| Objectives:Ischemic heart disease is a kind of morbid state,which refersto the heart blood perfusion reduce, leading to reduce cardiac oxygen,myocardial energy metabolism is not normal, it can’t support the normal workof the heart.Heart failure often arises as a consequence of coronary arterydisease. This most common form of heart diseases is the leading cause ofdeath in humans worldwide because in many patients it results in theoccurrence of acute myocardial infarction （AMI）. AMI is characterized by thesudden occlusion of a coronary artery and leads to limited oxygen supply（ischemia）, irreversible muscle damage and cardiomyocyte death.Ischemia inthe heart subsequently initiates a remodeling process that involvesmodifications of the cellular metabolism in surviving myocytes and changesof the left ventricular （LV） structure leading to cardiac hypertrophy, fibrosisand enlarged chambers. These alterations are accompanied by an adverseeffect on cardiac function and eventually result in the progression of cardiacdecompensation and heart failure.Mesenchymal stem cells represent a kind of multipotent somatic stemcells, and all kinds of stem cells have been detected and isolated from bonemarrow,adipose tissue, umbilical cord blood, peripheral blood,etc. Just likeembryonic stem cells, bone marrow-derived mesenchymal stem cells（MSCs）have capacities of extensive self-renewal potential, and differentiate into avariety of cell types such as cardiomyocytes, neurons,osteoblasts, adipocytes,and smooth muscle cells under the appropriate microenvironment. AfterMSCs transplantation,the soluble factors secreted by MSCs also played animportant role in cardiac repair.MSCs transplantation was shown to preventacute myocardium against ischemic injury and promote vascular regenerationvia promoting the release of protective cytokines.Moreover, recent studies also uncovered that transplanted MSCs produced the protective effects on ischemic hearts through immunosuppressive actions.It was also suggested thatthe potential stimulating influences of MSCs on cardiac stem cell niche wasone novel mechanism of cardiac repair.In summary, accumulating evidencefrom animal experiments and clinical trials confirms that MSCs celltransplantation lead to a global improvement of the myocardial functions viathe anti-apoptosis,the actions of the immunosuppression,neovascularization,differentiation,etc.There is no very exact evidence,however,to prove whethermesenchymal stem cells can protect the cardiomocytes from prematuresenescence. Several investigations have demonstrated that transfusion ofautogenic or allogeneic MSCs in the acute phase signi ficantly improves themyocardial damage.Accordingly,MSCs attenuate myocardial injury throughdifferentiation into a variety of lineages including vascular smooth musclecells, endothelial cells and cardiomyocytes, depending on the milieu.There isalso evidence that the trophic mediators secreted by MSCs can improvecardiac function via a combination of multiple mechanisms such as reducinginflammation, activating host tissue stem cells,inducing angiogenesis andinhibiting fibrosis remodeling.Thus, MSCs are able to ameliorate heart injuryand compensate stress by both cardiac cell replacement and also supplyinglarge amounts of anti-apoptotic and mitogenic factors.Many studies have studied the method of rats myocardial cell primitiveculture. In vitro separation, culture of myocardial cells, not only can keep theoriginal structure and function.For experiments in which differentiated cardio-myocytes are not required, neonatal cells may provide a simpler experimentalsystem.In comparison with adult cardiomyocytes, Milk rat myocardial cell hasthe ability to split.It also have the advantage of being easily cultured and aresuitable candidates for non-viral gene transfer, as will be described in thefollowing sections.Isolation of neonatal cardiomyocytes is also technicallymore straight-forward than cell isolation from adult hearts,as it does notrequire the rather difficult procedure of aorta cannulation and perfusion.Instead,a simpler two-step procedure can be employed, consisting of enzyme digestion and mechanical agitation of the ventricular tissue followed bypurification of the cardiomyocyte population.And the proof of myocardialcells can’t transfer of culture,in many times after the transfer of culturequantity can be reduced or even disappearNKX2-5is NK type homologous box gene families NK2type members,NKX2-5on myocardial differentiation,the whole heart tube formation andcyclization,and plays an