雄激素非依赖前列腺癌细胞系代谢组学的初步研究

摘要

探讨雄激素依赖性前列腺癌LNCaP细胞系向雄激素非依赖性前列腺癌细胞系(LNCaP-AI和LNCaP-AI+F)转变过程的代谢组学变化,寻找前列腺癌发生雄激素耐药的可能发生机制.利用超高效液相色谱-四极杆-飞行时间质谱,在电喷雾电离源在正离子模式下,采用60%(V/V)甲醇-0.85%(w/V)NH4HCO3溶液在-40 ℃淬灭,40%(V/V)乙醇-0.8%(w/V)NaCl溶液在-20 ℃淬灭,提取平行培养3组的细胞内代谢物上清液测定,获得代谢指纹图谱.数据经Masslynx,SIMCA-P 12.0和SPSS软件进行主成分分析(PCA)、偏最小二乘分析判别(PLS-DA)分析、正交校正的偏最小二乘分析判别(OPLS-DA)分析和非参数检验;根据模型变异权重参数(VIP)、S图、载荷矩阵和P值进行筛选不重复且组间变异小的标志物.雄激素依赖到非依赖转变过程中,酰胺类、溶血磷脂酰胆碱、类固醇激素及植物鞘氨醇呈现减少趋势,鞘磷脂呈增加趋势;含高不饱和度脂肪酰基的磷脂酰胆碱(总不饱和度之和大于10)在转变过程中呈现减少的趋势,而含低不饱和度脂肪酰基的磷脂酰胆碱(总不饱和度之和小于4)则呈现增加的趋势.这些代谢标志物的变化,说明在雄激素非依赖性前列腺癌转变过程中与脂质代谢发生异常密切相关.%To investigate the biological mechanisms at the basis of androgen resistance, a comparative metabolic approach based on ultra performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) was applied to the human epithelial prostate cancer cell lines LNCaP and its androgen non-dependent counterpart LNCaP-AI/LNCaP-AI+F. Three kinds of cells were quenched by 40％ ethanol-0.8％ sodium chloride solution (-20℃) and 60％ methanol-0. 85％ammonium bicarbonate(-40℃). The cellular metabolism was studied by UPLC-Q-TOF-MS using electron spraying ionization with positive mode to find potential metabolic biomarkers and reveal their physiological changes. After UPLC Q-TOF MS analysis, principal components analysis (PCA),projection to latent structures discriminant Analysis(PLS-DA), orthogonal projection to latent structures discriminant analysis (OPLS-DA) and nonparameter test were done using the Masslynx,SIMCA-P and SPSS for marker selection and identification. Potential biomarker screening was performed according to VIP value, Scatter plot, loading plot and P value. According to the data, most of biomarkers were identified as phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, palmitic amide, steroid hormones, phytosphingosine and SM. Lysophosphatidyl choline,phosphatidylcholine with lower unsaturated acyl chains (n＞10) and taurocholat higher time-dependent increasing trends, while phosphatidylcholine with lower unsaturated acyl chains (n＜ 4) and Palmitic amide,steroid hormones, Phytosphingosine show decreasing trends. The results indicate that there are differential metabolic biomarkers between androgen-dependent and androgen-independent cells of human prostate cancer especially in lipids.