| Research backgroundWith the deep study of stem cells,Adult stem cell for the potency which differentiate into cardiomyocyte lineage has a perspective for the therapy of myocardial damage.Bone-derived mesenchymal stem cells(BMSCs)has considered as a promising candidate for their some characteristics such as obtain easily,the less immunogenicity,easiness to separation and amplification in vitro,because it accepted exogenous gene importing and expression,gene would be modified,differentiation into the myocardial cell, endotheliocyte and vascular smooth muscle cell.Therefore,BMSCs are thought to be a suitable source of cell transplantation for the therapy of infarcted myocardium.There are many induced methods that made BMSCs differentiate into myocardial cells.5-azacytidine is frequently used the chemical inductor.Besides,the myocadial environment is the most important factor that affect the survival and differentiation of BMSCs in the local heart,including the contact between BMSCs and jumping myocardium,kinds of cytokine secreted by myocardial cell.It' s confirmed than BMSCs Transplantation improved the heart function.But,researchers hope that using the contractile cell substitute the damage myocardial cell,and then generate the synchronous contraction with myocardium,synchronous excitation would be the key foundation.This must be verified firstly whether BMSCs has this foundation for produce bioelectricity,but there were less exploration.The normal heart electrophysiological activity and rhythmic contraction depend on the integrity of message transmit between myocardial cell.The gap junction is the important message channel.Connexin 43(Cx43) is the main connexin in the gap junction,act as the structural and functional coupling of cardiomyocytes.In recent years,researchers had discovered that transplantation cells would have lower survival rate in the local host after transplantating single BMSCs,that method might affect the long term therapeutic efficacy.The therapeutic efficacy would be improved if BMSCs transplantation combine the cytokines including SCF,G-CSF and AKT and so on,or modified BMSCs before transplantation.After BMSCs transplanting into the infarcted area of myocardium,the microenvironment might effect on the BMSCs for its survival and differentiation,as for electrophysiological property,the cell factor of myocardium secreted would be a important factor,salvia miltiorrhizae is the most commonly used Chinese materia medical to treat cardiovascular disease clinically.TanshenoneⅡA is liposoluble constituent of salvia miltiorrhizae. TanshenoneⅡA have some pharmacological action of cardiovascular system, such as relaxing coronary artery,anti-myocardium ischemia acutely, anti-arrhythmia,anti-myocardial hypertrophy and anti-platelet aggregation. So,we would aim to study the effects of myocardial microenvironment in vitro on the ion channels and Cx43 of BMSCs membrane,meanwhile,to observe the effects of TanshenoneⅡA on Cx43.Research objective1,To study the separation,purification and amplification of the BMSCs of Sprague Dawley(SD) rat in vitro,to establish the mature and stable culture system of BMSCs,to provide sufficient cell support for posterior experimental research.2,To study the expression of some gene mRNA on the undifferentiated of BMSCs membrane induced by 5-aza,the normal and hypoxia myocyte supernatant during myocardium cultured,respectively,including SCN2a,sodium channel;Kv1.4, transient outward current channel;Kv2.1 and EAG-1,voltage-gated potassium channel;Kir1.1,inward rectifier potassium channel;CCHL2α,L-type calcium channelα-2 subunit.To infer the effects of myocardial microenvironment on the electrophysiological property of BMSCs,to provide the experimental evidence for the change of ionic channel on the transplanting cell affected by myocardial local infarcted environment.3,To study the Cx43 expression of BMSCs induced by 5-aza,the normal and hypoxia myocyte supernatant respectively,to observe the effects of TanshenoneⅡA on the Cx43 expression of BMSCs and the synergistic effect between TanshenoneⅡA and BMSCs for the treatment of infarcted myocardium.Research methods 1,The methods of the separation,purification,amplification and identification of the BMSCs of SD rat in vitro.To culture the rat BMSCs using the adherence screening method,to detect the antigenic expression of CD34,CD29,CD44 and CD45 using respectively the immunofluorescence staining method and flow cytometry.2,The effects of myocardial supernatant on the ionic mRNA expression of BMSCs.The cadiocyte of neonate rat were isolated by enzyme and cultured,and then make the hypoxia myocardial cell model.BMSCs were induced by the normal and hypoxia myocyte supernatant during myocardium cultured.,10μmol/L 5-aza, After 3 weeks,to detect the expression of ionic channel gene mRNA that concerned with the action