miR-640在腰椎间盘退变中的作用及相关机制研究
Effect and mechanism of miR-640 in lumbar disc degeneration
王坤 1李登宇 1李文庆 1闫维罡 1李家怡1
作者信息
- 1. 河南省洛阳正骨医院(河南省骨科医院)脊柱外科 471002洛阳市
- 折叠
摘要
目的:探讨miR-640在腰椎间盘退变(lumbar disc degeneration,LDD)中的作用及相关机制.方法:将自正常大鼠髓核组织分离的髓核细胞分为4组:对照组、TNF-α组(用50ng/mL TNF-α处理细胞48h)、TNF-α+miR-640-NC 组(50ng/mL TNF-α 处理已转染 miR-640-NC 的细胞 48h)及 TNF-α+miR-640-inhibitor 组(50ng/mL TNF-α处理已转染miR-640-inhibitor的细胞48h);RT-qPCR法检测髓核细胞中miR-640表达水平,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Western blot法检测细胞中Bcl-2、Bax、Cleaved caspase-3、Collagen Ⅱ、Aggrecan表达,免疫荧光法观察细胞中MMP-9表达.以椎间盘注射TNF-α法构建LDD大鼠模型.将25只大鼠随机分为5组(每组5只):假手术组(Sham组)、模型组(LDD组)、Sham+miR-640-NC 组、LDD+miR-640-NC 组和 LDD+miR-640-inhibitor 组.其中,Sham+miR-640-NC 组、LDD+miR-640-NC组和LDD+miR-640-inhibitor组大鼠分别于造模前1周将携带miR-640-inhibitor或miR-640-NC的慢病毒载体注射入大鼠椎间盘间隙内,然后进行假手术或LDD造模.RT-qPCR法检测髓核组织中miR-640表达水平,MRI及HE染色观察大鼠腰椎间盘形态,免疫荧光法观察髓核中MMP-9表达,免疫组织化学法观察髓核中Cleaved caspase-3、Collagen Ⅱ、Aggrecan表达.结果:TNF-α法构建的LDD大鼠髓核组织及TNF-α处理的髓核细胞中 miR-640 的表达增高(P＜0.05).与 TNF-α+miR-640-NC 组比较,TNF-α+miR-640-inhibitor 组细胞活力以及细胞中Collagen Ⅱ、Aggrecan、Bcl-2表达均显著性增高(P＜0.05),而细胞凋亡、Bax、Cleaved caspase-3及MMP-9表达均显著性降低(P＜0.05)o LDD+miR-640-NC组大鼠腰椎间盘出现明显的退行性改变,髓核组织中 Collagen Ⅱ、Aggrecan 表达显著性降低(P＜0.05),Cleaved caspase-3 及 MMP-9 表达显著性增高(P＜0.05);而LDD+miR-640-inhibitor组上述改变得到一定程度的逆转(P＜0.05).结论:在LDD大鼠模型和体外细胞模型中,miR-640的表达呈上调状态.敲降miR-640可减轻LDD,其机制可能与抑制髓核细胞凋亡和细胞外基质降解、促进细胞外基质合成有关.
Abstract
Objectives:To investigate the effect and mechanism of miR-640 in lumbar disc degeneration(LDD).Methods:The nucleus pulposus cells isolated from normal rat nucleus pulposus tissue were allocated into four groups:control group,TNF-α group(50ng/mL TNF-α treated the cells for 48h),TNF-α+miR-640-NC group(50ng/mL TNF-α treated the cells transfected with miR-640-NC for 48h)and TNF-α+miR-640-in-hibitor group(50ng/mL TNF-α treated the cells transfected with miR-640-inhibitor for 48h).The expression of miR-640 in nucleus pulposus cells was detected by RT-qPCR.The cell viability was detected by CCK-8 assay.Cell apoptosis was examined by flow cytometry.The expressions of Bcl-2,Bax,Cleaved caspase-3,Collagen n and Aggrecan were detected by Western blot.The expression of MMP-9 was observed by im-munofluorescence staining.The LDD rat model was established through the intradiscal injection of TNF-α.The 25 rats were randomly allocated into 5 groups(each consisting of 5 rats):sham group,LDD group,Sham+miR-640-NC group,LDD+miR-640-NC group,and LDD+miR-640-inhibitor group.Specifically,one week pri-or to sham surgery or LDD modeling,rats in the Sham+miR-640-NC,LDD+miR-640-NC,and LDD+miR-640-inhibitor groups were administered with the lentivirus vector carrying miR-640-inhibitor or miR-640-NC into the intervertebral disc space.The expression of miR-640 in nucleus pulposus tissue was detected by RT-qPCR.The morphology of lumbar intervertebral discs was observed by MRI and HE staining.The expres-sion of MMP-9 in nucleus pulposus was observed by immunofluorescence.And the expressions of Cleaved caspase-3,Collagen Ⅱ and Aggrecan in nucleus pulposus was observed by immunohistochemistry.Results:The expression of miR-640 was increased in nucleus pulposus tissue of LDD model rats and nucleus pulpo-sus cells treated with TNF-α(P＜0.05).Compared with TNF-α+miR-640-NC group,cell viability and the ex-pressions of Bcl-2,Collagen Ⅱ and Aggrecan were all increased in TNF-α+miR-640-inhibitor group(P＜0.05),while cell apoptosis,and Bax,Cleaved caspase-3 and MMP-9 expressions were decreased(P＜0.05).The LDD+miR-640-NC group showed obvious degenerative changes in lumbar intervertebral disc,the expressions of Collagen Ⅱ and Aggrecan in nucleus pulposus were decreased(P＜0.05),and the expressions of Cleaved cas-pase-3 and MMP-9 were increased(P＜0.05),however,the above changes were reversed to a certain extent in LDD+miR-640-inhibitor group(P＜0.05).Conclusions:In the LDD rat model and in vitro cell model,miR-640 expression is upregulated.Inhibition of miR-640 can alleviate LDD by potentially suppressing nucleus pulpo-sus cell apoptosis,reducing extracellular matrix degradation,and promoting extracellular matrix synthesis.
关键词
腰椎间盘退变/miR-640/髓核/细胞外基质/凋亡/大鼠Key words
Lumbar disc degeneration/miR-640/Nucleus pulposus/Extracellular matrix/Apoptosis/Rat引用本文复制引用
出版年
2024
NETL
NSTL
万方数据