细胞周期蛋白依赖性激酶9 (CDK9) 在椎间盘退变过程中的作用及机制研究

Degeneration of the intervertebral disc is believed to be the main cause of low back pain, however, the pathogenesis of intervertebral disc degeneration is still controversial. Inflammation has recently been proved to be closely associated with the onset of disc degeneration. Under exogenous stimulation, IVD cells secrets a range of pro-inflammatory cytokines such as IL-1b and TNFa, these inflammatory cytokines further induce the synthesis of matrix catabolic enzymes including MMPs and ADAMTSs via NF-kB signal pathway, finally result in disc degeneration. Cyclin-dependent kinase 9 (CDK9), a transcriptional factor, was lately reported to have the property of regulating the expression of pro-inflammatory cytokines and catabolic genes in acute knee injury. We have demonstrated that inhibition of CDK9 results in suppressing the expression of pro-inflammatory cytokines (IL-1b and TNFa) and catabolic enzymes (MMP13 and ADAMTS4) in an IVD cells in vitro culture system. However, whether the inhibition of CDK9 has protective effect in IVD from inflammation-induced degeneration, as well as its basic mechanism is still left undetermined. It was reported that the NF-kB dependent immediate-early inflammatory response genes are regulated via P65 ser276 phosphorylation coupled to CDK9 recruitment, and researches have also demonstrated that CDK9 correlates with P65 and is a crucial participant in the NF-kB signal pathway. Based on the roles that CDK9 plays in the inflammation and NF-kB pathway, we firstly plan to investigate CDK9 in IVD degeneration by regulating inflammatory genes expression directly and inducing these downstream target genes via its co-operation with P65, the NF-kB subunit to clarify the mechanism; then we are going to test the roles that CDK9 plays in protecting IVD explants from inflammation stimulation by CDK9 silencing; we further propose to demonstrate the CDK9 inhibition in preventing or delaying the onset of disc degeneration in mouse tail loop model. Our study may provide a theoretical foundation for understanding of the inflammatory pathways involved in intervertebral degeneration and may help to identify potential biological treatments in the future.

椎间盘炎症反应是椎间盘退变最主要的诱因：椎间盘异常分泌大量促炎因子，并激活NF-kB信号通路合成基质分解酶（MMPs或ADAMTS）分解细胞外基质最终导致退变。我们先前的研究发现抑制CDK9可以显著抑制椎间盘细胞分泌促炎因子；此外CDK9的活性与NF-kB信号通路关键蛋白P65的磷酸化密切相关，且抑制CDK9显著减少基质分解酶的合成，提示CDK9通过影响P65的磷酸化调控NF-kB信号通路。综合前期研究基础我们认为在椎间盘炎症过程中调控CDK9不仅抑制促炎因子的合成，而且通过抑制NF-kB信号通路减少分解酶的合成，起到关键的“双保险”作用,该观点未见研究报道。本研究将系统分析调控CDK9在椎间盘细胞炎症反应以及保护细胞外基质的作用以及分子机制，并进一步在体内研究通过调控CDK9来干预、延缓椎间盘退变的治疗效果，为开发以CDK9为靶点的椎间盘退变综合治疗方案提供理论依据。

本研究意在探讨转录因子CDK9在NF-kB信号通路介导的椎间盘退变发生发展过程中的作用和机制。着眼于该转录因子CDK9在椎间盘细胞炎症反应过程中的作用，在动物椎间盘退变模型建立以及模拟治疗过程中的作用，以及调控CDK9在预防甚至治疗椎间盘退变发生发展过程中可能的作用及其分子机制。.本研究首先在细胞层面研究了调控CDK9能够有效干预椎间盘细胞在炎症信号刺激下的应激反应过程，并且在椎间盘体外培养试验中，进一步验证了调控CDK9不仅可以干预验证环境下椎间盘细胞的活性，也能有效减少椎间盘细胞自分泌基质分解蛋白酶的活性及功能。信号通路的研究显示，调控CDK9能显著抑制P65的磷酸化，并且能有效遏制P65的入核过程。由此本研究基本证实了CDK9通过调控NF-kB信号通路调控椎间盘细胞炎症应激反应及生物学活性的分子机制。在体外细胞研究的基础上，本研究进一步采用了非侵入性造模方法成功在小鼠模型上获得椎间盘退变损伤模型。并在体内注射CDK9抑制药物，在短期试验中观察到调控CDK9能有效遏制椎间盘退变。据此，本研究基本证实转录因子CDK9通过NF-kB信号通路抑制椎间盘细胞炎症反应及影响椎间盘退变的作用。