| Objective:Obstructive sleep apnea-hypopnea syndrome(OSAHS) is the main clinical characteristics of sleep state of intermittent hypoxia, and result in many cardiova-scular diseases, including atherosclerosis, the independent risk factors.In the clinical observation found that patients with OSAHS,whose RAGE content is significantly higher than normal peripheral blood mononuclear cells, continuous positive airway pressure treatment can reduce the patient's RAGE expression of peripheral blood mononuclear cells. The mechanism links OSAHS to arteriosclerosis is unclear.The present study suggests that RAGE and its ligand system play an important role in the development of atherosclerosis.To investigate RAGE and its ligand in the role of OSAHS caused atherosclerosis, this study intends to use the THP- 1(human leukemia mononuclear cells) in vitro cell model of simulated hypoxia bring about sclerosis of arterial congee appearance, use lentivirus- mediated silence of gene RAGE of THP 1 cells, detect the different of monocyte chemotactic cells, macrophages transformation and foam cell formation in THP-1 by inter intermittent hypoxia.Methods:1.The relationship between RAGE, NF-k B p65 and AGEs expression of THP-1 cells after hypoxia for 0h,12 h,24h,48 h was detected.2 RAGE cells were transfected into THP-1 cells by targeting shRNA to construct the RAGE, and the stable cell lines with the highest silencing efficiency were screened for subsequent experiments. Cells were divided into four groups: Control group:RAGE(+/+) + normal culture; experimental control group A:RAGE(-/-) and normal culture; experimental control group B:RAGE(+/+) +IH culture. D: the experimental group: RAGE(-/-) +IH culture.3.THP-1 cells and RAGE(-/-)THP-1 cells were divided into four groups. After 24 hours, the expression of MCP-1, CD14, CD68 and CCR2 were compared between the four experimental groups.4. THP-1 cells and RAGE(-/-) THP-1 cells treated with PMA(100ng / ml) induced differentiation into macrophages, induced differentiation proceeds in THP-1 Cell source of macrophages in the experimental groups and culture medium with final concentration of 50 ug / ml ox-LDL induced macrophage foam cells and 24 hours after oil red O staining evaluation between the four groups of foam cell formation differences.result:1.The expression of RAGE,AGEs,NF-kB were increased with the time of IH. RAGE activation peaks 24h(RAGE/GAPDH 1.043±0.10) after IH exposure which can sustainable to IH 48 h(RAGE/GAPDH 1.289±0.18,P<0.05) test by WB. NF-k B activation peaks 12h(NF-kB p65/GAPDH 0.30±0.04,P<0.05) after IH exposure which can sustainable to IH 24 h(NF-kB p65/GAPDH 0.32±0.03,P<0.05) test by WB.The expression of AGEs in supernatant increased with the increase of hypoxia time in 48h(P < 0.05).2. Monocyte recruitment was determined by measuring the expression of MCP-1 and CCR2, which compared with the blank control group, the expression of IH group were increased(P < 0.05);while compared with IH group, the expression of MCP-1,CCR2 were decreased(P < 0.05).3. Macrophage differentiation markers CD14, CD68, and blank compared to the control group, training group THP-1 monocytes with IH, CD68 expression was increased(P < 0.05); IH culture and culture group THP-1 monocytes RAGE(-/-) +IH, CD68 expression decreased(P < 0.05).4, IH can promote the formation of macrophage foam cells, RAGE gene can promote IH induced macrophage foam.Conclusion:1The expression of RAGE, NF-kB p65 and AGEs in THP-1 cells increased with the increase of IH time in a certain time range.2 RAGE can promote THP-1 cell aggregation, macrophage differentiation and foam cell formation that induced by IH. |
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