| Objective: Myocardial infarction (heart attack) is greatly harmful to human health. In myocardial infarction, the loss of functional myocardial cells and the formation of scar tissue occurs, which makes the living cardiomyocytes become less and less. The loss of functional myocardial cells and the following ventricle reconstruction are the main reasons of heart failure, even death of the patients. Myocardial cells neither be regenerated nor differentiated once the damage is done. Therefore, clinical medicine treatment, interventional therapy and surgery usually do not produce satisfactory outcome. Owing to the complexity of the surgical procedure, rare donors and expensive expenditure, heart transplantation can not be generalized clinically. The key point in treating myocardial infarction is how to increase the number of functional cardiomyocytes in the infarcted area. Currently, cell transplantation is becoming a new trend to treat heard attacks. So the major challenge is to find suitable seed cells, which can substitute for dead cells to prevent heart failure and improve heart function.The bone morphogenetic protein (BMP) belong to the transforming growth factor-βfamily. There are at least 40 members in BMP family. Up to now, more than 15 of them have been identified. Based on the homology of functional domain, it is divided into several BMP superfamilies, such as BMP 2 and 4 superfamily, BMP 5, 6, 7 and 8 superfamily, growth and differentiation factor (GDF-5), GDF-6(BMP-13) and GDF-7(BMP-12) super family, and BMP-3 and GDF-10(BMP-3b) super family, among which BMP-2 and BMP-4 super family are highly bioactive. Researches show that BMP-2 and BMP-4 are involved in regulating cardiogenesis and myocardial differentiation. Researches show that the BMP2 expression which can be detected in the heart develoment area were improved at early stage in chick embryo. Some evidence show that exogenous BMP2 protein can induce the differentiation of marrow stroma cells to cardiomyocyte. Evidence also show that BMP signal is the upper stream effector molecule of some of heart's early transcription factor( such as GATA-4 and NKx2.5). These researches show that BMP2 plays an important role in heart development and myocardial differentiation at the early stage of embryo. However, the mechanism of BMP2 in myocardial differentiation, and especially the interaction between BMP2 and myocardial transcription factors such as NKx2.5 and GATA-4 is not unclear after a thorough literature review. Moreover, whether the exogenous expression of BMP2 can induce the differentiation of P19 cell to cardiomyocytes still remains unknow. As such, our experiment chose gene transfection technique to transfect BMP2 into P19 cell and observe the role of BMP2 in myocardial differentiation.P19 embryonal carcinoma cells (P19ECCs) are amplified rapidly in vitro and can continue to differentiate after many generations. P19 cells can be differentiated into three different types of cells, which contains endoderm, mesoderm and ectoderm. Under different extraorgan induction conditions, it can be induced into many kinds of cells including cardiomyocytes, skeletal muscle cells and neuron. For this reason, it is a relatively ideal instrumental cell that can be used to study cell differentiation. If transplanted after extraorgan induction towards myocardial differentiation, it may become a relatively ideal seed cell to replace the necrotic cardiomyocytes. Experiments show that during the differentiation of P19 cells to cardiomyocytes, the related gene expression about cell growth and electrophysiological feature mainly imitate the myocardial growth of the normal mouse. So P19 cells are considered as a good model in vitro to study heart development and myocardial differentiation. However, during the differentiation of P19 cells to cardiomyocytes, the formation of cell aggregates and the inductor are two important and necessary processes. Up to now, it has not been clearly proved that P19 cells can differentiate to cardiomyocytes in monolayer culture. Without the formation of cell aggregates and the inductor, P19 cells can't differentiate to cardiomyocytes.Therefore, there are two main project in the study. First is designed to transfect pEGFP-N2-BMP2 into P19 cells, and observed the differentiation of P19 cells to cardiomyocytes without inductor. Second is to study the differentiation of P19 cells which stable expressing of BMP2 gene in monolayer culture with DMSO induction. We also would be explore a better way of P19 cells differentiate into cardiomyocytes. It would be better, if we can search for a suitable seed cell for cell transplantation in the treatment of myocardial infarction.Methods:1 The construction and identification of eukaryotic expression plasmid pEGFP-N2-BMP2We used plasmid pEFSA- BMP2 as a template and transformed it into competent E. coli DH5αby the conventional CaCl2 method. The BMP2 gene sequence was amplified with PCR. Up stream primer: 5'-GCCAGATCTAAAGGTCGACCATGGT-3', the introduction of endonuclease sites BglⅡ; down stream primer: 5'-TTAGAATTCGCGACACCCACAACC-3', the introduction of endonuclease sites EcoRI. After the reaction completed, PCR products were subjected to agarose gel electrophoresis and target fragment was recovered from the agarose gel. The Coding sequence of BMP2 and vector pEGFP-N2 were digested by BglⅡand EcoRI overnight and recovered respectively. Restriction enzymesBglⅡand EcoRI were used to purify BMP2 DNA and pEGFP-N2 vector. BMP2 DNA was amplified by PCR and both the BMP2 and pEGFP-N2 will have coupled reaction through T4 DNA ligase. The ligation product was transformed into competent E. coli DH5α.cells and coated to the solid LB medium plate containing kanamycin and cultured inverted for 16-24h at 37℃. Single positive colony is chosen to extract recombinant plasmid and detect if there is target fragment through PCR, then by BamH I and HindⅢdouble digestion