Studies On The Effect Of Overexpression Of Mpr5 On Cardiac Differention Of P19 Cells And Its Establishment Of Transgenic Zebrafish Strains

At present,heart disease has become the disease with the highest mortality rate.Genetic factors play an extremely important role in the occurrence of heart disease.Mutations in related genes or changes in protein expression may lead to the imbalance of the entire regulatory network and thus heart disease.Therefore,the study of cardiomyocyte formation and gene function during heart development will provide an important reference for its treatment.Previous studies in our laboratory revealed that mpr5（maternal protein regulator 5）is one of the candidate genes for heart development.The zebrafish mpr5 knockout strain has been established using CRISPR/Cas9 technology.The zebrafish knockout with mpr5 showed abnormalities in embryonic heart development and heart function,suggesting that this gene can regulate heart development.Therefore,studying this gene will be helpful to explain the pathogenesis of heart disease.P19 cell is a cell line isolated from mouse malignant teratoma and can be differentiated into cardiomyocytes under the induction of DMSO.At present,the cell line has been widely used as an in vitro model for studying the development of cardiomyocytes.In order to verify the important role of the mpr5,in this paper the P19 myocardial differentiation model was used to conduct a preliminary study on the function of this gene during myocardial differentiation.A large number of embryoid bodies gradually appeared in P19 cells after DMSO induction.Compared with the control group,the expression of Nkx2.5 in the induced group showed an upward trend at different time nodes detected by RT-PCR and western blot techniques.Cellular immunofluorescence showed that in the induced group MF20（myosin heavy chain protein）signal was significantly enhanced,indicating that the myocardial differentiation model was constructed.Next,in this paper the role of the Mpr5 in the formation and differentiation of cardiomyocytes was explored.The results by RT-PCR analysis showed that the expression of Gata4,a myocardial specific marker factor,was significantly increased in the Mpr5 gene overexpression group compared with that of control.The results of immunofluorescence on the cells on day 8 and 12 showed that overexpression of Mpr5 gene significantly increased the MF20 signal with time.At the same time,morphologically irregular polygonal cells similar to cardiomyocytes gradually appeared.Western blot detection showed that,compared with the control group,the protein expression levels of myocardial differentiation marker GATA4 and myocardial specific protein CTNT in the Mpr5 overexpression group were significantly increased on days 6,8,10,and 12.These results indicate that the Mpr5 gene promotes the differentiation of P19 cells into cardiomyocytes.In vivo gene overexpression research is an important research tool to explore gene function.Therefore,in this paper a mpr5-GFP plasmid was further used to construct zebrafish strain with overexpressed fluorescent protein.The results of F₁ juvenile fish screening showed that a zebrafish mpr5 over-expression fluorescent strain was successfully constructed.Based on in situ hybridization and F₁ fluorescence tracking results,the mpr5 gene was expressed in the developing head,eyes,ear capsule,back,heart,yolk sac,and yolk extensions.Phenotype analysis showed that there were abnormal developing hearts in F₁ zebrafish during embryonic development at 48 hpf（hours post fertilization）,such as enlarged peripheral pericardial cavity and blood accumulation.These results suggest that overexpression of the mpr5 gene may lead to abnormal heart development.In summary,in this paper the P19 cell model was first usedto initially study the effect of Mpr5 on myocardial differentiation,and further the zebrafish model was used to explore the role of overexpressing the mpr5 gene in cardiac development.Using RT-PCR,cellular immunofluorescence,and western blot techniques,it was initially demonstrated that overexpression of Mpr5 increasedthe formation of cardiomyocytes and led to the abnormal development of the heart.These findings laid the foundation for further exploration of the regulatory mechanism of mpr5 in heart development.