Objetive:Through setting up big mouse's myocardial ischemia reperfusion injury animal's model,then using four kinds of different methods to deal with the corresponding group, and observe the change of myocardial infarction area ,myocardium cell apoptosis and hemodynamics,to discuss the changes of apoptosis of myocardial cell and expression of Bcl-2 and Bax of rats with myocardial ischemic postconditioning,then offer the theory materials for further studying the mechanism of the myocardium cell's protective action of Bcl-2 family in the myocardial ischemic postconditioning.Methods: 40 experimental Sprague-Dawley rats,weight 280-300g, no-divided male and femal, were randomly divided into 4 groups, 10 every groups。I.e.①Ischemic reperfusion group(Group I/R):Tighten up the line of ligature,the left anterior descending was occluded for 30 minutes,relaxed the line of ligature and reperfused for 120 minutes.②Ischemic postconditioning(Group IPO): Tighten up the line of ligature,the left anterior descending was occluded for 30 minutes,relaxed the line of ligature and reperfused for 120 minutes,then 3 cycles of 20 second of ischemia,each followed by 20 seconds of reperfusion,followed by 118 minutes's repersusion.③ HA14-1(Group HA14-1): 30 minutes before the left anterior descending was reperfused , Injected HA14-1 into the peritoneum. Tighten up the line of ligature,the left anterior descending was occluded for 30 minutes ,Then relaxed the line of ligature and reperfused for 120 minutes.④Ischemic postconditioning + HA14-1(Group IPO + HA14-1):30 minutes before the left anterior descending was reperfused , Injected HA14-1 into the peritoneum. Tighten up the line of ligature,the left anterior descending was occluded for 30 minutes,relaxed the line of ligature and reperfused for 120 minutes,then 3 cycles of 20 second of ischemia,each followed by 20 seconds of reperfusion,followed by 118 minutes's repersusion.All rats were fasted 3 hours and were prohibited water 30 minutes before operation. After the general anesthesia, lay on the back ,head and limbs were fixed, and four limbs joined the heart electrode, tracheostomy, the breathing machine assists breath. Median incision of manitruncus, cut off the brestbone, the heart appears, offered Heparin 500u/kg respectively to prevent thrombosis. On no blood vessel district ,put into the telescopic needle of puncture in the department of apex of the heart of left room by the thoracic cage wall, and connected the pressure transducer and monitoring instruments. Observe the index:①The Index of myocardium organization's morphology :After reperfusion,fetched right amount of size of cardiac muscle of left room,and fixed,wraped up and buried,HE dyed regularly ,then were observed under light microscope.②The myocardium infarction area:After 120 minutes reperfusion,take down the heart rapidly,blocks LAD in situ, irritated Even' s blue through the aorta, isolated the cardiac muscle of left room and crosscuted they into 5 slices.Put they into TTC Phosphate Buffered solution(PH 7.4) and 37℃water bathed for 15min,then no necrosis area was dyed brick-red,necrosis area does not dye ashen.Put they into 10℅neutral formaldehyde for one night to fixed.Separate the necrosis and no-necrosis district and Weighed separately. Infarction range expresses with the percentage of the myocardium quality of necrosis in myocardium quality of left room.③Cardiac muscle cell apoptotoc index:After 120 minutes reperfusion,cut the heart completely, the appropriate amount of the whole layer of cardiac muscle was fethed in the fixed position of the left room's apex to prepare myocardium wax sample, according to the regular histopathology's method .Adopted terminal deoxynucleotidy l transferase_mediated dUTP nick end labeling (TdT-mediated dUTP NickEnd Labeling, TUNEL) law,detected myocardial cell's apoptosis,according to the directions of the kit of testing-apoptosis in situ.Both the nucleus submitting brown or buffy grains and having apoptosis'cell morphology characteristics were judged for apoptosis'cell.According to the distribution situation of apoptosis,each slice shooted 5 positive visual fields,each visual field counted to apoptosis's cell from 300 myocardium cell.Take the proportion of apoptosis's cell with normal myocardial cell as apoptosis rate, i.e. myocardial cell apoptotoc index(AI) = (the count of myocardial apoptosis cell nucleus / the count of normal myocardial cell)×100℅.④The positive expression index of Bcl-2 and Bax (PEI):By immunohistochemisty's method detected the expression of Bcl-2,Bax, each rat obtained 4 sheets of slices,and observed positive cells under 200 double light microscope.It is positive that pale brown particle appears inside of the hyalomitome and intramembrane. According to the distribution situation of apoptosis,each slice shoot 5 positive visual fields randomly,each visual field counted to positive cell and total cellular score.Take the proportion of positive cell count with total cellular score as the positive expression index of Bcl-2 and Bax(PEI).