The Role Of BMP9and BMP13on C3H10T1/2Cells Differentiation To Cardiomyocyte-like Cells In Vitro And Improving Cardiac Fuction Of Infact Rats

PART ONE: AMPLIFICATION OF THE RECOMBINANTADENOVIRUS BMP9，BMP13,TRANSFECTION OFC3H10T1/2CELLSObjective: Obtained high titer of recombinant adenovirusBMP9,BMP13and GFP by repeated amplification with HEK293, transf-ected C3H10T1/2cells with AdEasy-BMP9, AdEasy-BMP13, AdEasy-GFP which over expressed BMP9and BMP13gene. Taking C3H primarycultured myocardial cells of neonatal mice as experimentalpositive control.Methods: Obtained high titer of recombinant adenovirusBMP9,BMP13and GFP by repeated amplification with HEK293. TakingC3H primary cultured myocardial cells of neonatal mice(1-3D) asexperimental positive control. Observed C3H10T1/2cells under invertedmicroscope after recovery. when cell growth vision fusion was about 70%, transfected AdEasy-BMP9, AdEasy-BMP13and AdEasy-GFP intothe culture medium of C3H10T1/2cells, then observe the transfectionefficiency by fluorescence microscope and flow cytometry technique, anddetect expression of BMP9and BMP13in each group by real-timefluorescence quantitative PCR.Results:1After amplified with HEK293cells, obtained high titer of recombinantadenovirus BMP9, BMP13and GFP.2Taking C3H primary cultured myocardial cells of neonatal mice(1-3D)as experimental positive control., cells were fusiform or polygon and auto-nomous beating.3Observed a high transfection efficiency of C3H10T1/2cells transfectedwith recombinant adenovirus by fluorescence microscope and flowcytometry, Detected expression of BMP9and BMP13in each groupby real-time fluorescence quantitative PCR. Expression of BMP9in BMP9group was120-125times over GFP group and blankcontrol group, expression of BMP13was125-130times over the amount ofGFP group and blank control group.(p<0.05)Conclusion: the repeated amplification with HEK293can obtain hightiter recombinant adenovirus BMP9, BMP13and GFP. transfectedAdEasy-BMP9, AdEasy-BMP13, AdEasy-GFP into C3H10T1/2cells canobtain higher transfection efficiency, and C3H10T1/2cells in BMP group expressed high level of BMP9and BMP13. PART TWO: THE FEEECTION AND DIFFERENCE OF BMP9AND BMP13DURING C3H10T1/2CELLS DIFFERENTEDINTO MYOCARDIOCYTESIN VITROObjective:To study and compare the effection of BMP9(bonemorphogenetic protein9), and BMP13（Bone morphogenetic protein13)on the differentiation ofC3H10T1/2cells into cardiomyocytes in vitro.Methods: The experiment grouped as followed: blank controlgroup (untreated C3H10T1/2cells), GFP group(C3H10T1/2cellstransfected with AdEasy-GFP), BMP9group (C3H10T1/2cellstransfected with AdEasy-9), BMP13group (C3H10T1/2cellstransfected with AdEasy-9),myocardial cell group (C3H primary culturedmyocardial cells of neonatal mice). Selected1weeks,2weeks,3weeks,4weeks as experimental time point. Expression and differencesof cardiac specific gene MEF2C, GATA4and ion channel encoding genesKCNA5, KCNJ12, CACNA1H,SCN5A in cells were detected byreal-time fluorescence quantitative PCR; Detected myocardialcells specific protein cTnT, Cx43in each group by immunofluorescence andWestern-blot; to observe the expression of cardiac specific ultrastructurewith transmission electron microscopy and Masson’s trichrome stain;detected calcium current, sodium currentand internal, outward potassium current of cell membrane usingsingle cell patch clamp; detected expression of ion channelencoding genes by real-timefluorescence quantitative PCR.Result:1. After randomization groups of cells,observed the cells in each groupby inverted phase contrast microscope, compared with the blank controlgroup, the shape of C3H10T1/2cells transfected with AdEasy-9andAdEasy-13were spindle, and its intercellular connection is morecompact, cells arranged more orderly consistent.2. Expression and differences of cardiac specific gene MEF2C, GATA4were determined by real-time fluorescent quantitative PCR. Theexperimental results show that in the beginning of the1week after idifferentiation, cardiac specific gene MEF2C, GATA4can be detected inBMP9group and BMP13group, was3times and2.5-3.5times over theGFP group and the blank control group (p<0.05).3. Expression and differences of cardiac specific protein cTnT, Cx43were determined by immunofluorescence and Western blot technique.Detected with immunoblotting technique, there was no expression of Cx43and