LPA及其受体在调控腰椎黄韧带增生纤维化中的作用及机制研究

The pathomechanism underlying central lumbar spinal stenosis is predominantly related to hypertrophied ligamentum flavum （LF）and accumulation of inflammation-related scar tissue contribute to LF hypertrophy and fibrosis. Our previous study showed that hypertrophy and fibrosis of LF in lumbar spinal stenosis is associated with increased expression of Lysophosphatidic acid (LPA) and LPA receptors (LPARs), however, the regulation mechanisms have not yet been elucidated. The current project attempts to elucidate the mechanism how LPA and LPARs can regulate the pathogenesis of LF fibrosis. Firstly, the expression of LPAR subtypes will be detected by molecular biological methods in the LF from patients. Secondly, the possible cellular and molecular mechanisms of LPA-LPARs action will be investigated through the use of in vitro and in vivo experiments. Finally, anti-fibrotic therapies by down-regulation or blockade of LPA receptors will also be investigated in vitro and in vivo. The results of this project will help to better understand the pathogenesis of LF fibrosis and may provide a potential biological method for lumbar spinal stenosis.

黄韧带的增生肥厚是腰椎退变性管狭窄症主要的病理变化之一。国内外学者研究显示黄韧带增生肥厚可能与多种损伤引起的慢性炎症相关的损伤修复过程有关，本课题组前期研究发现溶血磷脂酸（LPA）及其受体在黄韧带的增生纤维化过程中起了重要的作用，但其详细机制不明确。为进一步探讨LPA及其受体在腰椎黄韧带增生纤维化中的调控作用，本课题组拟首先通过对腰椎黄韧带组织样本LPA受体表达谱的研究，筛选与腰椎黄韧带增生纤维化相关特异性受体亚型；其次观察外源性LPA作用后体外培养的黄韧带细胞增殖、迁移、凋亡等情况以及生物因子TGF-β1、CTGF、MMP-2,9等的表达变化，分析参与此过程的可能信号通路；最后通过体内外实验观察LPA受体拮抗剂或基因沉默对黄韧带增生纤维化的影响。通过本课题试图阐明LPA调控黄韧带纤维化的作用机制，为黄韧带退变相关疾病的药物、生物治疗提供新思路和新途径。

主要研究内容：为检测溶血磷脂酸（Lysophosphatidic acid，LPA）及其受体（LPAR1）表达与黄韧带肥厚的相关性，明确LPA-LPAR1影响黄韧带肥厚的作用机制，我们进行了一下研究：（1）检测人黄韧带细胞的LPAR1表达水平，与黄韧带厚度行线性回归分析；（2）在LPAR1沉默或过表达的黄韧带细胞中检测细胞活力，细胞周期，细胞凋亡率和细胞的分子机制；（3）在人体标本和体内实验中验证LPA促进黄韧带肥厚及其分子机制。（4）测量国人正常腰椎椎间隙的高度及其与上位腰椎椎体高度的比值，明确国人正常腰椎间隙高度的分布并明确腰椎高间隙的定义，进行腰椎的影像解剖学形态学研究。.重要结果：（1）LPA及其受体LPAR1在黄韧带肥厚组的组织以及细胞中的表达明显高于非肥厚组；（2）LPA-LPAR1启动了Akt的磷酸化从而促进黄韧带细胞的增殖以及抗凋亡能力，LPAR1沉默以及过表达可以改变LPA-LPAR1-Akt信号轴对黄韧带细胞增殖以及抗凋亡的影响；（3）冻干后的LPA通过LPAR1-Akt信号通路显著诱导黄韧带肥厚，靶向性抑制LPAR1药物（Ki16425）可有效降低LPA刺激引起的黄韧带肥厚。LPA-LPAR1-Akt激活加强了黄韧带增殖以及抗凋亡能力，是导致腰椎椎管狭窄的重要原因。（4）为个体化腰椎椎间融合器的设计及临床应用提供可靠参数。.科学意义：LPA及其受体信号通路的激活对诱导黄韧带纤维化至关重要，阻断其信号转导对抑制纤维化有效。LPA及受体可作为治疗黄韧带纤维化的新的潜在候选药物靶点，为研发疗效明确、副作用少，能直接抑制、减轻或延缓纤维化的药物提供了参考。