| Part I：Gene screening for Cardiomyocyte differentiationObjective： Analysis for gene chip which is the result of subset includSCA-1+CD45+CD31+、 SCA-1+CD45+CD31-、 SCA-1+CD45-CD31+、SCA-1+CD45-CD31-， find out the highest gene expression in subset ofSCA-1+CD45+CD31+between the other three subsets and BMSC by Real-time PCR.Methods：According to the gene chip results showed，choose the genes which arehighest expression of SCA-1+CD45+CD31+subsets，consist of myl3、Itga4、Epor、Rock2、Cxadr，design primer for such genes。Extract RNA from SCA-1+CD45+CD31+、SCA-1+CD45+CD31-、SCA-1+CD45-CD31+、SCA-1+CD45-CD31-four subsets andBMSC，and reverse transcription of cDNA，then detect the gene expression in four subsetsand BMSC through Real-time PCR.Results：Theresult of Real-time PCR showed that highest expression of Itga4is in BMSC；the highest expression of Rock2is in SCA-1+CD45-CD31-；the highest expression ofCxadr is in SCA-1+CD45-CD31-； the highest expression of myl3is inSCA-1+CD45+CD31+，and there is significant differences compared with other threesubsets and BMSC（P<0.05）；the highest expression of Epor is in SCA-1+CD45-CD31+.Conclusion：There is the highest expression of myl3in SCA-1+CD45+CD31+。Myl3is amember ofthe family of myosin light chain，and also is the part of the sarcomere thickfilaments，research shows that myl3plays an important role in muscle regeneration andpromoting muscle contraction. Part II The establishment of mouse myocardial infarction modelObjective：Ligation of the mouse left anterior descending branch of coronary artery toestablish the mouse myocardial infarction model.Methods：C57BL/6male mice,24, weight:14-22g were randomly divided intoexperimental group and control group.Experimental group,12, tracheal intubation,thoracotomy direct vision mouse coronary anterior descending artery (LAD) ligation.Results：After ligation, the limb leads ST segment elevation of more than0.2mv. After4weeks, test of echocardiography: compare with the control group (sham-operation group),in experimental group, mice left ventricular ejection fraction (LVEF) and left ventricularfractional shortening index (FS) significantly reduced in myocardial infarction modelgroup (P <0.01, P <0.01). left ventricular end diastolic diameter (LVEDd) and leftventricular end systolic diameter (LVEDs) increased significantly (P<0.01), Cardiac HEstaining confirmed that the mouse myocardial infarction model is successfully established.Conclusion：Successfully established the model of mouse myocardial infarction.Part III Myl3promotes the mouse mesenchymal stem celldifferentiation into cardiomyocyte like cells in vitroSection A Culture of Mesenchymal Stem Cells from mouse Bone MarrowObjective：The separation of multilineage differentiation potential of bone marrowmesenchymal stem cells from mouse bone marrow，to provide reliable cells for furtherstudy.Methods：whole bone marrow culture，Identification of cell surface antigen expression byflow cytometry.Results：In vitro，after10-14days cultured primary MSCs achieved fusion，the resultsshowed that over90%of mouse bone marrow mesenchymal stem cells express CD29(cellintegrin molecules), CD90(adhesion molecule) and CD44(adhesion molecule); but did notexpress the hematopoietic progenitor cell surface marker CD11b、CD133. Conclusion：Successfully isolation,culture and amplification of the murine bone marrow mesenchymal stem cells in vitro。Section B Detecting the expression of cardiac specific factor in myl3-siRNAtransfected bone marrow mesenchymal stem cellsObjective：Thetransfection of myl3-siRNA in mouse bone marrow mesenchymal stemcells，and detecting the expression of cardiac specific gene after transfection。Methods：Using lipofactaminTM2000transfected myl3-siRNA into the mouse bonemarrow mesenchymal stem cells as the experimental groupA，then transfected siRNAblank vector into mouse bone marrow mesenchymal stem cells as control groupB，after24hour GFP expression was observed under inverted fluorescence microscope to detect thetransfection efficiency，myocardial cell lysate induced group A and group B differentiationinto cardiomyocyte like cells，collection of cells after48hours，design the primer of myl3、GATA-4、Cx43、Nkx2.5、cTnT、a-actin，detecting the gene expression of myl3、GATA-4、Cx43、Nkx2.5、cTnT、a-actin after transfected by Real-time PCR。Results：The efficiency of transfection is more than60%。Detecting by Real-time PCR，there is a significant decreasing in expression of myl3compared with control group，thesame result in expression of cardiac specific gene。Conclusion：After transfected myl3-siRNA，the expression of cardiac specific gene issignificant decreased compared with control group。Section C Construction of overexpressed plasmid myl3Objective：Construction of overexpressed plasmid myl3，and identification sequencing ofoverexpressed plasmid myl3。Methods：Isolated target genes by PCR method from Plasmid cloning template whichcontaining target gene，enzyme digest the target genes and target vector，Purified enzymeproducts after connection，the bacterial cells are rendered competent，to certify the targetgene had been directed into the target carrier by enzyme diges，and certify constructed target gene plasmid successfully by identification sequencing.Results： Successfully constructed overexpressed plasmid myl3by identificationsequencing and double enzyme digestion.Conclusion：Successfully constructed overexpressed plasmid myl3for functional study ofthis gene。