cTnT in GFP group and blank control group. the expression ofCx43and cTnT in BMP9group, BMP13group and myocardial cell gro-up was significantly higher than that in GFP group and blank controlgroup(p<0.05). Expression of BMP9group was higher than BMP13group (p<0.05). And this is Consistent with the results detected byimmunofluor-scence.4. To observe the expression of cardiac specific ultrastructure:myofilaments and intercalated discs with transmission electronmicroscope and Masson’s trichrome stain. Using transmission electronmicroscopy, Observed myofilaments and intercalated disclike structurein myocardial cell group, BMP9group and BMP13group; using Massontrichrome staining, observed filament like structures stained red in BMP9group, BMP13group. While no such structures in the GFP group andblank control group.5. Detected calcium current, sodium current and internal, outwardpotassium current of cell membrane using single cell patch clamp at4experimental time points. Differentiation after4weeks, the T typecalcium current detection (IT-Ca),ultrarapid delayed rectifier potassiumcurrent (Ikur), inward rectifier potassium current (Ikir) expression wasdetected in BMP9group and BMP13group. Sodium current (Ina) canbe detected in a few cells in the BMP9group. All these currents canbe detected in myocardial cell group, and failed to detect in GFP group and blank control group.6. Expression of ion channel encoding genes was detected by real-timefluorescent quantitative PCR. In BMP9and BMP13groups, theexpression of KCNA5is3-3.5times higher than that of controlgroup (P <0.05), the expression of KCNJ12is1.5-4times higher thanthat of control group (P <0.05), the expression of CACNA1H is2-6times higher than that of control group (P <0.05), whilethe expression of SCN5A in BMP9patients was4.5times group andBMP13control group (P <0.05).Conclusion: In vitro, BMP9and BMP13can promote thedifferentiation of C3H10T1/2cells into cardiomyocyte-like cells, and theeffction of BMP9is better than BMP13. PART THREE:DIFFERENT EFFECT OF C3H10T1/2CELLSTRANSFECTED WITH BMP9AND BMP13ON CARDIACFUNCTION OF MYOCARDIAL INFARTION RATSObjective: To study and compare the effect ofBMP9and BMP13on heart function of SD rat after myocardialinfarction.Methods:24male SD rats were randomly divided into four groups: MIgroup(MI group, myocardial infarction, n=6), MI+GFP group(MI injected with pAdEasy-GFP transfected C3H10T1/2cell, n=6), MI+BMP9group (MI injected with pAdEasy-BMP9tran-sfected C3H10T1/2cell, n=6),MI+BMP13group (MI injected withpAdEasy-BMP13transfected C3H10T1/2cell,n=6). Regularmonitoring weight of rats in each group every week; Using heart dopplerultra sound to detect LVEDD (Left ventricular end systolic diameter)，LVESD（left ventricular end systolic diameter），FS（Left ventricularfractional shortening) to evaluate and compare the heartfunction of rats; Using fluorescence microscopy trace implanted cellswith GFP green fluorescent in frozen sections of the heart; Observe cellmorphology of the implantation site with HE staining; Using Masson’strichrome stain to observe and compare the rats myocardial infarctionarea; Using immunofluorescence to observe the expression of cardiacspecific protein cTnT in implanted cells.Result.1.Heart color Doppler ultrasound at4weeks after operation: Heartfunction of MI+BMP9group,MI+BMP13group, MI+GFP group were allimproved, MI+BMP9group, MI+BMP13group improved were betterthan MI+GFP group, MI group,and MI+BMP9group were better than inMI+BMP13group (P<0.05).2.Fluorescence microscopy trace implanted cells with GFP green fluorescent in frozen sections of the heart, the transplantedcells with GFP green fluorescent tracer distributed mostly along thevascular.3.HE staining:In MI+BMP9and MI+BMP13group, large accumulationof implanted cells arranged more orderly.4.Masson trichrome staining suggested that percentage of myocardialinfarcttion area in three groups implanted with cells were less than MIgroup (P<0.05). But the difference between the three groups was notsignificant (P>0.05)5.Fluorescence observed the expression of cardiac-specificprotein cTnT in myocardial implantation site of MI+GFPgroup, MI+BMP9group and MI+BMP13group were weak, and unevendistribution.Conclusion: BMP9and BMP13transfected C3H10T1/2cellscan improve the cardiac function of myocardial infarction rats, and theeffect of BMP9is better than BMP13.