Using CRISPR/Cas9 Technology To Investigate The Role Of Nrf2 In Pulmonary Vascular Cell Senescence

Background:The incidence of chronic obstructive pulmonary disease(COPD)is caused by active or passive smoking,and pulmonary vascular remodeling often causes pulmonary hypertension,leading to ventricular remodeling leading to pulmonary heart disease.The nuclear transcription factor E2-related factor 2(Nrf2)involved oxidative stress pathway Nrf2/ARE(antioxidant response element).Nrf2/ARE is a classic defensive Transduction pathway.In recent years,it has been found that the expression of Nrf2 is significantly reduced in senescent cells,and senescent cells can promote the proliferation of adjacent cells through a paracrine pathway.Objective:To explore the role and mechanism of Nrf2 in pulmonary vascular cell senescence.Methods:1.The Nrf2 knockout lentivirus was constructed using the CRSPR/Cas9 technology.2.Lentivirus infection of HPAECs and HPASMCs and Western Blot experiments to verify the knockout effect of Nrf2,selected monoclonal stable passage for subsequent experiments.3.The expression of Nrf2 and its pathway-related proteins(HO-1,NQO1)and senescence-associated proteins(P16,P21,P53)in different cells were detected by Western Blot.4.Different cell senescence levels were measured by ?-glucosidase staining and cell cycle assays and compared.5.The effects of senescence cell secretion on normal cell proliferation and migration were examined by cck-8 cell proliferation assay and migration assay.Results:1.Western Blot experiments showed that the expression of downstream proteins HO-1 and NQO1 decreased and the expression of P16,P21 and P53 increased after intracellular Nrf2 gene knockout.2.The aging-related experiments showed that the cell senescence after Nrf2 knockout was more obvious,and the ?-glycosidase senescence-stained blue-stained cells increased,and the cell cycle arrest was obvious.3.The secretion of HPAECs senescent cells stimulates the proliferation and migration of normal HPASMCs,and the secretion of HPASMCs senescent cells stimulates the proliferation and migration of normal HPAECs.Conclusion:1.The CRISPR/Cas9 gene editing technology knocks out the Nrf2 gene and establishes a stable Nrf2 knockout cell line.2.Knockout of Nrf2 enhances the expression of cellular senescence-associated proteins and promotes cell senescence.3.HPAECs and HPASMCs with SASP play an important role in cell proliferation and migration,and may be one of the mechanisms of pulmonary vascular remodeling.