Section D Detecting the cardiac specific gene expression in transfected overexpressmyl3plasmidObjective：Thetransfection of overexpressed plasmid myl3in mouse bone marrowmesenchymal stem cells，and detecting the expression of cardiac specific gene aftertransfection.Methods：Using lipofactaminTM2000transfected overexpressed plasmid myl3into themouse bone marrow mesenchymal stem cells as the experimental groupA，then transfectedblank vector plasmid into mouse bone marrow mesenchymal stem cells as control groupB，bone marrow mesenchymal stem cells as empty groupC，after24hour GFP expression wasobserved under inverted fluorescence microscope to detect the transfection efficiency，5-aza-2’-deoxycytidine induced group A and group B differentiation into cardiomyocytelike cells，and myocardial cell lysate induced group A and group B differentiation intocardiomyocyte like cells，collection of cells after48hours，design the primer of myl3、GATA-4、Cx43、Nkx2.5、cTnT、a-actin，detecting the gene expression of myl3、GATA-4、Cx43、Nkx2.5、cTnT、a-actin after transfected by Real-time PCR.Results：The efficiency of transfection is more than60%。Detecting by Real-time PCR，there is a significant increasing in expression of myl3compared with control group both by5-aza-2’-deoxycytidine and myocardial cell lysate induced，the expression of cardiacspecific gene GATA-4、Cx43、Nkx2.5are increased in group A compared with group Bwhich is induced by5-aza-2’-deoxycytidine，but decreased in group C，however，theexpression of cTnT in group A is significant increased compared with group B and group CWhen induced by myocardial cell lysate，the expression of cardiac specific gene GATA-4、 Cx43、Nkx2.5、cTnT are significant increased in group A compared with group B andgroup C，and also the expression of cardiac specific gene from high to low is group A、group B、group C（P<0.05）.Conclusion：Transfected overexpress myl3plasmid which is induced by myocardial celllysate can promote cardiac differentiation.Section E Immunofluorescence detection of cardiac specific factorObjective：To further verify the capacity of cardiac differentiation of overexpressedplamisd myl3From the protein level.Methods：Using lipofactaminTM2000transfected overexpressed plasmid myl3into themouse bone marrow mesenchymal stem cells as the experimental groupA，then transfectedblank vector plasmid into mouse bone marrow mesenchymal stem cells as control groupB，myocardial cell lysate induced group A and group B differentiation into cardiomyocyte likecells，fluorescence detection after4weeks.Results：14days after induced by myocardial cell lysate，overexpressed myl3mouse bonemarrow mesenchymal stem cell expressed Cx43、Desmin more than transfected emptyvector BMSC.Concluison:Testify myl3can promote bone marrow mesenchymal stem cellsdifferentiation into cardiac-like cells in protein level。Part IV Myl3promotes the mouse mesenchymal stem celldifferentiation into cardiomyocyte like cells in vivoObjective：Detecting whether overexpress plamisd myl3can promote mice bone marrowmesenchymal stem cells differentiation into cardiac like cells in mouse myocardialinfarction model，Improve the cardiac function.Methods：C57BL/6male mice,24, weight:14-22g tracheal intubation, thoracotomydirect vision mouse coronary anterior descending artery (LAD) ligation，and are randomlydivided into four groups：transfected overexpressed plasmid myl3into the mouse bone marrow mesenchymal stem cells as the experimental groupA，then transfected blank vectorplasmid into mouse bone marrow mesenchymal stem cells as control groupB，bone marrowmesenchymal stem cells as empty groupC，PBS for group D，injection the four groups intomyocardial infarction respective，echocardiography testing on the day of operation and4weeks after operation，evaluation the cardiac function，four weeks after the mice werekilled，cardiac tissue also be detected.Results：After four groups are injected4weeks later，results of echocardiography showsthat left ventricular ejection fraction in group A is higher than group B、group C and groupD，but no significant differences（P>0.05）,when compared with difference in leftventricular ejection fraction before and after injection，there is significant higher in group Athan group B、group C and group D，the same result also saw in difference in leftventricular fractional shortening index before and after injection，difference in difference inbefore and after injection，and difference in left ventricular end systolic diameter beforeand after injection（P<0.05）。Cardiac Masson staining measure the myocardial infarctionarea，there is the smallest area of myocardial infarction in group A，and also the lightestfibrosis （P<0.05）。 Cardiac specific antigen Cx43, desmin were detected byimmunohistochemistry，Cx43, desmin expression were detected in tissues of the heart inmice，Immunofluorescence also confirmed the above results.Conclusion：The bone marrow mesenchymal stem cells which overexpress plasmid myl3can significant improve cardiac function， Reduce the myocardial infarction area，Immunohistochemistry and immunofluorescence certify myl3can promote bone marrowmesenchymal stem cell differentiation into cardiac like cells. |
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