本文選題：HDL + ABCG1　； 參考：《南華大學(xué)》2015年碩士論文

【摘要】：目的:膽固醇是細(xì)胞膜的主要組成部分,在破骨細(xì)胞的形成及生存中發(fā)揮重要作用。破骨細(xì)胞本身幾乎不合成膽固醇,因此細(xì)胞內(nèi)膽固醇容易失衡而影響破骨細(xì)胞形成或生存。研究發(fā)現(xiàn)HDL水平升高可促進(jìn)破骨細(xì)胞膽固醇流出并促進(jìn)其凋亡,但其促進(jìn)膽固醇流出的機(jī)制及對破骨細(xì)胞形成的影響尚不清楚。本研究選取RAW264.7單核/巨噬細(xì)胞作為破骨前體細(xì)胞,以RANKL及M-CSF誘導(dǎo)其形成的破骨細(xì)胞為研究對象,探討HDL促進(jìn)破骨細(xì)胞膽固醇流出的機(jī)制及對其形成和生存的影響。方法:用含HDL的培養(yǎng)基培養(yǎng)RAW264.7細(xì)胞,加入RANKL及M-CSF刺激其分化形成破骨細(xì)胞,在不同的時(shí)間觀察TRAP陽性的多核細(xì)胞的數(shù)目、大小和核固縮情況;用含不同濃度的HDL的培養(yǎng)基培養(yǎng)RAW264.7細(xì)胞,加入RANKL及M-CSF刺激其分化形成破骨細(xì)胞,液體閃爍計(jì)數(shù)儀檢測其膽固醇流出情況;用含HDL的培養(yǎng)基培養(yǎng)RAW264.7細(xì)胞不同時(shí)間,加入RANKL及M-CSF刺激其分化形成破骨細(xì)胞,液體閃爍計(jì)數(shù)儀檢測其膽固醇流出情況。HDL3、HDL2、Aop A1處理破骨細(xì)胞,觀察其膽固醇流出情況及TRAP陽性的多核細(xì)胞的數(shù)目、大小和核固縮情況。用含HDL的培養(yǎng)基培養(yǎng)RAW264.7細(xì)胞3天,加入RANKL及M-CSF刺激其分化形成破骨細(xì)胞,熒光定量PCR檢測破骨細(xì)胞ABCG1、SR-B1 m RNA的表達(dá),Western blot檢測ABCG1、SR-B1、Cav1蛋白表達(dá)。si RNA沉默ABCG1表達(dá),觀察其膽固醇流出情況及TRAP+的多核細(xì)胞的數(shù)目。結(jié)果:1)HDL處理細(xì)胞后,形成的破骨細(xì)胞最大直徑減小,融合指數(shù)減小,核固縮的破骨細(xì)胞增多;2)HDL促進(jìn)破骨細(xì)胞膽固醇流出,且呈濃度及時(shí)間依賴性,細(xì)胞內(nèi)游離膽固醇明顯減少;3)不同的HDL亞型促進(jìn)破骨細(xì)胞膽固醇流出的能力不同,以HDL3能力最強(qiáng);4)HDL處理使破骨細(xì)胞表達(dá)ABCG1增多而SR-B1減少;5)ABCG1 si RNA處理使HDL促進(jìn)破骨細(xì)胞膽固醇流出的能力下降,破骨細(xì)胞形成恢復(fù),凋亡減少;6)HDL處理使破骨細(xì)胞磷脂流出增多,Cav1表達(dá)減少。結(jié)論:HDL通過上調(diào)ABCG1的表達(dá)促進(jìn)破骨細(xì)胞膽固醇流出,破壞破骨細(xì)胞內(nèi)膽固醇平衡從而影響破骨細(xì)胞形成并促進(jìn)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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R580

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