神经胶质瘤相关癌基因同源蛋白1在椎间盘退变中的作用及相关机制研究  

Research on the role and mechanism of glioma-associated oncogene homolog 1 in intervertebral disc degeneration

作　　者：王天灵 吕巧 翟羽 卞志群 李长青 WANG Tianling;LU Qiao;ZHAI Yu(Department of Orthopedics,the Second Affiliated Hospital of Army Military Medical University,Chongqing,400037,China)

机构地区：[1]陆军军医大学第二附属医院骨科,重庆市400037 [2]陆军军医大学第二附属医院临床医学研究中心,重庆市400037

出　　处：《中国脊柱脊髓杂志》2025年第3期294-302,共9页Chinese Journal of Spine and Spinal Cord

基　　金：国家自然科学基金面上项目(82172488);陆军军医大学科技创新能力提升专项项目(2022XQN32)。

摘　　要：目的:探讨神经胶质瘤相关癌基因同源蛋白1(gioma-associated oncogene homolog,Gli1)对髓核细胞(nucleus pulposus cells,NPCs)的影响及其在椎间盘髓核组织纤维化改变中的作用。方法:针刺法构建SD大鼠尾椎间盘退变模型,组织学染色评估未处理(negative control,NC)组和针刺(puncture-induced intervertebral disc degeneration,PIDD)组的椎间盘退变及髓核纤维化改变水平,Western blot评估组织Gli1蛋白表达水平;通过慢病毒在NPCs中过表达Gli1蛋白并进行鉴定,实验分三组:对照(Blank)组、对照慢病毒处理(Lv-Ctrl)组、Gli1过表达(Lv-Gli1)组,通过免疫荧光和Western blot检测纤维化标志蛋白FAP、FSP-1表达水平;添加Gli1抑制剂GANT61,实验分四组:空白(blank)组、Gli1过表达(Lv-Gli1)组、Gli1过表达抑制(Lv-Gli1+GANT61),Gli1抑制对照组(Lv-Gli1+DMSO),通过Western blot检测FAP、FSP-1蛋白表达水平;动物体内实验,分组:针刺(PIDD)组,针刺治疗(PIDD+GANT61)组和治疗对照(PIDD+DMSO)组,通过苏木精伊红、番红固绿、天狼猩红染色和Western blot评估椎间盘退变及纤维化改变水平。结果:组织学染色结果显示,退变椎间盘组织学评分明显上升(P<0.01),Western blot结果显示,退变组Gli1蛋白表达水平明显增高(P<0.05),细胞实验结论与其一致;构建Gli1蛋白过表达NPCs,免疫荧光染色结果显示,相较于对照组、病毒对照组、Gli1过表达组的FAP、FSP-1表达明显上调,Western blot结果进一步显示,Gli1过表达组纤维化标志蛋白表达明显升高(P<0.05);GANT61干预细胞实验中Gli1过表达抑制组,较Gli1过表达组,Gli1抑制对照组,Western blot提示FAP、FSP-1蛋白表达下调(P<0.05),体内实验中,PIDD+GANT61组,组织学评分及胶原构成明显改善(P<0.01)。结论:退变髓核组织和细胞中的Gli1蛋白表达明显上调;抑制Gli1基因表达可以下调FAP、FSP-1蛋白表达。椎间盘纤维化退变机制可能与Gli1的激活有关。Objectives:To explore the impact of Gli1(gioma-associated oncogene homolog)on nucleus pulposus cells(NPCs)and its role in the fibrotic changes of the nucleus within intervertebral discs.Methods:A rat tail intervertebral disc puncture model was established.Histological staining was used to evaluate the levels of intervertebral disc degeneration and fibrotic changes in the untreated(negative control,NC)group and the puncture(puncture-induced intervertebral disc degeneration,PIDD)group.Western blot was used to assess the expression levels of Gli1 protein in the tissues.Gli1 protein was engineered to overexpress by lentivirus in NPCs and verified.The experiment was divided into three groups:control(Blank),Gli1 overexpression(Lv-Gli1),and virus control(lv-Ctrl).Immunofluorescence and Western blot were used to detect the expression levels of fibrotic markers FAP and FSP-1.Adding Gli1 inhibitor GANT61,cellular experiment was grouped into control(blank),Gli1 overexpression(Lv-Gli1),Gli1 overexpression+GANT61(Lv-Gli1+GANT61),and Gli1 overexpression+DMSO(Lv-Gli1+DMSO).Western blot was used to detect the expression levels of FAP and FSP-1 proteins.In vivo animal experiments were conducted in groups of puncture(PIDD),puncture treatment(PIDD+GANT61),and treatment control(PIDD+DMSO).Histological staining was used to evaluate the levels of intervertebral disc degeneration and fibrotic changes.Results:Histological staining results showed the histological score of degenerated discs significantly increased(P<0.01).Western blot results showed a significant increase in Gli1 protein expression in the degenerated group(P<0.05),which was consistent with the conclusions of the cellular experiments.NPCs overexpressing Gli1 protein were constructed,and immunofluorescence staining results showed that the expression of FAP and FSP-1 in the Gli1 overexpression group was significantly upregulated compared to the control and virus control groups.Western blot results further showed a significant increase in the expression of fibrotic markers in

关 键 词：纤维化 椎间盘退变 髓核细胞 神经胶质瘤相关癌基因同源蛋白1(Gli1) 

分 类 号：R681.5[医药卫生—骨科学]