Embryonic-like Stem Cell Derived From Bone Marrow Induced Into Pacemaker-like Cell

Sick sinus syndrome,atrioventric?lar block is a common sinoatrial node function disorder,the treatment is electronic pacemaker implanted.But electronic pacemaker still has many shortcomings which can't be overcomed,such as pouch hematoma,infection,and so on.Biological pacemaker becomes a hotspot in research of arrhythmia.As gene transfection to build biological pacemaker has many shortcomings,cell transplantation and tissue engineering pacemaker,conducting tissue transplants,may be the only or the important way to apply biological pacemaker into clinical.Whether cell transplantation or tissue-engineered pacemaker,conducting tissue transplants to build biological pacemaker,the first problem need to be solved is the seed cells.In recent years,studys found that there is a small,stemness embryonic-like stem cells.It is the source of autologous,no immune rejection,has the characteristics of embryonic stem cells and easy to differentiation;It should be a differentiation one of the ideal seed cells for heart pacemaker cells.But till now,there has no study on its differentiation on heart pacemaker cells.The isolation and culture of ELSC,is the material base to heart pacemaker cells differentiation.So far,the methods of isolation and culture of ELSCs is complicated and needs expensive equipment,high technical requirements,multi-purpose serum.To get a good activity,high purity ELSCs,it is necessary to carry out ELSCs isolation and culture methods of research.Differentiation of stem cells is a major means of get transplanted cells and constructing tissue-engineered tissues and organs seed cells.As the differentiation of gene editing more not clear problem,the genetic differentiation method of interference,worth further discussing.Based on the above point of view,this study proposes ELSC as the seed cells.Use bone marrow cell culture,gelatin-package and serum-free culture method to culture ELSC.Chemistry and micro environmental sim?lation method to induced ELSC into the pacemaker cells differentiation,in order to investigate feasibility for heart pacemaker cells in vitro,for the clinical application of biological pacemaker experimental basis.Part 1.The isolation culture and identification of Embryonic-like stem cellsObjective:To explore the purification and identification methods of ELSC,study its biological characteristics,access can be used to induce the seeds of pacemaker cells,and compared with MSC stemness,determine its degree of naive,and its source is discussed in this paper.Material and Method:By whole bone marrow culture,gelatin-package,and the method of serum-free culture,cultivate 1 month old SD rat bone marrow cells,after 5 days for the first time to extend,once every 1.5 days to extend.Observe the morphological structure,flow cytometry and immunofluorescence,Westorn-Blot detecting the expression of stemness markers,and compared with MSC.Result:The ELSC present long spindle,small size and uniform form.While MSC were isolated from the volume is bigger,have long fusiform,triang?lar,circular shape,extend the cells become flat,aging characteristics.Immunofluorescence,Western Blot detection,ELSCs compared with MSCs,have high gene expression of SSEA-4,Sox,Oct-4,Nanog,and high content of telomerase.Conclusion:By whole bone marrow cell culture,gelatin package and serum-free culture,obtained are more naive,embryo-stem cell morphology ELSC,between ELSC may be present in the bone marrow mesenchymal stem cells in a more naive,original,subgroup with embryonic stem cell properties.Part 2.Differentiation of Embryonic-like stem cells to pacemaker cellsObjective:Induced ELSC to heart pacemaker cells differentiation,discussed the non-genetic method of interference to the possibility of a heart pacemaker cells differentiation.Material and Method:With 5-azacytidine impurity cytidine,medium of sinoatrial node,sinoatrial node cracking liquid,coculture with sinus node and combined method,induction ELSC into pacemaker cells differentiation,observation after differentiation cell morphology,immunofluorescence,rt-pcr,western blot detection such as cTnT,HCN4 and NKX2.5 related gene and protein expression.Result: Sinus cell culture supernatant on the induction to the pacemaker ELSC cell differentiation,three weeks after immunofluorescence showed that both expressed cTnT and Cx45,5-aza and combination that group HCN4 supernatant fluid for weakly positive expression,Western blot also shows HCN4 and Cx45 expression induced joint >cocluture>sinoatrial node cracking liquid >cracking fluid> 5-aza;PCR showed that joint induced pacemaker genes expression is higher than the other group,the expression of flow cytometry to detect HCN4 from 4% of 5-aza and coculture in the 15% to 25%.But each differentiation cells,after all did not appear spontaneously beating cells.Conclusion:A single method is difficult to the ELSC differentiation of pacemaker cells,combined the method of differentiation,although does not appear spontaneously beating cells,but has the characteristics of the early heart cells,high expression of pacemaker cells HCN4 and CX45,is the most likely ELSC differentiation method for pacemaker cells.