CAJ | 학술논문

目的建立一种高效液相色谱(HPLC)测定血清-巨噬细胞间胆固醇净流动的方法,用以分析与动脉粥样硬化危险因素的关系.方法培养小鼠巨噬细胞J774,用含5％个体血清的培养基进行孵育8h,HPLC测定孵育前后细胞胆固醇,计算个体血清介导的细胞胆固醇净流动,建立血清-巨噬细胞间胆固醇净流动的测定方法.2010年10至12月征集75名北京医院健康志愿者,男38例、女37例,采集空腹静脉血,用所建方法测定胆固醇净流动；Spearman相关分析胆固醇净流动与血脂、脂蛋白、超敏C反应蛋白(hsCRP)等动脉粥样硬化危险因素的关系.结果发现个体血清孵育细胞时导致细胞胆固醇内流,细胞胆固醇随培养基中血清浓度和孵育时间的增加而增多.HPLC测定细胞胆固醇净流动的平均批内变异和总变异分别为0.81％～4.03％和4.25％～6.58％.75例健康志愿者胆固醇净流动均值为10.27 μg/mg蛋白,标准差2.69 μg/ mg蛋白,中位数10.12 μg/mg蛋白.胆固醇净流动与血清总胆固醇(TC)、三酰甘油(TG)、hsCRP,载脂蛋白(Apo)CⅡ、ApoCⅢ、小而密低密度脂蛋白胆固醇(LDLb-C)、高密度脂蛋白(HDL)胆固醇摩尔酯化速率(MERHDL) (P ＜0.001),HDL胆固醇分数酯化速率FERHDL、Preβ-HDL(P＜0.01)等呈显著正相关,但与HDL及其亚类胆固醇无相关性(P＞0.05).结论本研究建立了血清-巨噬细胞间胆固醇净流动的细胞分析体系和HPLC准确测定净流动的方法,为进一步研究血清-巨噬细胞间胆固醇净流动、HDL功能评价和心血管病危险分析提供了方法学基础.
목적 건립일충고효액상색보(HPLC)측정혈청-거서세포간담고순정류동적방법,용이분석여동맥죽양경화위험인소적관계.방법 배양소서거서세포J774,용함5％개체혈청적배양기진행부육8h,HPLC측정부육전후세포담고순,계산개체혈청개도적세포담고순정류동,건립혈청-거서세포간담고순정류동적측정방법.2010년10지12월정집75명북경의원건강지원자,남38례、녀37례,채집공복정맥혈,용소건방법측정담고순정류동；Spearman상관분석담고순정류동여혈지、지단백、초민C반응단백(hsCRP)등동맥죽양경화위험인소적관계.결과 발현개체혈청부육세포시도치세포담고순내류,세포담고순수배양기중혈청농도화부육시간적증가이증다.HPLC측정세포담고순정류동적평균비내변이화총변이분별위0.81％～4.03％화4.25％～6.58％.75례건강지원자담고순정류동균치위10.27 μg/mg단백,표준차2.69 μg/ mg단백,중위수10.12 μg/mg단백.담고순정류동여혈청총담고순(TC)、삼선감유(TG)、hsCRP,재지단백(Apo)CⅡ、ApoCⅢ、소이밀저밀도지단백담고순(LDLb-C)、고밀도지단백(HDL)담고순마이지화속솔(MERHDL) (P ＜0.001),HDL담고순분수지화속솔FERHDL、Preβ-HDL(P＜0.01)등정현저정상관,단여HDL급기아류담고순무상관성(P＞0.05).결론 본연구건립료혈청-거서세포간담고순정류동적세포분석체계화HPLC준학측정정류동적방법,위진일보연구혈청-거서세포간담고순정류동、HDL공능평개화심혈관병위험분석제공료방법학기출.
Objective To develop a safe and reliable method for the measurement of cholesterol net flux between macrophage cells and human serum by high performance liquid chromatography,and to investigate the relationship between cholesterol net flux and traditional risk factors of atherosclerosis.Methods J774 (murine cell line) cells were plated in growth medium containing 5％individual human serum and cell cholesterol before and after incubation were measured by HPLC.Serum induced cell cholesterol net influx was calculated.Fasting blood samples of 75 apparently healthy volunteers (38 males and 37 females) from Oct to Dec 2010 were collected.Cholesterol net flux was measured by the newly established method.Spearman correlation analysis was used to evaluate the relationship between cholesterol net flux and lipid,lipoprotein,high-sensitivity C-reactive protein (hsCRP) and other CVD risk factors.Results When J774 cells were incubated with individual serum,cell cholesterol levels were elevated with the increase of serum concentrations and incubation time.J774 cells were incubated with medium containing 5％ serum for 8 hours,the within assay and total CVs for the net movement of cellular cholesterol measured by HPLC were 0.81％-4.03％ and 4.25％-6.58％,respectively.The average cholesterol net efflux of 75 healthy subjects was 10.27 μg/mg,with a standard deviation of 2.69 μg/mg protein,and a median of 10.12 μg/mgprotein.Cholesterol net efflux was significantly positively correlated with total cholesterol (TC),triglycerides (TG),hsCRP,apolipoprotein (apo) C Ⅱ,apoC Ⅲ,small-dense low density lipoprotein cholesterol (LDLb-C),molar esterification rate of HDL cholesterol (MERHDL) (P ＜0.001),fractional esterification rate of HDL cholesterol (FERHDL) and Preβ-HDL(P ＜ 0.01).Cholesterol net efflux was not related with HDL as well as its subfractions.Conclusions A new method for the measurement of the net movement of cellular cholesterol between cells and serum by HPLC has been established.This method is safe,precise and reliable and may be used in the assessment of HDL function and cardiovascular diseases risks.