The Early Studies In Vitro Of The Role Of RAGE-its Ligands System In The Mechanisms For The Effect Of Chronic Intermittent Hypoxia On The Development Of Atherosclerosis

Objective:Obstructive sleep apnea-hypopnea syndrome(OSAHS) is one of the significant pathogenic factors for atherosclerosis(AS), but the definite mechanism is still unclear.Chronic intermittent hypoxia(IH) is one of the typical features of OSAHS, may also be a key factor for OSAHS to cause AS. The receptor for advanced glycation end products(RAGE)and its ligands system may play an important role in AS, and IH can also activate RAGE-its ligands system. Therefore, this project will explore the mechanisms of chronic IH on the development of AS, based on its effect on RAGE-its ligands system. Firstly, using RNAi to suppress RAGE In vitro, we will detect the effect of IH on THP-1 macrophage transformation, foam cell formation and cholesterol transport protein, etc.we aim at finishing the following two tasks: first,At the early stage of the experiment,explore the optimum condition for PMA inducing differentiation of THP-1 for the subsequent observation of AS pathogenesis.second,buid and select the best sh RNA/RAGE lentiviral vector in order to reach the effect of gene RAGE silence.It is necessary for subsequent experiment.Methods:Culture THP-1 in vitro;PMA was used to induce macrophage differentiation in THP-1 and the extent of differentiation have been identified by Flow cytometry and Morphological observation; Construct sh RNA/RAGE lentiviral vectors;sh RNA/RAGE lentiviral vectors infect THP-1 cells, RTQ-PCR and Western blot identify the silence of RAGE;Results:1. The expression of Cell surface marker CD14 and CD68 of THP-1 cells untreated was 3.5% and 1.3%, and the group treated with PMA was 37.7% and46.8%,the difference was statistically siginificantly(P<0.01),the differentiation of THP-1 was successful.2. sh RNA/RAGE lentiviral vectors have been successfully constructed.RT-PCR showed that KD1、KD2 and KD3 inhibition of RAGE is not obvious,the inhibitionrate were47.8%、 30.2% and 29.4%;Western-blot showed that the protion inhibition rate of KD1 、KD2 and KD3 were 41.2%、14.9% and 11.3%; KD1、KD2 and KD3 group sh RNA/RAGE lentiviral Vectors can successfully infect THP-1 but failed to silence the gene RAGE.Conclusions:1.Macrophage differentiation in PMA-stimulated THP-1 cells was successful.2. sh RNA/RAGE lentiviral vectors have been successfully constructed, KD1、KD2 and KD3 group sh RNA/RAGE lentiviral Vectors can successfully infect THP-1but failed to silence the gene RAGE,the method to silence RAGE needs to be further explored.