important role in atrioventricular separation,transmission system and the mature of the normal functioning of the heart tomaintain, therefore has become the heart during the process of developing themost important transcription factor.Nkx2-5is highly expressed in the earlyheart progenitor cells in both primary and secondary heart fields duringmurine embryogenesis and continues to be expressed at a high level in theheart through adult hood.Especially,the transient elevation of the Nkx2-5expression is observed in specialized myocardial conduction cells during theperiod of conduction system formation, suggesting a significant role of Nkx2-5in the development of the conduction system.Microenvironment consists of two aspects: cells surrounding influence toexcitement and inhibition activity of trace elements; The cells themselves inthe differentiation process of formation of the special structure and receptor.Cell reaction depends on the trace for quality and quantity, and its function inthe related parts of the state, the comprehensive effect of these two aspectstogether, their mutual influence and mutual coordination, make the cell growth,proliferation and directional differentiation to meet the needs of the body.Intramyocardial MSCs also in the interaction type of microenvironment inpromoting regulation, including from a host of myocardial cells of thephysical and chemical factors and their differentiation induction of structures.Mainly studies the role of the environment, the physical and chemical factorsin the environment of researchers divided into direct and indirect factors: theformer refers to the host cell mechanical, electrical stimulation, and the effectof the MSCs via "channel" direct transfer of ions and small molecules; Thelatter refers to the host cells secrete a variety of soluble factors. Tissue engineering aims to regenerate tissues and organs by using cell andbiomaterial-based approaches.One of the current challenges in the field is topromote proper vascularization in the implant to prevent cell death andpromote host integration.Bone marrow endothelial progenitor cells （BM-EPCs）and mes-enchymal stem cells （MSCs） are bone marrow resident stem cellswidely employed for proangiogenic applicationsExperimental research shows that has to bone marrow mesenchymal stemcells in NKX2-5gene transfection, simply through the genes of theinstantaneous expression induced to myocardial sample cell differentiation,through the induced MSCs cell differentiation into myocardial samplecell.Therefore,we try to use liposome instantaneous transfect NKX2-5gene toSD rat bone marrow mesenchymal stem cells,observed differentiation intocardiomyocytes under the action of myocardial trained.And compare with thesimple NKX2-5transfection of MSCs to myocardial cell differentiationsituation.We try to find the process of cardiac cell differentiation,provide aneffective strategy for the stem cells transplantation.Method：1The conversion,amplification and activation of eukaryotic expressionvector PEGFP-N1-NKX2-5Department store containing PEGFP-N1-NKX2-5plasmid of e. coliDH5alpha competent bacteria,and coated them to the LB medium platecontaining Ampicillin,cultured inverted at37℃for1216h.Picked a singlepositive bacteria to amplification.Extracted using plasmid extraction kit anddetecting concentrations of plasmid.2The SD rat bone marrow mesenchymal stem cells isolated,purified andamplifiedBMSCs were isolated from the tibia and femur of SD rats with adherentculture method.BMSCs were at L-DMEM/F12complete mediumcultured with10%fetal calf serum.The first exchanged of medium at the third day,mediumwas changed once after3days.Passaged when cells covered the bottom.Weused the second generation of MSCs for experiment. 3The neonatal rat cardiomyocytes’ isolation and culture1-3-days-old SD neonatal rat were disinfected the skin with75%alcohol,then operated the chest to take the ventricle out and cut into smallpieces aboout1mm3,dispersed the tissue by trypsin until it was digestedcompletely.Collected the digestive juice and then filtrated, centrifugaed andresuspended the cells,purified cardiomyocytes through differentially adherentand add to0.1mmol/L Brdu. After48h, observed with inverted microscope.We used the fifth day of the myocardial cells for experiment.4The eukaryotic expression vector PEGFP-N1-NKX2-5transfect MSCsBefore transfection,our preliminary experiment found the appropriate cellseeding density,the best transfection system.Used the cationic liposomereagent,Lipofectamine2000,the plasmid NKX2-5-PEGFP were