| How to accurate evaluate of the ability of the myocardial regeneration is currently the technical problems facing the by cardiovascular regeneration medicine. Its reason for this is the special biological charateristics of the myocardial cells. As we all know, the cells to complete the cell division are must be re-enter the cell cycle. The cell proliferation is composed mainly of2parts:caryokinesis and cytokinesis. Cytokinesis is located in the late M phase in the cell cycle, cell only experiencing the cytokinesis is considered to have truly complete mitosis process, reaching division and proliferation. However, the cardiomyocytes in the the process of of mitosis are lack of effective cytokinesis after the the completion of caryokinesis, and ultimately remain in caryokinesis period. Therefore, it is the result of the phenomenon of binucleation and polyploid in the normal heart. Prior evaluation of cardiomyocytes proliferation including some of the indicators, such as Ki67, Brdu (5-Bromo-2-deoxyUridine), PCNA (proliferating cell nuclear antigen) and H3P (phosphorylated histone H3), which are mainly directed against the DNA replication and the the caryokinesis, can’t reflect the the cytokinesis process of cardiomyocytes. Accordingly, the the above-mentioned methods are can not truly evaluate the cardiomyocyte proliferation. Because of no ideal method of evaluation of proliferation of cardiomyocyte, the controversy regards to cmyocardial regeneration capability existed. And this siutation also limits the the development of of cardiac regenerative medicine. If we find an accurate method of evaluation of cardiomyocyte proliferation, then it is bound to promote the development of the research of the heart regeneration, and bring the hope for the clinical application of myocardial regeneration. Previous studies showed that the cell cycle-related genes score (Cell Cycle Progression Score, CCP-Score) could be able to accurately evaluate the proliferation of the tumor cells, thereby effectively predicted the prognosis of of the tumor patients. Learning from the successful experience of multi-gene score (Muti-gene Score) in the tumor cell proliferation research, and considering the the particularity of of cardiomyocyte proliferation, in accordance with database gene information provided by the United States National Center for Biotechnology Information (NCBI) and the past the literature, our research group preliminary selected the10cytokinesis related genes, which all of the selected genes can regulate and control the process of cardiomyocyte cytokinesis, and express the the structure of protein during the cytokinesis process. By Real-time PCR method we calculated relative expression of myocardial cells one by one. Based on PCR results we set up the multi-Gene sysyrem by mathematic formula. First, we established the cytokinesis gene score (CC-Score) system in vitro cells experiment. Second, we confirmed the CC-Score could be able to effectively distinguish between DNA replication, caryokinesis and cytokinesis these three kinds of the different states. Ultimately using this score we assessed the changes of the capacity of myocardial regeneration in postnatal mice.The main research results are as follows:First part:the establishment of cytokinesis gene score (CC-Score) systemAccording to the the gene annotation of NCBI and reported literature, we have selected10cytokinesis related genes including:ANLN, AURKB, Cenpa, KIF4, Kif23, PRC1, RhoA, Spin1, TACC2and Cdc42, which are used to establish the multi-gene scoring system of evaluation of the proliferation of cardiomyocyte. H9C2myocardial cells were, respectively, treated with GHRH and FGF-land DMSO and IFN-alpha, as well as as the angiotensin Ⅱ and endothelin. Meantime, useing the traditional approaches of proliferation we compared with the CC-Score. We found that in the proliferation promotion group, the expression of these genes were up-regulated (P<0.01), and down-regulated in the proliferation inhibition group (P<0.01), not difference in the hypertrophy group (P>0.05). By matching with the traditional method analysis, we found the values of CC-Score in GHRH and FGF-1groups were positive (P<0.01), wheras in DMSO and IFN-alpha groups values of CC-Score is negative (P<0.01). The negative value indicated the cell proliferation ability was suppressed and postive value indicated the cell proliferation ability was prompted. In the cell hypertrophy group there was no significant difference (P>0.05). Description CC-Score could be able to effectively reflect the the cell proliferation condition and distinguish between proliferation and hypertrophy of cardiomyocyte. Second part:CC-Score could effectively distinguish between DNA replication, caryokinesis and cytokinesis of H9C2myocardial cellsIn order to further study the CC-Score which could be able to evaluate the different situation of the the cardiomyocyte proliferation. We take advantage of the colchicine, the the cytochalasin D and the GHRH, respectively, dealing with H9C2myocardial cells ti establish the three different cell models of the DNA replication and caryokinesis and cytokinesis. By calculating the the CC-Score, we found that the CC-Score in the cytokinesis group (GHRH) was obviously elevated (P<0.01), wheras in the the DNA replication group (colchicine) and the caryokinesis group (cytochalasin D) the CC-Score showed no significant change (P>0.05). Comparing with the traditional MTT method, BrdU method, ICC and flow cytometry method, the CC-Score could be able to the effectively distinguish between DNA replication, caryokinesis and cytokinesis of H9C2myocardial cells. This study further verified that the CC-Score could be able to more accurately reflect the true world of cardiomyocyte proliferation.Third Part:the application of CC-Score to assess the changes of regeneration capacity of cardiomyocyte in postnatal micePrevious studies reported after the birth of the1day the newborn mice possessed myocardial complete regeneration after injury. However, after7days of the abilitiy of myocardial complete regenerationin mice lost, which indicated that the1day newborn mice has a strong myocardial proliferating capacity,wheras lost this after7days. Nevertheless, the previous studies subjecting to the technical limitations of proliferate evaluation method, often could’t accurate assess the ability of proliferation. Therefore, we take advantage of CC-Score to assess the changes of regeneration capacity of cardiomyocyte in postnatal mice form1day to7days. We found that CC-Score gradually declined from1to7days after the birth of mice and didt’t appear peak of proliferation in daq4reported in the previous literature, which demonstrated the linearly weakness of cardiac regeneration. The results suggested that CC-Score as a simple and easy evaluation method of cardiomyocyte proliferation owned the incomparable advantage comparing with traditional methods, which was able to truly and effectively evaluate the cardiomyocyte proliferation, could provide a reliable method for the study of heart regenerative medicine. |
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