The Study Of Ischemia/Reperfusion Injury Repair And The Sources Of Repair Cells By Ischemic Postconditioning

Objective:Ischemic postconditioning (IPO) can reduce the reperfusion injury. The signal transductionprotective mechanism not only can prevent apoptosis, but also involves cell cycle regulation. Ourgroup has proven that the IPO can induce cardiomyocyte proliferation by the expression ofspecific marker5–bromodeoxyuridine (BrdU), phosph-Histone H3(H3P), and Aurora B. It isevident that the IPO not only can reduce apoptosis, but also can promote the proliferation ofmyocardial cells, so as to compensate for the damaged cardiomyocytes. The purpose of thestudy is to clear the source of proliferation cells, which is from normal mature cardiac cells ormultipotent stem cells. Through the detection of multi-potential stem cell surface specific markerc-kit,Gata-4, Mef2C, determine the cell source. If the c-kit, Gata-4, Mef2C are all positive, wecan determine that the presence of pluripotent stem cells. Or they are Cardiac mast cells. Investigatethe number of mature cardiac myocytes by immunofluorescence detection of α-sarcomeric actin(α-SMA). Make correlation analysis between the number of mature cardiomyocytes and thenumber of pluripotent stem cells, and speculate the possible sources of myocardial proliferationcells. In addition, observe the changing trend of the stem cells by the expression of c-kit at fivepoints.Methods:1. The establishment of the model. The healthy adult male SD rats, weighting250±30g arerandomly divided into3groups: Sham operation (S), open the chest and braid withoutligation; Ischemia/reperfusion (I/R), reversibly ligated left coronary artery (LAD) andreperfused after ischemia for30min; Ischemic postconditioning (IPO), reversibly ligatedLAD and ischemiced for30min, then reperfused for10s and ischemia for10s beforereperfusion.2. The observation of cardiomyocytes proliferation. Observe DNA synthesis of cardiomyoctyesby specific marker5-bromodeoxyuridine (BrdU) and α-sarcomeric actin (α-SMA) withimmunohistochemistry SABC method.3. The source of proliferation cells. It is proved by the c-kit with Western Blot andimmunofluorescence and the expressions of c-kit, Gata-4and Mef2C with RT-PCR. Makesure the source of proliferation cells by the correlation analysis of the positive cells of c-kitand α-SMA. 4. The examination of hemodynamic. Observe whether IPO can protect damaged heart.Results1. The proof of cardiomyocyte proliferation. Observe the expression of BrdU byimmunohistochemistry SABC method. BrdU is located in the myocardial nuclear and stainedbrown. The brown positive protein particles are scattered in myocardial tissue. The numberof positive cells of Sham group is less and I/R, IPO groups increase. IPO group(PU value:7.81±1.39), I/R group(PU value:5.27±1.35), Sham group (PU value:4.39±1.22). Thestatistical analysis shows that there is significant difference between IPO group and Shamgroup(P <0.05). The PU values of IPO and I/R group are higher than the Sham group, butwithout difference.2. The proof of the source of proliferating cells2.1The detection of c-kitThe positive cells of c-kit are observed in IPO group by Immunofluorescence labelingmethod. It reaches peak at3day (PU value:2.91±0.29,5.39±0.42,4.37±0.35,3.02±0.67,2.14±0.25) and there is significantly difference (P <0.05); The most positive cells ofc-kit are at7d (PU values are:1.29±0.12,2.64±0.274.71±0.59,2.85±0.27,1.71±0.38).The difference is significant (P <0.05). There is no c-kit positive cells in other groups.The expression level of c-kit in IPO group reaches peak at3d by Western Blot and thedifference is significant (P <0.05). However, the expression of c-kit is most at7d and there issignificantly difference (P <0.05).There is no expression in other groups.The curve of the expression level of c-kit which is speculated by RT-PCR in IPO andI/R group is similar to the trend with Western Blot. Determine the maximum expression ofc-kit pluripotent stem cells of IPO group appears at3d, whereas I/R group is at7d. There issignificantly difference in two groups.(P <0.05)2.2The detection of c-kit is consistent with the results of Gata-4, Mef2C by RT-PCR2.3Distinguish the stem cells and mast cells. Determine the number of c-kit pluripotent stemcells in myocardial tissues through the detection of piuriotent stem cell surface specificmarker c-kit, Gata-4and Mef2C. If they are all positive and the curve of the expression issame to them, they are the c-kit pluripotent stem cells. The number of cardiachypertrophy doesn’t affect it.2.4The speculation of the source of proliferation myocardial cells. Determine the number ofmature cardiac cells and pluripotent stem cells by immunofluorescence. The former isrelated to the latter through the correlation analysis. Speculate that the proliferation cells may be from stem cells.3. IPO can improve cardiac function. The hemodynamic postoperative14d and30d reveals: thevalue of LVSP and±dp/dt max in I/R and IPO groups is significantly lower than the Shamgroup. However, the value of LVEDP is higher than Sham group. Compared with I/R grou p,the value of LVSP and±dp/dt max in IPO group is significantly higher at different point (P<0.05), while there is no difference in LVEDP.Conclusions:1. The methods of IPO and I/R can increase the number of the cardiomyocytes, and they ma ybe from c-kit positive pluripotent stem cells.2. The IPO and I/R can promote c-kit positive pluripotent stem cells proliferation. The numberof them in I/R group reaches peak at3d, while at7d in IPO group. It proves that IPO canpromote c-kit positive pluripotent stem cells proliferation in advance.3. The number of c-kit positive pluripotent stem cells is related to the number of maturemyocardial cells. Maybe c-kit positive pluripotent stem cells after damage differentiate intothe mature myocardiac cells to repair damaged cardiac tissue.4. IPO can promote myocardial tissue hemodynamic recovery and improve cardiac function.