⑤Hemodynamics's index:10 minutes before LAD blocked,30minutes after LAD blocked,15 minutes ,30 minutes 60 minutes 120 minutes after reperfused,total 6 time point. Record the heart rate (HR),left ventricular systolic pressure(LVSP), left ventricular end diastolic pressure;left ventricular end-diastolic pressure(LVEDP),left ventricular pressure ascensive maximum velocity (+dp/dtmax) and left ventricular pressure descending maximum velocity (﹣dp/dtmax) separately By RM6240C physiological signal acquisition system ,then analyzed the change of all index before ischemia and after regulation.Result:①the results of myocardium cell histomorphology: Under light microscope, the change of Group I/R in myocardium muscle tissue pathomorphology was obviously aggravated,and Group IPO's change in myocardium muscle tissue pathomorphology was obviously lessened than Group I/R,but the change of Group IPO,Group HA14-1,Group IPO+HA14-1 were alike or similar in pathomorphology.②the results of myocardial infarction size: Respectively Group I/R(76.63±4.84),Group IPO(46.99±2.82),Group HA14-1(76.51±3.47),Group IPO + HA14-1(75.67±4.57). The infarct sizes of Group IPO was obviously less than Group I/R, The difference was significant ( p<0. 01); The infarction area of Group IPO+HA14-1 was obviously larger than Group IPO, The difference was significant (p<0. 01);There was no significant difference among Group I/R,Group HA14-1 and Group IPO + HA14-1 in the above parameters (p>0.05).③The results of cardiac muscle cell apoptosis index:TUNEL positive cells obviously increased in the Group I/R'myocardium tissue,and was distributed intensively.The AI of four groups were respectively Group I/R ( 42.32±1.48 ),Group IPO(20.48±1.20),Group HA14-1(41.21±3.63),Group IPO + HA14-1(40.17±1.94).The tunel positive cells of Group IPO is obviously less than Group I/R and the difference was significant (p<0. 01). There was no significant difference among Group I/R,Group HA14-1 and Group IPO + HA14-1 in the above parameters( p>0.05).④The positive expression index of Bcl-2 and Bax :The PEI of four groups were respectively Group I/R(0.48±0.03,1.07±0.01),Group IPO(1.10±0.03,0.30±0.03),Group HA14-1(0.47±0.05,1.06±0.04),Group IPO + HA14-1(0.47±0.05,1.06±0.04). In the normal cardiac muscle tissues, the expressions of Bcl-2 and Bax mRNA are relatively low or not expressed; In Group I/R The Bcl-2 mRNA expression descended , Bax mRNA of expression rised,Bax/Bcl-2 ratio rised;In Group IPO ,Bcl-2 mRNA expression rised, Bax mRNA expression descended, Bax/Bcl-2 ratio descended.The difference between Group IPO and group I/R was significant (p<0. 01) ;The Bcl-2 mRNA expression level of Group HA14-1 was lower than Group I/R obviously, Bax mRNA expression level was nosiganificant with Group I/R,Bax/Bcl-2 ratio increased than Group I/R;Between Group IPO+HA14-1 and Group I/R,the Bcl-2 mRNA and Bax mRNA expression level were similar.⑤The index of Hemodynamics : Before LAD was blocked,the difference among the HR,LVDP,LVEDP,+dp/dtmax,-dp/dtmax of four groups was no significant meanings (p>0.05); After LAD was blocked for 30 minutes, the LVEDP, +dp/dtmax, - dp/dtmax of experimental animal in every group had downward trends, HR had the tendency to rise,but but the difference of change was no significant (p>0.05) among any group;the LVDP,LVEDP,+dp/dtmax,-dp/dtmax of Group IPO was obviously higher than Group I/R's obviously and the difference was significant (p<0.01) on the reperfusion time 15 minutes,30 minutes,60 minutes,120 minutes,but HR was obviously lower than Group I/R;Among Group HA14-1,Group IPO+HA14-1 and Group I/R,the change of every hemodynamics'index in the above three groups was no significant (p>0.05). The LVEDP of every group rapidly rised in the early 15 minutes of reperfusion, afterward with reperfusing time to lengthen,LVEDP droped gradually ,and Group IPO were obviously less than Group I/R ,the difference was significant (p<0. 01).Conclusion: 1.The ischemic postconditioning could lessened the change of myocardial pathomorphology that was happened after ischemia reperfusion injury, reduced myocardium infarction area and lessened myocardial ischemia reperfusion injury,thus had certain protection effects on myocardial ischemia reperfusion injury. 2.The ischemic postconditioning can inhibit myocardium cell's apoptosis that is induced by myocardial ischemia reperfusion injury. 3.The ischemic postconditioning reduces myocardial ischemia reperfusion injury and reduces the intensity of myocardium cell's apoptosis, may relate to the fact that Bcl-2 albumen's expression was up-regulated, Bax albumen's expression was down-regulated. 4. The ischemic postconditioning can improve obviously myocardial contractibility and the ability of myocardial diastole,after ischemia reperfusion injury.thus could improve heart function after Myocardial Ischemia Reperfusion Injury. |