potential of myocardium on BMSCs,which include SCN2a,Kv1.4,Kv2.1,Kir1.1,EAG1 and CCHL2αusing RT-PCR.3,The effects of myocardial supernatant and TanshenoneⅡA on Cx43 expression of BMSCs.RT-PCR technique was used to detect Cx43 mRNA expression of BMSCs that induced by 5-aza,the normal and hypoxia cadiocyte supernatant during myocardium cultured,to study the protein expression of Cx43 on BMSCs using westernblotting and immunofluorescence staining methods after applying the different concentration TanshinoneⅡA,and to proof the promoting effects of TanshenoneⅡA on the Cx43 establishment inside BMSCs.Results1,BMSCs were isolated by the whole bone marrow adherent cultivation.We operate the first medium change after 48 hours,to removal unadherent blood cells and a part of hemopoietic stem cell.The cells begin to enter the exponential phase of growth phase after 3days,cell begin rapid proliferation, as well as the majority of cells displayed spindle-like shape.The cell would spread out fully at the ninth or tenth day for original generation,above 90% cells get fusion,they have radiation or swirl shape.After the cell carried out passage,they adhere the culture dish quickly,no eclipse period,entering the growth period,at the fifth day cell might enter the next passage.The BMSCs array of shows a regular pattern,most are whirlpool shape.With continuous passage,other non-BMSCs were decreased gradually.The cell have been depurated at passage 3.The reproductive activity of BMSCs began to descend at passage 6,cell' s shape grow into thin and flat,poor refractive activity. Part of BMSCs have a trend of differentiating into adipose cell.cells of passage 3 were used identification.The results of CD44 and CD45 using the immunofluorescence staining are positive expression of CD44,negative expression of CD45(this is a cell-surface marker of hematopoietic cell) on BMSCs.The cell-surface marker of CD29(expressed BMSCs) and CD34 antigen (positive expression on the hemopoietic stem cell and endothelia cell)using flow cytometry,the results are 99.27%positive cell of CD29 and 1.28%negative cell of CD34.2,Passage 3 were used to experiment through RT-PCR amplification.The results show that there were basic expression of SCN2a(sodium channel gene),Kv2.1,Kir1.1,EAG1 and Kv1.4 mRNA(potassium channel gene),CCHL2αmRNA of L-type calcium channelα-2 subunit on the undifferentiating BMSCs.There were lower expression level on BMSCs than myocardial cell as for the same gene(p<0.01).After 10μmol/L 5-aza induced,The morphous of BMSCs changed gradually,BMSCs were larger in size,from fusiform shape change into rod-shape or irregular form.After 2 weeks,the BMSCs began to fusion into each other,3 weeks later,myotulule-like structure formed,a part of BMSCs form some global-like shape.The results of ionic channel mRNA expression detected by RT-PCR are that SCN2a,Kv2.1,Kir1.1,EAG-1 and CCHL2αmRNA increase significantly at the expressing level(p<0.01),no obvious variance on Kv1.4 mRNA expression.The shape of BMSCs have no more change induced by normal myocyte and hypoxia myocyte culture fluid.In contrast of control group,the expression of SCN2a,Kv2.1 and Kv1.4 mRNA respectively increase induced by normal myocardial supernatant after 3 weeks,that had statistically significant(p<0.05),and the expression of Kir1.1,EAG-1 and CCHL2αmRNA also increase obviously(p<0.01).The expression of ionic channel mRNA have the same variation as the normal myocardial supernatant after induced by hypoxia myocardial supernatant,at expressing level just lower,but no statistically significant between two groups.There are no spontaneous contraction phenomenon on the BMSCs during the whole experimental process.3,The result of RT-PCR show that the expression of Cx43 mRNA on undifferentiating of BMSCs is lower.However,the level of Cx43mRNA had been increased after induced by 5-aza,normal and hypoxia myocardial supernatant respectively(P<0.01).The effects of different TanshenoneⅡA on the Cx43mRNA of BMSCs are great,that are the Cx43 expression had more raise treated by 1.5×10-7mol/L TanshenoneⅡA(P<0.05),significant increase treated by 1.5×10-8mol/L TanshenoneⅡA(P<0.01).To compare with another groups,the increasing degree of Cx43 mRNA change very significantly after allying treated by 1.5×10-8mol/L TanshinoneⅡA and hypoxia myocardial supernatant.The expression level of Cx43 after combining hypoxia myocadial supernatant and low-dose Tanshinone got close to the myocardium group(p>0.05).Certainly, there are obvious expression of Cx43 on myocardium.Meanwhile,The results detected by Westernblotting are there are special straps of different intensity on each group,molecular weight is 43KD.further,the Cx43 protein straps of BMSCs had increased after affected 5-aza,normal and hypoxia myocardial supernatant respectively contrast to the undifferentiating BMSCs. The effects of