基于代谢组学的子宫内膜异位症发病机制和诊断生物标志物研究

The pathogenesis and mechanism of endometriosis still remains unclear. Results of many studies suggest the existence of many differentially expressed genes and proteins involved in endometriosis. Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or proteinic changes. In our previous research, serum metabolic profiling employing time-of-flight mass spectrometry (TOFMS) was utilized to investigate the complex physiopathologic regulations in endometriosis. It was revealed that metabolic differences between endometriosis patients and healthy controls could be observed, so it will be informative to investigate the potential machanisms and biomarkers of endometriosis by metabolomics. In the current study, different conditions for metabolomics analysis will be performed and optimized on TOFMS, and we will make metabolomic research on serum, urine and endometrium samples from endometriosis patients and healthy controls. The different metabolite and related metabolic pathway will be identified. In order to validate the results of metabolomic on endometriosis, we will isolate and culture stromal cells from endometria of EMs patients and control patients without EMs, and evaluate their proliferation, apoptosis, adhesion, invasion in vitro. In addition, these functions will be examined after RNA interference used to silence related metabolic genes. Finally, transgenic stromal cells and maternal stromal cells will be implanted in female nude mice, and evaluate proliferative activity, VEGF expression, apoptosis and invasion, this vivo models of endometriosis will be used for further validate the results of metabolomics.

子宫内膜异位症（EMs）中存在大量差异表达的异常基因和蛋白，这些差异表达的异常基因和蛋白必然会引起其下游产物也是最终产物即小分子代谢产物的变化，因此我们前期采用高分辨质谱测定了EMs患者和正常对照者血清中的代谢产物，发现EMs患者确实存在大量差异代谢产物，因此进行基于代谢组学的EMs研究,可能会为进一步阐述EMs的发病机制和生物标志物奠定基础。为此，拟建立基于MS的代谢组学方法，绘制EMs患者血清、尿液和组织的代谢图谱，进行统计学分析，找到相关差异代谢产物及其代谢通路。分离培养子宫内膜间质细胞，通过RNA干扰手段改变代谢通路相关基因观察其对子宫内膜间质细胞生物学功能的影响，对代谢组学结果进行体外验证。将相关代谢通路改变的转基因子宫内膜间质细胞及母本细胞接种至人工控制生理周期的裸鼠背部皮下，动态观察接种细胞的生长、增殖、凋亡与浸润以及微血管密度等情况，以对代谢组学结果进行体内验证。

子宫内膜异位症（EMs）中存在大量差异表达的异常基因和蛋白，而这些大量差异表达的异常基因和蛋白必然会引起其下游产物也是最终产物即小分子代谢产物的变化，因此进行基于代谢组学的EMs研究,可能会为进一步阐述EMs的发病机制和发现EMs早期诊断标志物奠定理论和实验基础。我们采用基于UHPLC-Q-TOF-MS技术的代谢组学方法，检测子宫内膜异位症患者和对照者谢轮廓图谱，同时获得一级质谱和二级质谱数据，随后采用XCMS对数据进行峰提取和代谢产物鉴定，利用PCA、PLC-DA等统计方法对所得数据进行分析，发现与对照者相比，子宫内膜异位症患者存在大量差异代谢产物，与正常对照比，子宫内膜异位症患者血清溶血磷脂酸（LPA）水平显著升高，免疫组化结果显示与正常对照组相比，子宫内膜异位症 患者子宫内膜组织中溶血磷脂酸受体1、4、5（LPA1，LPA4，LPA5）表达显著升高，而溶血磷脂酸受体2、3（LPA2，LPA3）表达显著降低，未检测到溶血磷脂酸受体6（LPA6）表达。进一步采用以子宫内膜间质细胞作为体外细胞模型和裸鼠体内动物模型，对溶血磷脂酸及其受体在子宫内膜异位症发展发展中的作用进行了验证，结果显示溶血磷脂 酸（LPA）可以显著诱导子宫内膜间质细胞IL-8分泌增加，但对子宫内膜间质细胞细胞增 殖和VEGF分泌无影响，RNA干扰抑制LPA1表达后促进人子宫内膜上皮细胞增殖，溶血磷脂酸促进人子宫内膜上皮细胞裸鼠皮下移植瘤的生长，证实溶血磷脂酸及其受体参与了子宫内膜异位症的发生发展。