Study On The Role Of SDF-1/CXCR4 Axis In The BMSC Mediated Cardiac Repair In Rat Model Of Acute Myocardial Infarction

Experiment 1Experimental study on isolation and culture of bone mesenchymalstem cells and its CXCR4 expressionObjective To investigate the method of the isolation and culture of bone marrow-derivedmesenchymal stem cells(BMSC);To observe the biological characterization of BMSC andthe expression of Stromal cell-derived factor-1 (SDF-1) receptor-CXCR4 in the2th,3th,4th,Sth generation of BMSC.Methods Bone marrow mesenchymal stem cells were isolated from adult SD mice by thewhole bone marrow culture and were purified through several passages.The morphology ofBMSC was observed under inverted micro scope.Immunohistochemistry was used todetermine the expression of CD34,CD44,and CXCR4 in the 2nd,3rd,4th,5th generationof BMSC respectively.RT-PCR was used to measure the expression of CXCR4 in the 2nd,3rd,4th,5th generation of BMSC respectively.Results BMSC obtained by the whole bone marrow culture had good performances ofpurity,uniform and ability of proliferation in the process of culture in vitro.Immunohistochemical analysis revealed that part of BMSCS were CXCR4 positive andmost BMSCS were CD44 positive,while most BMSCS were CD34 negative.The mRNAof CXCR4 was successfully reversely transcribed by RT-PCR in every generation ofBMSC,and their quantity has no difference.Conclusions The whole bone marrow culture method is a helpful method that can obtainBMSC conveniently and effectively.BMSC exhibit CXCR4 expression. Experiment 2Study on rat model of acute myocardial infarctionObjective To find out how rat model of acute myocardial infarction can be establishedsuccessfully.Methods Left anterior descending coronary arteries were ligated to produce acutemyocardial infarction in SD rats.The cause of complications in and after operation and itsprevention and cure approaches were analyzed in 80 rats.Credible rat models wereevidenced by electrocardiogram,histology examination and ultrasonic cardiogram.Results Rats established model of acute myocardial infarction had elevation of ST segmentin ECG in early stage,patho-Q wave in late stage,scarring in ventricular wall and worseheart function.Intraoperative main complications and death causes were arrhythmia,hemorrhage,airway obstruction,injury of lungs,an so on;postoperative maincomplications and death causes were infection,pulmonary atelectasis and congestive heartfailure,an so on.Conclusion Our methods include decreasing lung injury,injecting anti infective and so on.They give a guarantee for making a successful rat AMI model. Experiment 3In vitro study on the role of SDF-1/CXCR4 axis in the migration ofbone mesenchymal stem cells into heart after acutemyocardial infarctionObjective To study the role of stromal cell derived factor 1/CXC chemokine receptor 4axis (SDF-1/CXCR4 axis) in the migration of bone mesenchymal stem cells into the heartafter acute myocardial infarction.Methods Bone mesenchymal stem cells culture were performed with the wholemarrow-adherence way,Acute myocardial infarction (AMI) model was built by ligation ofleft anterior descending coronary in SD rats,cardiac tissular extract were made fromnormal heart and infracted heart.By using Boyden chamber in vitro migration assaysystem,the effects of different concentrations of SDF-1,cardiac tissular extract fromnormal heart and from infracted myocardium at different time point after acute infractionon bone mesenchymal stem cells migration were observed,the influence of AMD3100treatment on bone mesenchymal stem cells' migration induced by cardiac tissular extractwere also observed.Results SDF-1 had a concentration-dependent chemotaxis effect on bone mesenchymalstem cells.Cardiac tissular extract made from hearts one week after AMI can induce themigration of bone mesenchymal stem cells obviously,and this effect can be suppressed byAMD3100 which is a blocker of CXCR4.Conclusion Cardiac tissular extract from hearts one week after AMI can induce themigration of marrow stromal cells,SDF-1/CXCR4 axis play an important role in thisprocess. Experiment 4Effects of stromal-derived factor 1 on proliferation and apoptosis ofrat bone mesenchymal stem cells in vitroObjective To detemrine the effect of SDF-1 on in-vitro proliferation of bone mesenchymalstem cells (BMSC);To investigate the effects of stromal-derived factor 1 preconditioning(PC) on apoptosis of bone mesenchymal stem cell (BMSC) treated with hypoxia plusserum deprivation.Methods BMSC culture was performed with the whole marrow-adherence way;BMSCwere incubated with different doses of SDF-l(lng/mL,10ng/mL,100ng/mL)for 48h,thenproliferation of BMSC were measured by MTT assays.BMSC were incubated in mediumfor 24 hours with 10ng/mL,100ng/mL SDF-1 respectively,then they were treated withhypoxia plus serum deprivation for 6 hour.Apoptic rate were detected by flow cytometerand TUNEL method.Results MTT assay showed that SDF-1 could enhance BMSC proliferation in dose dependmanner compared with the control group and the effect on proliferation of BMSC washighest in 100ng/ml group among all groups.The number of apoptotic cells wassignificantly reduced in SDF-1 PC group compared with the control group,and 100ng/mLSDF-1 PC group has the lowest level of apoptosis.Conclusion SDF-1 could enhance BMSC proliferation in dose depend manner.SDF-1preconditioning suppresses the apoptosis of BMSC treated with hypoxia plus serumdeprivation. Experiment 5Study about cellular transplant applied with rat bone mesenchymalstem cells preconditioned with stromal-derived factor 1 totreat acute myocardial infarctionObjective To observe therapeutic efficacy of cellular transplant with bone mesenchymalstem cells (BMSC) preconditioned with SDF-1 in rats with acute myocardial infarction.Methods BMSC cultures were performed with the whole marrow-adherence way;BMSCwere incubated in medium for 24 hours with 100ng/mL SDF-1.Acute myocardial infarction(AMI) model was built in SD rats,and BMSC preconditioned or non-preconditioned withSDF-1 were transplanted into border zone around infarct area,then heart function wasdetermined after 2 weeks by ultrasonography.Results We established AMI model successfully.Two weeks after BMSC transplant,significant improvement in cardiac function was observed in 100ng/mL SDF-1preconditioned group compared with the non-preconditioned group.Conclusion We conclude that SDF-1 PC is a novel approach for enhancing therapeuticefficacy of cellular transplant in rats with acute myocardial infarction.