| The decrease of cardiac muscle cells,hyperplasia of scar tissue andventricle reconstructions after myocardial infraction.It will be evolved intoheart failure,arrhythmia and even death.Nowadays,in clinic,those treatmentscould not replace the nectrotic cardiac muscle cells because of the impossibleregeneration of cardiomyocytes.Heart transplantation could not be spreadbeacause of its rejection and expensive expenditure.So it is important to finda effective treatment.The focus of the study has been put on the treatment of transplantationof mesenchyma stem cells.The mesenchyma stem cells is divided into twocategories:embryonic stem cells and adult stem cells.The focus of the studyhas been put on adult stem cells beacause of the rich sources and the easycultivation.Bone marrow mesenchyma stem cells(MSCs)is a kind of the AdultStem Cells.It develop from the mesenchymal layer of mesoderm.In certaincondition and actions of stimulation factors,it can be differentiated intomulti-histiocytes.As a important seed cell,people pay more and more attentionto MSCs in tissue engineering.To use the cardiac cells induced anddifferentiated by MSCs to cure myocardial infraction give some new theoreticbasis for the treatment of cardiovascular diseases by the combination of bothTCM and stem cells.MSCs belongs to nonhematopoietic cell and its basic character is adherentgrowth and looks like spindle fibrocyte in shape.It is considered that MSCsis a kind of stem cell which has potency of pluripotential progenitor and canbe differentiated into bones,cartilage,muscle,fat,tendon and nerve,etc.It has high plasticity.Scholars both here and abroad used 5-aza into induceMSCs after passage in culture in vitro.And the cardiac cell like ultrastructure,specific gene expression of cardiac cell and persistent actionpotential showed that they induced cardiac cells successfully.Some scholarsmedication experience of ten years,which has the function of promoting blood circulation directly injected MSCs into self cardiac muscle cells tissue and cardiac musclelike cytomorphosis was found.It had specific symbols,for example,thechromatin reconstitution of cardiac troponin I and myosin heavy chain,ofcardiac muscle cells.It showed that MSCs could be induced and differentiatedinto cardiac muscle cells in microenvironment in vitro.Icariin (ICA)is one of the constituents of epimedium,a traditionalChinese herbal medicine.It possesses many kinds of biological actions,particularly in cardiovascular function improvement,hormone regulation,immunological function modulation,anti-tumor and anti-virus activity.It hasbeen used for the treatment of heart disease.It could be fund that ES cells were remarkably induced into therhythmically beating EBs with ICA in a concentration-and time-dependent manner.So it will be considered that if the concentration-and time-dependent mannercan be fund with MSCs induced by ICA.This study was designed by the detection of gene and protein of cardiacmuscle such asα-MHC,β-MHC,Desmin,cTnT,VEGF and bFGF etc.This study can be divided into four parts.We hope that we can explore the relationshipbetween ICA and MSCs.ObjectiveExplore the best concentration-and time-dependent manner of MSCs beeninduced by ICA,and explore the improvement of myocardial infraction by thetransplantation in the treatment of MSCs induced by ICA.Method This study can be divided into four parts.Firstly:ICA was divided into 5 concentration gradient(10-6M,10-7M,10-8M,10-9M,10-10M).It can be studied the effects of MSCs induced by ICA withmethod of MTT.At the same time,MSCs can be identified withimmunohistochemistry method and Morphology.Secondly:It can be explore the concentration-and time-dependent mannerof the specific gene ofα-MHC,β-MHC and GATA-4 in myocardial cells whichdetected by reverse transcription-poly-merase chain reaction(RT-PCR)analysis.Thirdly:On the basis of the second part,It can be explore the expressionof VEGF and bFGF with Western Blot method compared with 5-aza further.Fourthly:On the basis of above experiments,the MSCs induced by ICA for2 weeks and 4 weeks was injected into MI rats through intravenous of tail. The specific Protein Desmin,cTnT,VEGF and BFGF can be detectived withimmunohistochemistry method.And we can observed the improvement of myocardialinfraction by the transplantation in the treatment of MSCs induced by ICA withstaining of HE and Masson.ResultsFirstly:MSCs were isolated from adult rat's gradient centrifugationand anchoring culture,the MSCs were purified by culture-expanded.Comparedwith control group,10-7M and 10-8M of ICA could promoted MSCs Proliferationobviously,P<0.01.Secondly:It showed that RT-PCR analysis of the expression ofcardiomyocyte-specific genes in differentiated rat MSCs cells.The expressionof the 10-7M of ICA group forα-MHC andβ-MHC were more strongly than othergroups,and that 10-7M of ICA could promoted MSCs proliferation obvirsly for24h and a 7-days culture in primary culture.Thirdly:It showed that Western Blot analysis of the expression of VEGFand bFGF in differentiated rat MSCs cells.the 5-aza group were used as apositive control.The expression of the 10-7M of ICA group for specificprotein---VEGF and bFGF were more strongly than other groups,that 10-7M of ICA could promoted MSCs proliferation obviously for 24h and a 7-days culturein primary culture.Fourthly:It was obviously that the expression of Desmin,cTnT,VEGF andbFGF in Myocardial infraction of 4-weeks of ICA group was strongly comparedwith 2-weeks of ICA group,model group and simpel MSCs group.ConclusionIt can be inferred that 10-7M of ICA could promoted MSCs differentiatedinto cardiomyocyto-like cells for 24h and a 7-days culture in primary culture.It can be concluded that the expression of the 10-7M of ICA of groupcardiomyocyte-specific genes and protein forα-MHC,β-MHC,Desmin,cTnT,VEGF and bFGF were more strongly than other groups than other groups.Thereare a lot of experimental research for bone marrow-derived mesenchymal stemcells (MSCs)transplantation in the treatment of myocardial infraction,moreresearchers think that MSCs through certain channels to myocardial infractionin rats in vivo transplantation can be effective in repairing damagedmyocardium.But,it was found that MSCs which had not been treated could notexpress Desmin,cTnT,VEGF and bFGF obviously.The most ideally treatment is that 10-7M of ICA could promoted MSCs differentiated into cardiomyocyto-likecells for 24h and a 7-days culture in primary culture.The method can inducedMSCs differentiated into cardiomyocyto-like cells. |
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