identification. At the same time, the recombinant plasmid pEGFP-N2-BMP2 was sent to Shanghai Sangon Co. Ltd. for sequencing identification.2 Cardiomyocyte differentiation from P19 cells with exogenous expression BMP2 gene can be induced by cell aggregate formation.The experimental subjects were divided into two groups- experimental group and control group. In the experimental group were P19 cells transfected with pEGFP-N2-BMP2. Before transfection, preliminary experiment was used to determine the suitable cell seeding density, the best transfection system, and the appropriate G418 selection concentration. Using the cationic liposome reagent, Lipofectamine 2000, the plasmid pEGFP-N2-BMP2 were transfected into P19 cells. After 14th days of stable selection by G418, the survival cells were suspension cultured to form aggregates for 4th days and the aggregates were transferred to the Petri dish for adherent culture. The cells were collected at 4th day, 8th day, 12th day and 16th day after adherent culture. The expression of cTnT andα-cardiac actin was detected with immunocytochemical staining method. RT-PCR was used to detect the expression of GATA-4 and NKx2.5 genes. Western blot was used to detect the expression ofα-cardiac actin and cTnT proteins. In the experimental group at 16th day after adherent culture, the ultrastructural changes of the cells were observed. In the control group, P19 cells were aggregated and cultured directly.The detection index and sampling time were same as that of experimental group.3 Dimethylsulfoxide induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2 to differentiate toward cardiomyocyteThere were experimental group and control group in the experiment. In the experimental group, DMSO induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2. In the control group, DMSO induced monolayer cultured P19 cells. 1% DMSO was used to induce the cells both in experimental group and control group. Cells were collected at 4th day, 8th day, 12th day and 16th day after induction in two groups. The expression of cTnT andα-cardiac actin was detected with immunocytochemical staining method. RT-PCR was used to detect the expression of GATA-4 and NKx2.5 genes. Western blot was used to detect the expression ofα-cardiac actin and cTnT ultrastructural changes of the cells were observed. In the control group, P19 cells were induced culture with 1% DMSO directly.The detection index and sampling time of control group were same as that of experimental group.Results:1 Construction and identification of pEGFP-N2-BMP2 eukaryotic expression plasmidModeled by pEFSA- BMP2 eukaryotic expression plasmid, a gene fragment of about 1.2 kb was obtained, whose size was as the same as the human BMP2 gene encoding frame (1191bp). Vector pEGFP-N2 and BMP2 gene were digested with restriction enzyme BglⅡand EcoR I respectively. About 1.2 kb BMP2 gene fragment and 4.7kb pEGFP-N2 vector with BglⅡand EcoR I restriction enzyme sites were obtained then they were retrieved and connected. A gene fragment of about 1.2kb was obtained by PCR from the recombinant plamid after transformation and connection. Two gene fragments of about 1.2kb and 4.7 kb were obtained from the recombinant plasmid after double enzyme digesting with BamH I and HindⅢ. With CMV promoter primer for sequencing, analyzed by BLAST after proofread, the sequence result showed that the nucleotide sequence was agreed with the gene encoding sequence of BMP2 mRNA(NM-001200) in GeneBank and the endonuclease sites were complete. There was an intact Kozak sequence in the gene start area. Terminator of gene was mutated and the reading frame was correctly identified. This means BMP2 gene encoding frame has been correctly inserted in MCS of pEGFP-N2 vector. It is confirmed that recombinant plasmid pEGFP-N2-BMP2 was constructed successfully in this experiment. 2 Cardiomyocyte differentiation from P19 cells with exogenous expression BMP2 gene can be induced by cell aggregate formation.The experiment confirmed that the stable selection concentration of G418 was 600μg/ml and the appropriate cell seeding density for transfection was 2×105/ml. The best transfection system was the plasmid DNA(μg):Lipofectamine2000 (μl)=1:2.5. In the experimental group, at the 24th hour after transfection with plasmid pEGFP-N2-BMP2, some cells expressed the green fluorescence. Most cells expressed green fluorescence after being selected with 600μg/ml G418 two weeks later, which were P19 cells with exogenous expression BMP2 gene. Cells in experimental group and control group were collected and cultured for 4th days to form aggregates. After adhesion of the aggregates, some cells grew out and formed outgrowth around the aggregates. In the outgrowth, the cell body grew bigger and longer, and evections reach out and connected with each other. Immunocytochemical staining results showed that, in experimental group, at 4thday two kinds of antibody,α-cardiac actin and cTnT, were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day, two kinds of antibody were all expressed, and the expression was gradually increased. Brown positive reaction products were in cytoplasm. With time going by, there was obviously brown filament-like structure in the cytoplasm of the cells at 12th day and 16th day. The statistical results of positive cell absorbance value showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference(P<0.01)between 8th day, 12th day and 16th day in experimental group. Positive expression of the two antibodies was not detected in control group. RT-PCR analysis showed that, in experimental group, GATA-4 and Nkx2.5 expressed at 4th day after adherent culture, but it is deficient in quantity. At 8th day, 12th day and 16th day, the expression of GATA-4 and Nkx2.5 was gradually increased. The statistical result showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference(P<0.01) between 8th day, 12th day and 16th day in experimental