TanshenoneⅡA on Cx43 of BMSCs using Western blotting had same changes as the findings of RT-PCR.That to say,TanshenoneⅡA might also up regulate the expression of Cx43 on protein level.Furthermore,we observed the Cx43 variation between each group through antibody immunofluorescence labeling.That' s to found vague cell contour under fluorescence microscope in each group,cell nucleus is blue(DAPI dyeing),there are spot or strip green fluorescence on the endochylema and cell membrane,no same fluorescence were found on the blank control group(no Cx43 antibody),the results confirmed the specificity fluorescence massage from Cx43.The distribution of fluorescence on the cytoplasm and membrane of undifferentiating BMSCs were thin and weak intensity.After induced respectively by 5-aza,normal and hypoxia myocardial supernatant and therapied by Tanshinone,the fluorescence of Cx43 grew dense, increased fluorescence intensity.The change of fluorescent intensity among various groups had the same as RT-PCR and westernblotting.Conclusion1,We have established a successful culture system for BMSCs of SD rat in vitro.The cell were rapid proliferation,good condition and stable biological character,more purification,the cell identified by cell appearance and cell-surface marker was BMSCs.2,5-aza would induce the gene mRNA expression increase on BMSCs membrane, which include SCN2a genefor sodium channel,Kv2.1,Kv1.4,Kir1.1 and EAG1 gene for potassium channel,CCHL2αof L-type calcium channelα-2 subunit.That indicate 5-aza would induce BMSCs differentiate into cardiomyocyte lineage on ionic channel respect.The normal and hypoxia myocardial supernatant respectively also promote the above-mentioned gene expression,and no depend on chemical induction.Our finding would infer that dissoluble cell factor secreted by local myocardium promote the ionic channel expression on BMSCs membrane,when BMSCs were transplanted into the infarcted myocardium.The occurrence of ionic channel on BMSCs membrane might contribute to generate resting potential and action potential.3,5-aza would induce the Cx43 expression on BMSCs,meanwhile normal myocardial supernatant promote the Cx43 expression,hypoxia myocardial supernatant did alsoo Solo TanshenoneⅡA could up regulate the Cx43 expression of BMSCs,If TanshenoneⅡA and hypoxia myocardial supernatant were associated,the stimulating action would more powerful.This experimental results indicate that might improve the therapeutic efficacy of cell therapy if BMSCs transplantation would combine with TanshenoneⅡA.That would provide the new pathway for Chinese materia medical applying the cell transplantation. Achievement and innovative pointsWe have detected the effects of chemical inductor,soluble factor secreted myocardium and Chinese materia medica-Tanshenone on BMSCs from ionic channel and connexin aspect in vitro.The results are as follows:①many studies focus on whether or not BMSCs differentiating into the myocardial cells and expressing the specific myocardial protein on BMSCs by 5-aza.Our results confirmed that 5-aza would induce BMSCs differentiate into myocardium on ionic channels relating to action potential of myocardium.This findings raise to the level of theoretical research on 5-aza inductor.②We have observed the effects on BMSCs using the normal and hypoxia myocardial supernatant,and to detect conveniently the effects of myocardial microenvironment on BMSCs in vitro,furthermore to understand the change of grafting calls in local host microenvironment.This experiment confirmed that the cytokine secreted myocardium would regulate the expression of ionic channel and connexion43 proteins,and this action didn't depend on the chemical inductor.So,the findings would provide the experimental evidence for the change of electrophysiological proteins of BMSCs in local myocardial factor,and provide the theoreticai reference for grafting cell creating the bioelectricity and inducing arrhythmia.③The study of Chinese materia medica monomers or compound recipe on BMSCs in the domestic researcher focus on the induced effects of materia medica as so as 5-aza,but the results indicate the lower induced rate for them.The innovative points of our findings is that proved the high expression level of BMSCs Cx43 after treated Tanshenone and hypoxia myocardial supernatant,and this action has be higher than other factors.The results would indicate that the therapy for method cell transplantation combined the drug activating blood circulation to dissipate blood stasis,not only increasing the grafting cell survive,but also improving the electrical signal communication between cells,that contribute to the synchronous excitation,furthermore,to increase the transplanting effect. Also,our findings might provide a new therapy pathway for improving the effect of BMSCs.
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