Study On Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Cardiomyocyte-like Cells Induced By Sodium Butyrate And FGF-2

Background: Ischemic heart disease(IHD)is not only a common disease in the elderly but also a trend of younger people.It has been widely concerned because it can be life-threatening.Myocardial ischemia can induce Cardiac myocytes(CMs)necrosis,CMs as terminal differentiation cells can hardly regenerate,so the CMs after necrosis,fibrous tissue gradually instead of necrotic tissue,the heart can cause irreversible damage,until the heart failure.At present,the hospital treats IHD therapy generally takes drugs to relieve,intervention,or heart transplant.Recent studies show that between bone marrow mesenchymal stem cells(BMSCs)transplantation to the heart of myocardial infarction,the cardiac function will improvement,and can improve the life quality of patients,which makes the regenerative medicine can be used for treatment of IHD.Mesenchymal stem cells can be obtained from bone marrow,amniotic fluid,fat and other tissues.BMSCs are widely used in regenerative medicine because of their relatively easy access,multi-directional differentiation potential,no immune rejection and no ethical issues.BMSCs can differentiate into neural cells,osteocytes,chondrocytes,cardiomyocytes,hepatocytes and other ectodermal and mesodermal cells.These characteristics make BMSCs have great clinical application potential in regenerative medicine.Sodium butyrate is a short-chain fatty acid in histone deacetylase inhibitor(HDACi).Its receptors are GPCR43 and GPCR41 in G protein-coupled receptors(GPCRs).Sodium butyrate can reduce the expression of HDAC1,acetylate histone and promote the differentiation of BMSCs into CMS.Basic fibroblast growth factor(FGF-2),as is common in human tissue growth factor has obvious effect on promoting proliferation and differentiation.Recent studies have shown that: first,sodium butyrate can inhibit HDAC,promote the activation of FGF promoter and induce differentiation;second,compared with FGF-2 alone,FGF-2 and 5-azacytidine can induce BMSCs to differentiate into cardiomyocytes more efficiently,although both 5-azacytidine and sodium butyrate belong to epigenetics,sodium butyrate can induce BMSCs to differentiate into cardiomyocytes more efficiently;third,GPCR and various members of receptor tyrosine kinase can form heterologous complexes together and trigger different intracellular signal transduction and cellular responses;fourth,sodium butyrate inhibition of proliferation,FGF-2 promote proliferation.Objective: In this study,we used sodium butyrate combined with FGF-2 to induce BMSCs to differentiate into CMs and compared with the induction of them alone.According to the expression of gene and protein,we found out whether they have synergistic effect,and explored how they promote BMSCs to differentiate into CMs,to provide the basis for cell transplantation in the treatment of heart disease.Method:BMSCs were isolated and purified by whole bone marrow adherent method from the bone marrow of 2-3 weeks old SD rats.the 3th generation cells with good growth were selected and identified by flow cytometry.Then the cryopreserved cells were resuscitated for induction and culture.The 3th generation cells with good growth were divided into four groups: negative control group,sodium butyrate group,FGF-2 group,and sodium butyrate combined with FGF-2 group(combine group).The activity and value of BMSCs were detected by MTT.RT-qPCR was used to detect cardiac transcription factors after BMSCs were induced for 1,2and 4 weeks.Immunocytochemistry and Western blotting(WB)were used to detect the expression of cardiac-specific proteins after BMSCs were induced for 4 weeks.Results: The morphological changes of BMSCs were observed under the inverted microscope: the primary cells were collected into the cell culture bottle,and they were round and suspended under the microscope.After 48 hours,they were round and flat and began to adhere to the wall.After 72 hours,most of the cells were spindle-shaped,and the BMSCs cultured for one week further extended,After 4 weeks of induction,BMSCs showed obvious directional arrangement between adjacent cells,and some cells even became vortex-like structures.Flow cytometry was used to identify BMSCs: the positive expression rates of CD29,CD45,and CD90 were 93.1%,1.7%,and 95%,respectively.The results showed that the isolated cells were purified BMSCs.MTT test: The viability of the BMSCs determined by MTT were increased after FGF-2 and combine group,however,sodium butyrate was falling(P<0.05).RT-qPCR results: GATA-4 had the highest expression at 2 weeks,and MEF-2c had the highest expression at 4 weeks.the combine group was significantly better than other induction groups(P<0.05).Western Blot(WB)test results: after BMSCs were induced for 4weeks,the positive expression of cardiac-specific protein connexin 43(Cx43),Desmin,and cardiac troponin I(cTnI)was detected,and?-actin was selected,as an internal reference protein,combine group was significantly higher than other induced groups,while the protein expression of non induced group was lower than that of the induced group(P<0.05).Immunocytochemistry results showed that the positive rates of cardiac troponin T(c Tn T),cTnI,and desmin,and the positive rates of combine group were higher than control group in BMSC after 4 weeks of induction(P<0.05).The experimental results show that:1.BMSCs can be purified by whole bone marrow adherence and density gradient centrifugation.2.Sodium butyrate and FGF-2 can be individually or together induced BMSCs to differentiate into cardiomyocyte-like cells,the combination of sodium butyrate and FGF-2 for induction has a better effect.