group. No positive expressions of the two kinds of genes was detected in control group. Western Blot result showed that, in experimental group, at 4thdayα-cardiac actin and cTnT, were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day they were all expressed, and the expression was gradually increased. The statistical result showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two proteins was not detected in control group. The observation of ultrastructure showed that after aggregation and adherent culture for 16th days, some cells became elongated cylindrical and there appeared cell junction between the adjacent cells in experimental group. Some microfilament-like structure appeared beside the cell junction. There was some dense myofilament-like structure arranged in parallel way in cytoplasm of some cells,but sarcomere-like structure was not founed. no differentiated cell was found in control group.3 Dimethylsulfoxide induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2 to differentiate toward cardiomyocyteCells both of experimental group and control group induced monolayer culture with 1% DMSO and grew slowly compared with the P19 cells without inducing. And cells accumulated in a state similar to adherent aggregates-like structure as increased in number. The cells in the center of the aggregates were small and dense, while the periphery cells became longer, crawling and radiating out in all directions with cell body elongated to be fusiformis and stretched processes. Beating cells were not observed in either of the two groups during culture. Immunocytochemical staining result showed that, in experimental group, at 4thday after being induced by 1% DMSOα-cardiac actin and cTnT were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day two kinds of antibody were all expressed, and the expression was gradually increased. Brown positive reaction products were in cytoplasm. With time going by, there was obviously brown filament-like structure in the cytoplasm of the cells at 12th day and 16th day. The statistical result of positive cell absorbance value showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two kinds of antibody was not detected in control group. RT-PCR analysis showed that, in experimental group, GATA-4 and Nkx2.5 expressed at 4th day after induced by DMSO, but it is deficient in quantity. At 8th day, 12th day and 16th day the expression of two genes was gradually increased. The statistical result showed that the expression of GATA-4 was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01 or P<0.05 ) between 8th day, 12th day and 16th day in experimental group. the expression of Nkx2.5 was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and in experimental group there was also significant difference (P<0.01 ) between 8th day, 12th day and 16th day, but there was no significant difference(P>0.05)between 12th day and 16th day. No positive expressions of the two kinds of genes was detected in control group. Western Blot result showed that, in experimental group, at 4thdayα-cardiac actin and cTnT were not expressed. At 8th day they were all expressed in a little amount. At 12th day and 16th day they were all expressed, and the expression was gradually increased. The statistical result showed that the expression ofα-cardiac actin was distinct difference (P<0.01 or P<0.05) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. the expression of cTnT was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, but there was no significant difference (P>0.05 ) between 8th day and control group. And there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two proteins was not detected in control group. The observation of ultrastructure showed that after induced by DMSO culture for 16th days, some cells became elongated cylindrical and there appeared cell junction between the adjacent cells in experimental group. Some microfilament-like structure appeared beside the cell junction. There was some dense myofilament-like structure arranged in parallel way in cytoplasm of some cells, but sarcomere-like structure was not founed. no differentiated cell was found in control group.Conclusions:1 The eukaryotic expression plasmid pEGFP-N2-BMP2 was successfully constructed, which would lay a foundation for the further studies on the role of BMP2 in myocardial differentiation and heart development.2 Eukaryon expression plasmid pEGFP-N2-BMP2 can be expressed in P19 cells and can be used for the transfection and stable selection of eukaryotic cells.3 Without inducing agents, exogenous expression of BMP2 gene induced P19 cells to differentiate towards cardiomyocytes only with aggregates formation. P19 cells transfected with pEGFP-N2-BMP2 induced the expression of cardiac-specific markers at the level of gene and protein which increased gradually with prolongation of culture time. It shows that exogenous expression of BMP2 gene can induce myocardial differentiation from P19 cells. This study would explore a reasonable induction way for myocardial differentiation from P19 cells .4 Without inducing agents, exogenous expression of BMP2 gene induced P19 cells to differentiate towards cardiomyocytes only with aggregates formation. This shows that BMP2 gene can promote myocardial differentiation from P19 cells and indicates that BMP signal path plays a very important role in the process of myocardial differentiation from P19 cells.5 With monolayer culture and induced by DMSO, P19 cells which transfected with pEGFP-N2-BMP2 can differentiate towards cardiomyo- cytes, which show the expression of cardiac-specific markers at the level of gene and protein. It suggests that BMP2 gene can promote P19 cells to differentiate towards cadiocyte under monolayer culture condition. It is proved further that BMP2 signal path plays a very important role in the process of myocardial differentiation from P19 cells.
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