Experimental Study Of Lentiviral Vector Mediated ShRNA-PHC1 Gene Infection On Human Embryonic Stem Cells

Objective: In recent years,the incidence of dilated cardiomyopathy gradually increased,still lack of specific treatment,5-year survival rate is low.In the basic study of cardiomyocyte development,PHC1 gene plays an important role in cardiac morphological development and dilated cardiomyopathy.Lentivirus carrying sh RNAPHC1 plasmid infected human ESC in order to obtain ESCs knocked down PHC1 gene.To lay the foundation for the next step to clarify the pathogenesis and provide the theoretical basis for clinical diagnosis and treatment of dilated cardiomyopathy.Methods: Sh RNA-PHC1 plasmid including sh RNA1 and sh RNA2 two experimental groups that have puromycin resistance;Using lentiviral vector packaged the sh RNA1,sh RNA2 and control plasmids,then transfected 293 T cells.Collect and concentrate the virus that infected human ESC.After 3 days,puromycin was added into ESC.After 6 days,By extracting RNA reverse transcription and PCR;by way of 3% agarose gel electrophoresis,quantitative real-time PCR,western Blot and cell cycle analysis to detect whether successful to get ESCs knocked down PHC1 gene or not.Results:(1)The constructed sh RNA-PHC1 gene sequence can effectively silence the PHC1 gene in human ESC;(2)Silencing PHC1 gene can slow the cell proliferation rate and increase the number of cells in G0 phase,but decrease the number of cells in S phase(P <0.05).Conclusion: The decrease of PHC1 gene level could decrease the proliferation and division ability of human ESC.