What's more,the notch pathway has a important role in the vascular regeneration.Notch signaling mybe has a significant role in the MSCs differentiation into endothelial cells.In this experiment,in order to provide perfect seed cells for the cell transplant therapy for coronary cardial disease,we studied the feasibility of MSCs differentiation into cardiomyocyte and endothelial cells in vitro,explored the effect of notch signaling on the differentiation of MSCs into endothelial cells.Aim1.Isolate and cultivate rat bone MSCs in vitro,study the biological characteristics of MSCs.Induced MSCs to differentiate into cardiomyocytes in vitro.Research the feasility of MSCs differentiation into cardiomyocytes.2.Induced MSCs to differentiate into endothelial cells by VEGF and bFGF in vitro.Research the feasility of MSCs differentiation into endothelial cells.Detecte the change of the differentiated cells' proliferation ability,migration ability and capillary-like structure' s informing ability.3.The mRNA expressions of notch signaling receptors and ligands on rat bone MSCs were detected by RT-PCR.Research the change of mRNA expression of Notch receptor and its ligand on the cells treated by VEGF and bFGF.the gene of VEGF was imported into cells which were treated by VEGF and bFGF to promote the proliferation and migration ability of the cells,mRNA expression change of the Notch receptor and its ligand on these cells also detected.The proliferation ability,migration ability and capillary-like structure's informing ability of these cells were mesured.Explore the effect of notch signaling on the differentiation of MSCs into endothelial cells.Moths1.Isolate the rat bone mononuclearcells by density gradient centrifugation,through change the culture fluid,non-adherent cells were removed.The form of the cells were observed under the microscope,surface antigen of MSCs were detected by flow cytometry;MSCs were induced to differentiate into cardiomyocyte by 5-aza,the form of the differentiated cells were observed under the microscope;in order to identificate the differentiated cells' nature, cardiomocyte specific markers of connexin 43 and troponin I were detected by immunofluorescence.2.Isolate the rat bone mononuclearcells by density gradient centrifugation,through change the culture fluid,non-adherent cells were removed.MSCs were induced to differentiate into endothelial cells by VEGF and bFGF,the form of the differentiated cells were observed under the microscope;in order to identificate the differentiated cells' nature,endothlial cells specific markers of Flk1 and CD31 were detected by immunofluorescence;inoculate MSCs and the differentiated cells on the semisolid gel to study the cells' ability of informing the capillary-like structure.The proliferation and migration ability of the differentiated cells were mesured by ³H-thymidine incorporation and by scarification test.3.The receptors and the ligands of the Notch signaling were detected by RT-PCR;There are four groups in the following experiment:group A: rat bone MSCs;group B:MSCs which treated by VEGF and bFGF; group C:the cells in group B were imported in the gene of VEGF₁₆₅; group D:the cells in group C which were inhibited the notch signaling.Notch signaling receptor and its ligand on these cells were detected by RT-PCR;the proliferation and migration ability of these cells were mesured by ³H-thymidine incorporation and by scarification test.Inoculate these cells on the semisolid gel to study theirs ability of informing the capillary-like structure.Results1.The shape of rat bone MSCs were mainly appeared as long fusiform and fusiform,it expressed antigens of CD29 and CD44,do not expressed antigens of CD34 and CD45,after treated by 5-aza,the cells form didn't has significant change and they expressed the cardiomyocyte' specific markers of connexin 43 and torponin I.2.The experiment showed that after induced by VEGF and bFGF,the cells' form mainly appeared as round and ellipse,they have the morphological characteristic of the endothlial cells and they expressed the endothelial cells' specific markers of Flk1 and CD31,at the same time,they also gained the ability of mature endothelial cells to inform the capillary-like structure on the semisolid gel.Compared to rat bone MSCs,the proliferation ability of the differentiated cells was decreased(P＜0.01 ) and theirs migration ability was increased(P＜0.01 ).3.The results RT-PCR experiment showed that there are Notch 1 and Jaggedl's mRNA expressed on rat bone MSCs;the mRNA expression of the receptor Notch1 and its ligand Jagged 1 have no significant change on the differentiated cells;After treated by VEGF and bFGF, the cells' proliferation ability were decreased(P＜0.01),and its migration ability were increased(P＜0.01),at the same time,they gained the ability to inform the capillary-like structure on semisolid gel.When imported the gene of VEGF₁₆₅ into the cells which have treated by VEGF and bFGF,the mRNA expression of notch signaling ligand Jaggedl was strenghened(P＜0.01) and the cells' proliferation ability,migration ability and informing capillary-like structure' ability were all increased(P＜0.05,P＜0.05,P＜0.05);when notch signaling were inhibited,these abilities of these cells were further increased(P＜0.05,P＜0.05,P＜0.05 ). Conclusions:1.The differentiation of rat bone MSCs into cardiomyocytes after treated by 5-aza in vitro is feasible,the defferentiated cells gained part of the phenotypes of the cardiomyocytes.2.The differentiation of rat bone MSCs into endothelial cells after treated by VEGF and bFGF in vitro is feasible,the defferentiated cells have part of the phenotypes of the endothelial cells and at the same time,the differentiated cells also gained the function of the mature endothelial cells.3.There are Notch signaling receptor Notch1 and its ligand Jaggedl's mRNA expressed on rat bone MSCs.After imported the gene of VEGF into the differentiated cells,the mRNA expression level of the Jagged 1 was upgrade.When rat bone MSCs were induced to differentiate into endothelial cells,its proliferation ability was decreased and its migration ability was increased,and they also gained the ability to inform the capillary-like structure on semisolid gel.VEGF can promote the differentiated cells' proliferation ablility, migration ablility and informing capillary-like structure' ability but notch signaling has the reversed effect.