脂质质谱分析用于卵巢癌疾病生物标志物及耐药机理研究

Ovarian cancer (OC) ranks as the most deadly cancer in gynecologic diseases. The development of new reliable diagnostic and prognostic molecular biomarkers for OC, to increase the 5-year survival rate, is very necessary. Many studies showed that the lipid metabolic disorder was involved in the occurrence and development of OC. For example, lysophosphotidic acid (LPA) was reported to enhance the proliferation, invasion and migration of OC cells, and plasma LPA levels are increased in OC patients vs. healthy controls; Ceramides (Cers) are known to induce apoptosis whereas glucosyl-ceramides (GluCers) induce the proliferation of cells, and recent evidence indicates that disrupting ceramide glycosylation can resuscitate wild-type p53 expression and p53-dependent apoptosis in OC cells. The endogenous LPA and GluCers synthesis are mainly regulated by phospholipase A2 (PLA2) and glucosylceramide synthase (GCS) and their relative enzymes,respectively. In addition, recent studies have also reported that inhibition of the phosphatidylinositol 3-kinase/protein kinase B （PI3K/PKB） pathway increases the efficacy of chemotherapeutic agents in human malignancies including ovarian cancers. Based on these studies, in this work, we will develop mass spectrometry (MS)-based methods for analysis of lipids, especially the lipids regulated by PLA2, PI3K and GCS and their relative enzymes. Furthermore, these methods will be applied to a large number of real samples, to systematically investigate the lipid metabolism in OC. Finally, utilization of these lipids data, combined with clinical information and statistical analysis, to explore new lipid molecular biomarkers for OC. So far, it is the first research to systematically study the bioactive lipid small molecules as biomarker for OC. Meanwhile drug resistance ovarian cancer cells model will be established for lipid profile to study the mechanism of drug resistance of cancer cells. The discovery of new molecular biomarkers and mechanism of drug resistance for OC, if successful, undoubtedly，is of significance.

卵巢癌在妇科肿瘤疾病中死亡率居首位,主要原因在于有效诊断标志物的缺失及癌细胞耐药现象，发展新的诊断生物标志物及研究癌细胞耐药机制,以提高卵巢癌患者存活率，是非常必要的。研究表明，脂质小分子代谢异常与肿瘤发生发展密切相关，系统研究肿瘤患者体液中脂质变化，有助于确定其中关键的脂质，为发现新的生物标志物提供重要依据；就卵巢癌而言，磷脂酶A2（PLA2）、磷脂酰肌醇3-激酶（PI3K）及葡糖脑苷脂合成酶（GCS）等调控的脂质代谢途径与卵巢癌发生发展及耐药关系密切。因此研究卵巢癌患者脂质的变化，尤其是这些酶调控代谢途径中涉及的脂质，探讨它们是否可作为卵巢癌分子生物标志物不仅具有创新性，而且颇具重要性。在本申请中，我们拟发展基于质谱的血液中脂质分析及酶活检测方法，进而系统研究卵巢癌的脂质代谢，并结合临床信息与统计分析，探索卵巢癌新的脂质分子生物标志物及耐药机理。

脂质代谢紊乱与很多疾病（包括心血管疾病、癌症等）发生发展密切相关。快速、全面地分析脂质，有助于研究疾病发生发展机理，提供新的药物靶点或治疗方式。通过3年的努力，结合质谱光谱等分析技术，开展了大量脂质的分析方法学以及应用研究。取得的主要成果包括：.1..基于质谱的脂质定性、定量及原位成像分析：神经节苷脂在生物样本中含量极低且被其它脂质抑制信号而难以检测，针对此问题，发展了样本前处理方法，实现了神经节苷脂质谱信号的捕获及提高，进而对神经节苷脂开展了定性、定量以及原位成像分析。(Sci. Rep. 2016, 6, 25289; Sci. Rep. 2016, 6, 37903)。.2..脂质作为卵巢癌生物标志物研究：建立了非靶向血浆中脂质质谱分析方法，研究了卵巢癌病人、巧囊病人及健康人血浆中大量脂质。数据可以有效区分卵巢癌病人、巧囊病人及健康人，提供了潜在的诊断标志物。另外，信号通路分析表明甘油酯代谢通路及PLA2酶可能与卵巢癌疾病发生发展密切相关，为疾病治疗提供了潜在的治疗靶点 (Talanta 2016, 150, 88-96)。.3..利用脂质组装纳米脂质体，进而构建基于FRET效应的比例型pH荧光探针：利用脂质组装纳米脂质体，包裹两种荧光染料构建基于FRET效应的比例型荧光探针。该探针生物相容性好，短时间内即可进入细胞，实现了细胞内pH高灵敏的荧光成像。该工作首次利用纳米脂质体发展新型荧光探针，为生物相容性好的探针的构建提供了新方法 (Anal. Chem. 2016, 88, 12380-12385)。