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

中文关键词:  腰椎间盘退变  miR-640  髓核  细胞外基质  凋亡  大鼠

英文关键词:Lumbar disc degeneration  miR-640  Nucleus pulposus  Extracellular matrix  Apoptosis  Rat

中文摘要:

  【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）。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。

英文摘要:

  【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-inhibitor 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 Ⅱ and Aggrecan were detected by Western blot. The expression of MMP-9 was observed by immunofluorescence 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 prior 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 expression 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 pulposus cells treated with TNF-α(P<0.05). Compared with TNF-α+miR-640-NC group, cell viability and the expressions 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 caspase-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 pulposus cell apoptosis, reducing extracellular matrix degradation, and promoting extracellular matrix synthesis.