丙酮酸激酶PKM2调控椎间盘退变的机制研究

Pyruvate Kinase M2 (PKM2) is a key regulator of aerobic glycolysis and also play a critical role for cell proliferation, survival, metabolism, matrix production. Intervertebral disc (IVD) is the largest avascular structure in the body. The nucleus pulposus (NP) cells reside in and accommodate to a hypoxic environment, and produce energy mainly through glycolysis pathyway. However, little is known about the relationship between PKM2 and IVD degeneration. Recently, not only the glycolytic but also the nonglycolytic functions of PKM2 have attracted a great deal of attention. Recent findings demonstrate that PKM2 can regulates HIF-1α Activity and hypoxia promotes energy metabolism through glycolysis by increasing the HIF-1α trans¬activation activity of NP cells. Our previous research shows that HIF-1α is related to the degeneration of IVD and NP cells could survive for a long time at low oxygen tension and the expression of PKM2 and HIF-1α increased in the degenerated disc. However, the effect and mechanism of PKM2 in the IVD degeneration is still unknown. In this study, we use human IVD and rat disc degeneration model to explore the dynamic expression and regulation mechanism of PKM2 involved in IVD degeneration with various molecular biology methods. Moreover, we use shikonin and siRNA technique to further inhibit the expression of PKM2 to discuss the role of PKM2 in degeneration process of IVD. In this research, we provide a systematic analysis of regulation mechanism of PKM2 in the disc degeneration. This study is a basis for finally elucidate the theory of disc degeneration and offer a new thinking and new way for prevention and treatment of disc degeneration related disease.

丙酮酸激酶PKM2是糖酵解途径的关键酶，在调节细胞增殖凋亡与能量代谢等方面起重要作用。椎间盘是人体最大的无血管组织，其髓核细胞在低氧环境中通过糖酵解方式获取能量。迄今对PKM2与椎间盘退变的关系知之甚少。研究表明PKM2能调控缺氧诱导因子HIF-1α的活性，我们前期发现HIF-1α参与低氧下椎间盘髓核细胞糖酵解过程，且PKM2与HIF-1α均同向表达增加。提示PKM2参与调控椎间盘退变代谢的过程，但其机制尚不明确。本课题采用人体标本和动物模型相结合，从分子、细胞、组织及动物水平来观察PKM2动态表达及对细胞增殖凋亡、基质和能量代谢、细胞因子与免疫应答的作用，并分析相关信号传导(HIF-1α等)，探讨RNA干扰及药物抑制PKM2表达对体内外椎间盘退变过程的影响，系统分析PKM2在椎间盘退变中的调控机制，为最终阐明椎间盘退变病理机制奠定理论基础和实验依据，为椎间盘退变的防治提供新思路和新靶点。

丙酮酸激酶PKM2是糖酵解途径的关键酶，在调节细胞增殖凋亡与能量代谢等方面起重要作用。椎间盘是人体最大的无血管组织，其髓核细胞在低氧环境中通过糖酵解方式获取能量。迄今对PKM2与椎间盘退变的关系知之甚少。研究表明PKM2能调控缺氧诱导因子HIF-1α的活性，我们前期发现HIF-1α参与低氧下椎间盘髓核细胞糖酵解过程，且PKM2与HIF-1α均同向表达增加。提示PKM2参与调控椎间盘退变代谢的过程，但其机制尚不明确。本课题的主要研究内容、重要结果和关键数据包括：①开发并验证了两种新型椎间盘退变造模装置，此装置通过异常应力可控、作用时间可控、穿刺深度可控等多个创新可以达到椎间盘均匀退变，简单经济可靠，重复性好，申请国家专利3项，已授权2项，本装置的研发和验证保证了后续研究的顺利进行，也是后续研究的基础；②提取人椎间盘和大鼠椎间盘髓核细胞进行传代培养，在低氧和常氧条件下，观察到退变椎间盘组织和细胞PKM2表达明显增加，且在低氧条件下PKM2和HIF-1α表达增加明显（P＜0.05），这种表达的变化是通过ERK的磷酸化上调实现的；③在低氧和常氧状态下，采用RNA干扰技术沉默PKM2的表达，通过westernblot和realtime-PCR来观察相关代谢基因的变化情况，结果表明，EGLN1和LDHA的表达没有差异，但是葡萄糖转运子基因SLC2A1的表达在低氧状态下沉默组比正常表达明显减小（P＜0.05）；④常氧和低氧条件下采用realtime-PCR观察两种慢病毒转染HIF-1α的mRNA的变化，进行筛选，后采用westernblot检测常氧或条件下，HIF-1α沉默后PKM2的表达明显下降（P＜0.05）；⑤采用针刺大鼠腰椎间盘退变模型，通过抑制 PKM2 表达组（紫草素喂养）与对照组比较观察椎间盘退变的X线和核磁共振的差异，结果表明抑制PKM2的表达可以部分延缓椎间盘的退变。本课题深入探讨PKM2 对体内和体外培养的椎间盘的影响，系统分析PKM2在椎间盘退变中的调控机制，为最终阐明椎间盘退变病理机制奠定理论基础和实验依据，为椎间盘退变的防治提供新思路和新靶点。