NF-κB调控血管内皮细胞-间质转化影响人鼻咽纤维血管瘤侵袭复发的实验研究

Local high invasive and recurrence of juvenile nasopharyngeal angiofibroma (JNA) is a research hot spot. Our previous experiments based on magnetic-activated cell sorting (MACS) demonstrated that increased migration and invasion capabilities of endothelial cells derived from juvenile nasopharyngeal angiofibroma(JEC) compared with normal endothelial cells. Furthermore, we found that: 1. different from normal endothelial cell's "cobblestone" appearance, JEC rendering a long fusiform class fiber-like morphology. 2 the mesenchymal markers α-SMA upregulation in accociated with the endothelial marker VE-cadherin downregulation. 3.reversal of JEC endothelial-mesenchymal transition could significantly inhibit the invasion and migration ability of JEC in vitro. These all suggested: JNA endothelial cells - mesenchymal cell transformation (EndMT) might be functional related to the local invasion and high recurrence of JNA. Based on the whole genome comparative analysis of JEC with different recurrence potential we found that the NF-κB pathway might be the leading pathway involved in JNA recrrcunce, thus we speculated the regulation of JEC's EndMT was related to the expression of NF-κB. Therefore, we planned to carry out the following studies: (1) analysis of NF-κB pathway's regulation on EndMT, and to clarify its relationship with JNA local invasion and recurrence; (2) analysis of "NF-κB-EndMT"'s contribution to clinicopathological characteristics and the establishment of effective clinical prediction model and intervention targets.

鼻咽纤维血管瘤（JNA）的高侵袭和易复发是其研究热点。课题组前期基于免疫磁珠分选发现：JNA血管内皮细胞（JEC）较正常内皮细胞具有更强的侵袭，运动能力。对比分选获得的JEC和正常内皮细胞的形态及生物学特性发现：1.不同于正常内皮"铺路石样"外观，JEC呈现长梭形的类纤维样形态 2.间充质标记物α-SMA表达上调，而内皮标记物VE-cadherin表达下调3.逆转JEC内皮间质转化（EndMT）能显著抑制其侵袭及运动能力。故提出：EndMT与JNA侵袭及复发可能有关。基于全基因组芯片对比分析不同复发潜能JEC，发现NF-κB通路差异最显著。故开展以下研究：(1)通过分析NF-κB 对EndMT 的功能调控，阐明后者对于JNA局部侵袭及复发的新机制； (2) 通过分析"NF-κB-EndMT" 功能轴与JNA临床病理特征及预后的相关性，建立有效的临床预测模型和干预靶点。

鼻内镜下肿瘤切除是目前治疗鼻咽纤维血管瘤（juvenile nasopharyngeal angiofibroma, JNA）的主要方法，但因为肿瘤侵袭范围广，术中出血多，所以手术难度大，术后复发率高（可高达50%）。我们通过高通量全基因表达谱筛选、对复发与不复发JNA肿瘤组织的各组基因表达谱进行分析，发现NF-κB通路表达差异最显著。NF-κB为一组含有特殊序列的转录因子，与DNA特定序列结合后，激活多种炎症因子、生长因子、趋化因子的表达。因此本课题以NF-κB为出发点，研究JNA术后复发率高的原因。.内皮细胞-间质细胞转化（Endothelial-mesenchymal transition, EndMT）表现为内皮细胞特异性标志物的表达减弱或者缺失，同时间质细胞特异性标志物的表达增强，并获得较强的增殖和迁移侵袭能力，这是目前肿瘤研究的热门领域。我们运用免疫组织化学方法检测NF-κB p65及内皮细胞-间质细胞转化相关因子（Snail-1、VE-cadherin和α-SMA）在70例JNA患者的肿瘤组织和10例中鼻甲黏膜中的表达水平，并分析其与临床预后的相关性, 发现NF-κB p65和Snail-1高表达组的无瘤生存时间较短（p值分别为0.028和0.025）。然后通过免疫磁珠法分选人鼻咽纤维血管瘤血管CD105+内皮细胞，并进行细胞传代培养，制作JNA内皮细胞模型。最后通过NF-κB特异性抑制剂吡咯烷二硫代氨基甲酸酯（pyrrolidine dithiocarbamate, PDTC）和NF-κB激活剂脂多糖（lipopolysaccharides, LPS）分别下调和上调NF-κB，发现PDTC抑制NF-κB p65的表达，Snail-1表达下调，VE-cadherin表达增高，α-SMA表达降低，同时MMP-2和MMP-9的表达下降，提示内皮细胞-间质细胞转化受到抑制，此时细胞增殖减少，凋亡增加，迁移侵袭能力减弱。而LPS 可以减弱了PDTC的药效。从而初步确定了NF-κB调控EndMT促进JNA血管内皮细胞侵袭、运动及血管生成的机制。.药物及靶向治疗是治疗JNA的新途径。NF-κB介导的内皮细胞-间质细胞转化的研究可为JNA的治疗提供新思路，为进一步探究JNA治疗的新方法提供干预靶点。