RAS/MEK/ERK通路介导NF-кB调节子宫内膜异位症细胞侵袭性的机制研究

Endometriosis (EMs) is a refractory disease with high recurrence; in the previous study, the EMs gene spectrum screening showed that RAS, MEK, ERK and NF- κB showed differential expression in the eutopic and ectopic endometrium, and the NF- κB pathway is an important part of changing cell invasion, but the regulation of NF-κB pathway upstream mechanism is not clear. In view of the research shows that the activation of RAS/MEK/ERK signaling pathway through the activation of NF-κB activation and activation of NF-κB induces activation and migration of tumor cells to promote cell activation and migration. From this, we hypothesized that RAS/MEK/ERK pathway dependent on MAPK kinase in EMs may be regulate the upstream pathway of NF-κB activity and enhanced cell invasion and recurrence;for this,the subjects will use drug intervention EMs primary culture of stromal cells、gene knockout cell model、eukaryotic expression vector and the changes of the pathway factors before and after the intervention of EMs model in rats, Invasion and adhesion of cells were detected by invasion assay and adhesion protein adsorption, and in vivo imaging to track rats adhesions,from the mechanism to provide the basis for reducing recurrence of endometriosis.

子宫内膜异位症(EMs)以其高复发成为难治性疾病；课题组前期对EMs基因谱筛查显示RAS、MEK、ERK和NF-κB呈现在位/异位内膜中差异表达，且NF-кB 通路是改变细胞侵袭重要环节，但调控NF-кB上游通路机制不清。鉴于研究显示RAS/MEK/ERK信号通路通过激活NF-κB促使肿瘤细胞的活化与迁移促进细胞活化与迁移。由此，我们推测EMs中依赖MAPK激酶的RAS/MEK/ERK通路可能是调节NF-кB活性的上游通路并增强了细胞侵袭与复发；为此课题组拟药物干预EMs原代培养的间质细胞、基因敲除细胞模型、真核表达载体及大鼠EMs模型观察干预前后各通路因子变化，借助侵袭实验和层粘连蛋白吸附技术检测细胞侵袭和粘附性，并活体成像仪追踪大鼠体内粘连情况。通过体内/体外观察验证RAS/MEK/ERK通路是EMs调节NF-кB活性上游通路并促进了细胞侵袭与粘附，为从机制上减少内异症复发提供依据。

子宫内膜异位症（EMs）虽被视为一种常见的良性病变，但其异位内膜的种植却具有增殖、迁移侵袭等恶性生物学特征。课题组前期研究证实核转录因子кB（NF-κB）可能是骨桥蛋白（OPN）诱导PI3K/ Akt激酶通路增强异位内膜侵袭性的关键因素。但活化NF-KB增强异位内膜增殖侵袭性的上游机制仍不清楚，课题组通过表达谱芯片测序分析了EMs中差异基因表达：发现MAPK、RAS、ERK、NF-κB，PI3K等因子显著上调，因此我们推测：具有高度侵袭能力的EMs中，依赖MAPK激酶的RAS/MEK/ERK通路可能是介导NF-кB表达增强异位细胞增殖侵袭性的关键。第一部分：差异性表达基因的筛选验证，即RAS/MEK/ERK通路因子及NF-κB在内异症患者组织中的表达及其相关性：通过临床组织样本检测RAS/MEK/ERK通路因子及NF-κB在内异症患者在位/异位内膜组织中的表达，免疫组化及Western-blot结果均显示：与对照组相比，内异症患者组织中上述各因子表达明显升高且呈正相关，揭示RAS/MEK/ERK通路因子及NF-κB与内异症的发生发展密切相关。第二部分：RAS/MEK/ERK信号通路介导NF-KB表达影响EMs患者在位间质细胞增殖侵袭性的机制研究：分离培养鉴定EMs患者在位间质细胞，利用慢病毒转染、基因沉默等技术手段在体外构建敲低RAS/MEK/ERK信号通路及NF-KB的细胞模型，Western-blot、RT-PCR检测干预前后RAS/MEK/ERK信号通路及NF-KB蛋白及基因表达变化，CCK-8、Transwell试验检测干预前后细胞增殖侵袭性变化，通过细胞分子水平证实：子宫内膜异位症患者间质细胞中RAS/MEK/ERK信号通路是调节NF-KB表达影响细胞增殖及侵袭性的上游机制。第三部分：构建EMs大鼠模型并给予上述通路抑制剂药物干预，检测干预前后异位病灶体积变化、Western-blot、IHC检测干预前后各因子表达变化及异位病灶增殖侵袭性变化，证实靶向抑制RAS/MEK/ERK信号通路通过下调NF-KB表达抑制异位病灶的增殖侵袭，即依赖MAPK激酶的RAS/MEK/ERK通路通过介导NF-кB表达增强异位细胞增殖侵袭能力。为子宫内膜异位症的发病机制和药物靶向治疗提供了可靠的理论和实验依据。