IGF-1通过激活IGF-1R/AKT/mTORC1信号通路促进黄韧带肥厚的机制研究

hypertrophy of ligamentum flavum (HLF) is the key factor to lumbar spinal stenosis. Recent studies suggest that HLF is closely related to endocrine and metabolic disorders, and endocrine factors play an important role in the regulation of HLF. Insulin growth factor 1 (IGF-1) is involved in the onset of many major diseases in the body, and its biological effects are regulated by the mTORC1 signaling pathway. So far, there has been no correlation study between IGF-1 and HLF. The previous study found that the expression of IGF-1 and pIGF-1R in the hypertrophic ligamentum flavum was significantly increased, the expressions of the upstream proteins pAKT and pS6 in the mTORC1 signaling pathway were increased, and the type I and III collagen fibers were proliferated. These results suggest that HLF may be related to endocrine secretion. The biological effects of factor IGF-1 are closely related and regulated by the mTORC1 signaling pathway. Based on this: This project is intended to further elucidate the relationship between the endocrine factor IGF-1 and the incidence of ligamentum flavum hypertrophy in the overall level; study the effect of endogenous and exogenous IGF-1 on the metabolism of the ligamentum flavum cells at the cellular level, and explore the molecular mechanism of IGF-1 regulating the metabolism of ligamentum flavor cells at the level of transcription in the use of the CRISPR-Cas9-based chromatin immunoprecipitation technology.

黄韧带肥厚（HLF)是造成腰椎管狭窄的关键。最新研究提示：HLF与内分泌代谢紊乱密切相关，内分泌因子在HLF发病起重要调节作用。胰岛素样生长因子1(IGF-1)参与体内许多重大疾病发病，其生物效应受mTORC1信号通路调控。至今尚未见IGF-1与HLF发病的相关研究。课题组前期研究发现：肥厚的黄韧带组织中IGF-1、pIGF-1R表达显著增加；mTORC1信号通路上游蛋白pAKT和pS6表达上调，Ⅰ型和Ⅲ型胶原纤维增生;上述结果提示HLF可能与内分泌因子IGF-1的生物效应密切相关，且受mTORC1信号通路调节。据此：本课题拟进一步在整体水平：阐述内分泌因子IGF-1与黄韧带肥厚发病之间的关系；在细胞水平：研究内源性及外源性IGF-1对黄韧带细胞代谢的影响；在转录水平：利用基于CRISPR-Cas9的染色质免疫共沉淀技术研究IGF-1调控黄韧带细胞代谢的分子机制。

机械应力促进人黄韧带细胞(LFCs)合成多型胶原蛋白，导致黄韧带肥大(LFH)。然而，机械应力对胶原蛋白形成的机制尚不清楚。因此，我们在本研究中研究了机械应力与胶原蛋白合成之间的关系。首先，从9例患者中分LFC，在不同时间进行机械应力培养。分别通过ELISA和qPCR检测IGF-1，I型胶原(col-I)，III型胶原(col-III)蛋白和mRNA水平。采用Western blot法检测pIGF-1R、pAKT和pS6的激活情况。为了进一步探讨其作用机制，我们使用了IGF-1中和抗体NVP-AEW541和雷帕霉素。与非应激lfc相比，应激LFCs中IGF-1、coli和应激coliii显著增加。此外，pIGF-1R、pAKT和pS6的激活应激lfc中明显增强。有趣的是，IGF-1中和抗体抑制了colI蛋白、colImRNA、IGFIII蛋白、colIIImRNA和IGF-1蛋白，而不是IGF-1mRNA。此外，NVP-AEW541和雷帕霉素均抑制colI和colIII蛋白和mRNA，而不是IGF-1。此外，IGF-1中和抗体和NVP-AEW541降低了pIGF-1R、pAKT和pS6的激活，而雷帕霉素降低了pS6的激活。综上所述，这些结果表明，机械应力促进LFCs产生IGF-1，IGF-1通过IGF-1R/AKT/mTORC1信号通路促进col-I和col-III的合成，从而缓解LFH。