肺组织细胞内胆固醇及代谢物水平检测方法初探

发布时间：2018-07-02 23:26

本文选题：高效液相色谱 + 气相色谱串联质谱　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:建立高效液相色谱(HPLC)和气相色谱串联质谱(GC-MS)的方法对肺癌细胞系A549细胞内胆固醇浓度和肺组织内27羟基胆固醇(27-OHC)水平有效进行定性定量分析,为以细胞内胆固醇含量作为指标鉴定组织细胞泡沫化程度以及探讨CYP27通路参与胆固醇代谢的后续研究奠定基础。方法:A549细胞超声裂解后,用KOH乙醇溶液和三氯乙酸分别除去细胞裂解液中的甘油三酯和蛋白,再以正己烷—异丙醇的二元提取剂将细胞内的胆固醇进行萃取。胆固醇含量采用HPLC法测定,色谱柱为ZORBAX SB-C18(4.6 mm×150 mm,5μm),流动相为乙腈—异丙醇(67:33),流速为1 mL·min~(-1),检测波长为206 nm。定量称取肺组织,施行高速匀浆法,获得匀浆液。按匀浆液:氯仿(3:5)加入氯仿萃取。有机相用水冲洗直至中性,氯仿在减压条件下移去。加入衍生化试剂0.1 ml三甲基氯硅烷/六甲基二硅氮烷/吡啶(1:2:3)60℃反应60 min,氮气吹干,氯仿复溶,进GC/MS分析。色谱柱为HP-5MS石英毛细管柱,电离方式(EI)正离子扫描,单次进样1.0μl,载气为氦气。利用气质联用方法分析肺组织内27-OHC的水平。结果:利用本实验所建立的HPLC检测方法,胆固醇标准品保留时间为8.2 min,5 ng·μL~(-1)—100 ng·μL~(-1)范围内呈良好的线性关系(R=0.9999);细胞内胆固醇和半数量胞内胆固醇出峰时间均与标准品溶液出峰时间一致,均在8.2 min处出峰。胞内胆固醇定量检测,进行方法学验证,其精密度、重复性、稳定性均良好,RSD均低于3.0%,平均加样回收率为94.89%,RSD为2.86%(n=6),检测限为2.665ng·μL~(-1)(S/N=3)定量限为10.197 ng·μL~(-1)(S/N=10)。利用气质联用方法检测,肺组织内27羟基胆固醇碎片总离子流在55.39 min出峰,衍生化后的碎片质荷比为m/z 456,m/z 546。可以将其定性测出。对其定量检测,进行方法学验证初步预测,所测单次样品的加样回收率部分数值在92%—105%范围之外。其精密度、重复性、稳定性等方法学验证指标有待进一步优化。结论:本文所建立HPLC检测的方法准确、精密度高、重复性好、准确度高、操作简便,回收率也在符合要求的范围内,经方法学验证,后续可用于A549细胞内的胆固醇水平的批量检测;为进阶探讨肺组织损伤与胆固醇代谢失调的病理进展关系奠定基础。气相色谱串联质谱检测肺组织内27-OHC;该法目前可以有效定性检测出组织内27-OHC。但还需要进一步进行方法学论证。
[Abstract]:Objective: to establish a high performance liquid chromatography (HPLC) and gas chromatography-tandem mass spectrometry (GC-MS) method for the qualitative and quantitative analysis of cholesterol concentration in lung cancer cell line A549 and the level of 27 hydroxycholesterol (27-OHC) in lung tissue. The results provide a basis for identifying cellular foaming degree and further study on the involvement of CYP27 pathway in cholesterol metabolism. Methods after ultrasonic cleavage of cell line: A549 cells, cholesterol was extracted by Koh ethanol solution and trichloroacetic acid, respectively, after removing triglyceride and protein from the cell lytic solution, and then the cholesterol was extracted with a binary extractant of n-hexane-isopropanol. The cholesterol content was determined by HPLC. The chromatographic column was ZORBAX SB-C18 (4.6 mm 脳 150mm) mobile phase was acetonitrile-isopropanol (67:33), the flow rate was 1 mL min ~ (-1), the detection wavelength was 206 nm. The lung tissue was taken quantitatively and the homogenate was obtained by high speed homogenate method. According to homogenate: chloroform (3:5) added chloroform extraction. The organic phase is rinsed with water until neutral, and chloroform is removed under the condition of decompression. The derivative reagent 0.1 ml trimethylchlorosilane / hexamethyldisilyl azane / pyridine (1:2:3) was added for 60 min, then the nitrogen was blown dry, chloroform was redissolved and analyzed by GC / MS. The chromatographic column is HP-5MS quartz capillary column, the ionization mode (ei) is positive ion scanning, the single injection is 1.0 渭 l, the carrier gas is helium gas. The level of 27-OHC in lung tissue was analyzed by GC-MS. Results: the HPLC method was established. The retention time of cholesterol standard was 8.2 min ~ 5 ng 渭 L ~ (-1) -100 ng 渭 L ~ (-1) (R _ (0.9999), and the peak time of intracellular cholesterol and half amount of intracellular cholesterol was the same as that of standard solution, both of which were at 8.2 min. The accuracy, repeatability and stability were all lower than 3.0. The average recovery was 94.89% (n = 6) and the detection limit was 2.665ng 渭 L ~ (-1) (S / N ~ (3) = 10.197 ng / L ~ (-1) (S / N ~ (10). The total ion current of 27 hydroxyl cholesterol fragment in lung tissue was detected by GC-MS at 55.39 min, and the fragment mass charge ratio after derivation was m / z 456 m / z 546. It can be determined qualitatively. The quantitative detection and the preliminary prediction of methodology verification showed that the partial recovery of the single sample was beyond 92% -105%. Its precision, repeatability, stability and other methodological verification indicators need to be further optimized. Conclusion: the HPLC method established in this paper is accurate, reproducible, accurate, easy to operate, and the recovery rate is within the range of requirements. The follow-up can be used for batch detection of cholesterol level in A549 cells, which lays a foundation for the advanced study of the relationship between lung tissue injury and cholesterol metabolism disorder. Gas chromatography-tandem mass spectrometry (GC-MS) was used to detect 27-OHC27 in lung tissue. However, further methodological arguments are needed.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

【参考文献】