The Study Of Myocardial Proliferation Induced By Ischemic Post-conditioning

ObjectiveIt is generally believed that the heart becomes a postmitotic and differentiated organ,atwhich point the cardiomyocytes withdraw from the cell cycle.Recenly,scientists have provedthat myocardial cells could reenter into the cell cycle on the condition of physiology andpathology.The cell signal mechanism of ischemic post-conditioning(IPO)to ischemicreperfusion injury,not only inhibits cell apoptotic,but slso regulates the cell cycle.The IPOprotect myocardial from apoptotic and may induce cell proliferation to damage repairing.Through the rat model of ischemic postconditioning,observe the specific indicators of cellproliferation and investigate whether ischemic postconditioning could promote proliferation.Itwould provide a new theory foundation and idea for further study of the myocardial protection.Methods1.The establishment of the model.The healthy adult male SD rats,weighting250±30g arerandomly divided into3groups:①Sham operation(S,n=6),open the chest and braid withoutligation;②I schemia/reperfusion(I/R,n=6),reversibly ligated left coronary artery(LAD)andreperfused after ischemia for30min;③Ischemic postconditioning(IPO,n=6),reversibly ligatedLAD and ischemiced for30min,then reperfused for10s and ischemia for10s before reperfusion.④Sign of ischemic:the left ventricular anterior wall changes from red to dark gray and STsegment elevates.Observe the indicators at1d,3d,7d,14d and30d respectively.2.Determination of cardiac function.Record the left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP),left ventricular pressure ascensive maximumvelocity(+dp/dtmax)and left ventricular pressure descending maximum velocity(-dp/dtmax)separately by RM6240C physiological signal acquisition system and analyze the data;3.Observation of morphological.Observe the myocardial morphological by HE stainingand compare the damage induced by I/R and IPO.4.Detection of cardiomyocyte proliferation indexes by immunofluorescence andimmunohistochemistry.①Observe DNA synthesis of cardiomyoctyes by5-bromodeoxyuridine(BrdU)and α-sarcomeric actin(α-SMA);②I nvestigate the mitosis by phosph-Histone H3(H3P)and α-SMA;③Detect the cardiomyocytes cytoplasmic division by Aurora B and α-SMA.Investigate the expression of Aurora B at different point by Western Blot.One of its objectives isto further confirm the proliferation of myocardial cells,and judge the trend of cardiomyocyte proliferation induced by IPO.Results1.IPO can improve cardiac function.The Hemodynamic at different time points revealsLVSP and±dp/dt max of I/R and IPO groups are significantly lower than the Sham group.At thesame time LVEDP is higher than the Sham group.Compared with I/R group,LVSP and±dp/dtmax of IPO group increase,while LVEDP has a downward trend.2.IPO can reduce reperfusion injury of myocardial tissue.HE staining shows thepathological changes of Sham group is not obvious and the muscle fibers arrange neatly andtravel naturally;I/R group myocardial cell deranges,swells and inflammatory cells infiltrate;IPO group muscle fibers arrange neater and the reduction of cell swelling and inflammatory cellinfiltration.3.Observe the cardiomyocyte proliferation of IPO3.1Obseve BrdU,H3P and Aurora B by immunohistochemical SABC method.BrdU,H3Pand Aurora B are stained brown.The brown positive protein particles are scattered in myocardialtissue.The number of positive cells of Sham group is less and I/R,IPO group increase.Thestatistical analysis shows:PU of BrdU,H3P and Aurora B respectively,IPO group(7.81±1.39,5.18±0.47,10.75±0.93);I/R group(5.27±1.35,4.23±0.57,8.95±0.68);Sham group(4.39±1.22,2.97±1.24,5.31±0.50).①BrdU:compared with Sham group,IPO group has a significantlydifference(P<0.05).The PU of IPO and I/R group,I/R group and S group increase,but nodifference.②H3P:analysis results are same to the BrdU.③Aurora B:IPO has a significantlyhigher than S group(P<0.01).Compared with I/R group,IPO has a significantlydifference(P<0.05).3.2Analyze the H3P and Aurora B by Immunofluorescence.The number of positive cells ofH3P and Aurora B respectively,IPO group(7.58±1.04，11.23±0.75),I/R group(5.53±0.64，9.70±0.44),S group(3.16±0.57，7.76±0.53).The number of IPO and I/R group are more than Sgroup,and has a significantly difference(P<0.01).The IPO group has a difference comparedwith I/R group(P<0.05).They have demonstrated the expression of H3P and Aurora B in IPO,I/R group rises.However,it is different between immunohistochemical SABC method andimmunofluorescence.Maybe it’s related to the sensitivity.4.Investigate the expression of Aurora B by Western Blot.The expression level of Aurora Bof I/R and IPO group is higher than S group,and reach the peak at3d and7d respectively.Thestatistical analysis shows:the expression of Aurora B of IPO group(3d:1.04±0.19;7d:0.91±0.05),I/R group(3d:0.73±0.01；7d:0.78±0.02)，Sham group(3d:0.29±0.04;7d:0.51±0.01).Comparedwith S group,IPO and I/R group have significantly difference at3d(P<0.05).IPO and I/R grouphave significantly difference compared with S group(P<0.01).It is of difference between IPO group and I/R group(P<0.05).Conclusion1.IPO can effectively improve cardiac function and reduce pathological changes ofmyocardial tissue,thereby protected the damaged myocardium;2.IPO can promote the expressions of BrdU,H3P and Aurora B.3.IPO not only reduces cell apoptotic,but also promotes myocardial cells to reenter into thecell cycle and compensates for the damaged cells by proliferation and differentiation.It canimprove heart function and reduce reperfusion injury.