transfectedinto MSCs.After6h,the expression of NKX2-5-PEGFP fusion protein wasdetected with epifluorescence microscope.5MSCs cells which had transfected by NKX2-5gene and cardiomyocytes inco-cultureMSCs cells which had transfected by NKX2-5gene were co-culturedwith cardiomyocytes as the first generation（F1）. This experiment is dividedinto common training group and not trained group, common training group forinstantaneous transfection NKX2-5gene MSCs and myocardial cell trained;Not common training group for simple transfection NKX2-5MSCs cells. Tocollect the cells for15d. And transfering to culture as the second generation（F2）. To collect the cells for15d. And transfering to culture as the thirdgeneration（F3）.In training F1,F2,F3cells with immunocytochemistry methodto detect myocardial troponin T （cTnT） in the expression of MSCs and HEstaining method to observe the shape of the cells and the expression of protein.6Statistical analysis:Use SPSS13.0statistical software.All data showed bymean±standard deviation（x±s）.Paired t test is used for statistical analysis. Takeα=0.05as testing standards,P<0.05was considered had a significantdifference. Results：1Primitive MSCs cells24h after inoculation, the cells is the main way toclone proliferation. Culture bottle bottom that appear a little adherent cells, aspindle or spindle, there is a small amount of cells has started to proliferationand fibroblasts sample, gather the growth, rare single decentralized state.Cultivation to the third day, the vast majority of cells are attached, cultivate to7days, adjacent cells reach90%of the fusion and divided into three forms:long spindle, polygon, small round. Transfer of culture and h after completelyattached, about5days appear confluent, the second generation later, cellsform a uniform started the long spindle cells arranged in obvious swirl, orradial.2Three days after the training of myocardial cells spindle, polygon andirregular form, out of apophysis interconnection interwoven into network, andthe emergence of pulsation, frequency for70-150times/min. Myocardial cell2-3h or so adherent cells out after pseudopods into diamond or polygon,occurrence regularity pulsation frequency, ranging from fifty to140times/min,2days later cells gradually in the bottle wall surface development,34daysout of the cell plasma, yea, and mutual contact mixed in mesh3After transfection6h fluorescence microscope to observe transfectiongroup PEGFP-NKX2-5fusion protein in MSCs have green fluorescentexpression. We can see the visible cells with green fluorescence, the nucleusand cytoplasm were existed; Instead of MSCs of nucleus and cytoplasm in thetransfection group were no expression of green fluorescent.4HE staining to observe some cells were gathered growth, which were tobe the cell mass, between cell mass there were some scattered cells whichwere long fusiform,and cell morphology is relatively uniform.5Cell culture F1，F2，F3, trained group and not trained group withimmunocytochemistry method were respectively detected myocardial troponinT （cTnT） in the expression of MSCs, and F2express quantity than F1expressvolume is high,F3express quantity than F2and F1express volume is high,each time point compared with significant difference （P <0.05）. Trained group and not trained group, compared to the F1，F2，F3three time points,total training group of cTnT two gene expression quantity are not trainedgroup than high. Statistical analysis results show that trained group of F1，F2，F3cTnT expression quantity are higher than not trained group, compared tothe three significant difference （P <0.01or P <0.05）Conclusions:1Neonatal rat cardiomyocytes were purified by using differentialadhesion and adding Brdu after1h, which can inhibit fibroblast proliferation,increased the purification of cardiomyocytes.2With the myocardial cell culture of total foreign transfection expressNKX2-5gene MSCs to myocardial differentiation situation is better thannot trained group of MSCs cells that transcription factor NKX2-5can promoteMSCs to myocardial differentiation, in protein level express myocardialmarkers and early transcription factor, and show that mechanical force ofguyed effect can effectively promote MSCs cell to myocardial celldifferentiation.3Myocardial cell trained method, can be in vitro microenvironment ofmyocardial cells induced MSCs to myocardial cell directional differentiation,express myocardial cell specific cTnT and express quantity with the extensionof incubation time and increased gradually. |
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