| Objective:Ischemic Preconditioning(IPC) is an endogenous protective mechanism, which can reduce the myocardial infarction areas mainly via inhibiting myocardial apoptosis. But it’s not clear whether IPC can complement myocardial cells to repair damaged heart tissue through stimulation of cell proliferation. Based on the establishment of ischemic preconditioning rats model, this study recorded the change of cell proliferation indexes Ki67 that reflects myocardial cell proliferation. Transcription factors Nkx2.5, GATA4,c-Kit was detected to reveal the source of the repair cells.Methods:1. The establishment of the model. The change of ST segment was adopted to verify the success of the model building. ST segment elevation indicated myocardial ischemia and the return to the baseline meant the success of reperfusion.2. Detection of myocardial tissue morphology. HE staining was used to determine the degree of injury of the Sham, I/R and IPC groups.3. The detection of Ki67. Double immunofluorescence staining and immunohistochemical SABC method are used to detect the expression of Ki67. Observed the results with microscope and select ten visions of each section to calculate the number of the positive cells.4. Detection of the new cells in myocardial tissue source. RT-PCR, western blot,immunochemistry were used to detect the expression of GATA4,Nkx2.5 and c-Kit.Results:1. The establishment of the model: ST segment elevation indicated myocardial ischemia and the return to the baseline meant the success of reperfusion.2. HE staining showed:(A)In the sham group there was little change in the myocardium and the myocardium was arrayed naturally;(B)In the I/R group, enlargement of the cardiac cells, partially dissolved membrane and nucleus and inflammatory cells infiltration were observed from tissue sections;(C)In the IPC group, the pathological phenomenon was significantly improved compared with the other two groups.3. The detection of cell proliferation index Ki67.(A)immunochemistry:the Sham group had little Ki67 positive cells, while the number of positive cells of IPC and I/R groups increased after surgery. Compared with the Sham group, there was significance difference in IPC group(P<0.01). There was significant difference at the 3rd day and 7th day between the I/R and IPC groups on the Ki67 positive units(P<0.01).(B)Double immunofluorescence staining: positive cells in IPC group is more than that in Sham group at 3rd day and 7th day(P<0.05). However, after treatment positive cells in IPC group is more than I/R group at 3rd day and 7th day. Maybe it is because of high sensitivity of double immunofluorescence staining.4. Source of the new cells in cardiac tissue.(A)RT-PCR: the expression trend of c-Kit,Nkx2.5 and GATA4 are similar; the peak expression of the genes happened on the3 rd day in the IPC group and on the 7th day in the I/R group(P<0.01).(B)Western blot:there was little expression of c-Kit,GATA4 in the sham group; the expression of the two increased in IPC and I/R groups(P<0.05);within the two groups, the expression differed at the 1st day, 3rd days and 7th days(P<0.05).(C)Immunochemistry: In the sham group,there were little positive cells of Nkx2.5,GATA4; the number of the positive cells increased in IPC and I/R groups(P<0.05), at the 1st day, 3rd day and 7th day; the IPC and I/R groups had significant difference at the 3rd day after the operation.(D)Double immunofluorescence staining which showed positive cells for c-Kit,Nkx2.5,GATA4, in Sham group was less than IPC and I/R groups(P<0.05); Within the groups, significant difference at the 1st day, 3rd day and 7th day were observed(P<0.05). The positive cell number in IPC group peaked at the 3rd day.Conclusions:1. IPC could reduce the myocardial infarction area.2. IPC could promote the expression of Ki67 and myocardial cell regeneration.3. IPC and I/R could increase the number of Nkx2.5,GATA4,c-Kit positive cells, but the peak time was different, with the IPC at the 3rd days, the I/R at the 7th day. Further more IPC could promote the related indicators more than I/R.4. c-Kit positive cells are likely the new myocardial cells which could differentiate into cardiomyocytes, and participate in the repair of injured cardiac issue. |
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