HDL上调破骨细胞ABCG1表达从而影响破骨细胞生成并促进其凋亡

发布时间：2018-07-11 14:56

本文选题：HDL + ABCG1　；参考：《南华大学》2015年硕士论文

【摘要】：目的:胆固醇是细胞膜的主要组成部分,在破骨细胞的形成及生存中发挥重要作用。破骨细胞本身几乎不合成胆固醇,因此细胞内胆固醇容易失衡而影响破骨细胞形成或生存。研究发现HDL水平升高可促进破骨细胞胆固醇流出并促进其凋亡,但其促进胆固醇流出的机制及对破骨细胞形成的影响尚不清楚。本研究选取RAW264.7单核/巨噬细胞作为破骨前体细胞,以RANKL及M-CSF诱导其形成的破骨细胞为研究对象,探讨HDL促进破骨细胞胆固醇流出的机制及对其形成和生存的影响。方法:用含HDL的培养基培养RAW264.7细胞,加入RANKL及M-CSF刺激其分化形成破骨细胞,在不同的时间观察TRAP阳性的多核细胞的数目、大小和核固缩情况;用含不同浓度的HDL的培养基培养RAW264.7细胞,加入RANKL及M-CSF刺激其分化形成破骨细胞,液体闪烁计数仪检测其胆固醇流出情况;用含HDL的培养基培养RAW264.7细胞不同时间,加入RANKL及M-CSF刺激其分化形成破骨细胞,液体闪烁计数仪检测其胆固醇流出情况。HDL3、HDL2、Aop A1处理破骨细胞,观察其胆固醇流出情况及TRAP阳性的多核细胞的数目、大小和核固缩情况。用含HDL的培养基培养RAW264.7细胞3天,加入RANKL及M-CSF刺激其分化形成破骨细胞,荧光定量PCR检测破骨细胞ABCG1、SR-B1 m RNA的表达,Western blot检测ABCG1、SR-B1、Cav1蛋白表达。si RNA沉默ABCG1表达,观察其胆固醇流出情况及TRAP+的多核细胞的数目。结果:1)HDL处理细胞后,形成的破骨细胞最大直径减小,融合指数减小,核固缩的破骨细胞增多;2)HDL促进破骨细胞胆固醇流出,且呈浓度及时间依赖性,细胞内游离胆固醇明显减少;3)不同的HDL亚型促进破骨细胞胆固醇流出的能力不同,以HDL3能力最强;4)HDL处理使破骨细胞表达ABCG1增多而SR-B1减少;5)ABCG1 si RNA处理使HDL促进破骨细胞胆固醇流出的能力下降,破骨细胞形成恢复,凋亡减少;6)HDL处理使破骨细胞磷脂流出增多,Cav1表达减少。结论:HDL通过上调ABCG1的表达促进破骨细胞胆固醇流出,破坏破骨细胞内胆固醇平衡从而影响破骨细胞形成并促进其凋亡。
[Abstract]:Objective: cholesterol is a major component of cell membrane and plays an important role in the formation and survival of osteoclasts. The osteoclasts themselves almost do not synthesize cholesterol, so the cholesterol in the cells is easily out of balance and affects the formation or survival of osteoclasts. It was found that the increase of HDL level could promote cholesterol efflux and apoptosis of osteoclasts, but its mechanism of promoting cholesterol efflux and its effect on osteoclast formation were unclear. In this study, RAW264.7 mononuclear / macrophages were selected as osteoclasts, and RANKL and M-CSF induced osteoclasts were used to investigate the mechanism of HDL promoting cholesterol efflux of osteoclasts and their effects on the formation and survival of osteoclasts. Methods: RAW264.7 cells were cultured in HDL medium and stimulated by RANKL and M-CSF to form osteoclasts. The number, size and pyknosis of trap positive multinucleated cells were observed at different time points. RAW264.7 cells were cultured in a medium containing different concentrations of HDL. RANKL and M-CSF were added to stimulate the differentiation of RAW264.7 cells to form osteoclasts. The cholesterol efflux of RAW264.7 cells was detected by liquid scintillation counter, and RAW264.7 cells were cultured in HDL medium for different time. RANKL and M-CSF were added to stimulate the osteoclasts to differentiate into osteoclasts. The cholesterol efflux. HDL3 and HDL2Aop A1 were detected by liquid scintillation counter. The cholesterol efflux and the number, size and pyknosis of trap positive multinucleated cells were observed. RAW264.7 cells were cultured in HDL-containing medium for 3 days. RANKL and M-CSF were added to stimulate the osteoclasts to differentiate into osteoclasts. The expression of SR-B1 mRNA in osteoclasts was detected by fluorescence quantitative polymerase chain reaction (FQ-PCR). Western blot was used to detect the protein expression of RAW264.7 cells. Si RNA silenced ABCG1 expression. Cholesterol efflux and the number of polymorphonuclear cells in trap were observed. Results the maximum diameter and fusion index of osteoclasts were decreased, and the number of osteoclasts increased after treatment with HDL. The HDL promoted cholesterol outflow of osteoclasts in a concentration and time dependent manner. The ability of different HDL subtypes to promote cholesterol efflux of osteoclasts was different. HDL3 (4) HDL treatment increased the expression of ABCG1 in osteoclasts, while SR-B1 decreased the expression of ABCG1. 5) ABCG1si RNA treatment decreased the ability of HDL to promote cholesterol efflux of osteoclasts, and the formation of osteoclasts recovered. Apoptosis decreased 6) HDL treatment increased phospholipid efflux in osteoclasts and decreased the expression of Cav1. Conclusion by up-regulating the expression of ABCG1, VHDL can promote cholesterol efflux of osteoclasts, destroy the cholesterol balance in osteoclasts, and thus affect the formation of osteoclasts and promote the apoptosis of osteoclasts.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580

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