摘要
目的探讨退变的人椎间盘髓核组织中miR-199及肿瘤坏死因子-α(TNF-α)的表达变化及生物学作用。方法收集人退变椎间盘及正常对照髓核组织标本各5例,高通量测序筛查2组样本中表达差异明显的微小RNA,ELISA方法检测TNF-α水平变化及RT-qPCR检测miR-199表达量变化;人髓核细胞株经体外培养后应用TNF-α(20 ng/mL)诱导建立体外细胞凋亡模型;miR-control及miR mimics-199转染至髓核细胞,然后应用TNF-α刺激,探讨miR-199的调控作用。结果与正常髓核组织比较miR-199在退变髓核组织中表达降低,TNF-α表达明显升高(P<0.05)。随着TNF-α诱导时间的延长miR-199表达及髓核细胞增殖下降(P<0.05),而乳酸脱氢酶(LDH)升高(P<0.05)。与正常组比较,TNF-α组细胞增殖能力下降(P<0.05),细胞凋亡率升高(P<0.05),凋亡相关蛋白(Bax、Bcl-2、caspase-3及cleaved caspase-3)表达水平升高(P<0.05);与TNF-α组比较,TNF-α+miR-199mimics组则逆转上述现象,且LDH升高。结论在退变髓核组织中miR-199表达降低,TNF-α表达升高;髓核细胞凋亡可由TNF-α诱导产生,且呈时间依赖性;TNF-α诱导的髓核细胞凋亡可通过上调miR-199表达而逆转,提示miR-199参与椎间盘退变的发病过程,可作为椎间盘退变的早期诊治标志物及治疗靶点。
Objective To investigate the expression levels of miR-199 and tumor necrosis factor-α(TNF-α)in human degenerated intervertebral disc nucleus pulposus tissue and their biological effects.Methods Five tissue samples of degenerated intervertebral discs nucleus pulposus and 5 tissue samples of normal intervertebral discs nucleus pulposus form patients were collected.Differentially expressed microRNA(miRNA)between the two groups was screened by high-throughput sequencing.Changes in TNF-αlevel between groups were detected by enzyme-linked immunosorbent assay(ELISA),and miR-199 level was detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR).In vitro nucleus pulposus cell lines were cultured and induced with TNF-α(20ng/mL)to establish an in vitro apoptosis model.Nucleus pulposus cells were transfected with miR-control and miR-199 mimics,followed by TNF-αinduction,thus exploring the regulatory effect of miR-199.Results Compared with normal nucleus pulposus tissue,miR-199 was significantly decreased in degenerated nucleus pulposus tissue,and TNF-αwas significantly upregulated(P<0.05).With the prolongation of TNF-αinduction,the expression of miR-199 and the cell proliferation ability of nucleus pulposus cells decreased significantly(P<0.05),and Lactate dehydrogenase(LDH)increased significantly(P<0.05).Compared with the normal group,nucleus pulposus cells induced with TNF-αshowed significantly lower viability(P<0.05),and the expression of apoptosis-related proteins(Bax,Bcl-2,caspase-3 and cleaved caspase-3)were significantly increased(P<0.05).While compared with the TNF-αgroup,the TNF-α+miR-199 mimics group reversed the experimental changes mentioned above,and LDH increased.Conclusion Down-regulated miR-199 and up-regulated TNF-αare seen in the nucleus pulposus tissue of degenerative intervertebral disc.Apoptosis of nucleus pulposus cells can be induced by TNF-αin a time-dependent manner,and TNF-α-induced apoptosis of nucleus pulposus cells can be reversed by overexpression of miR-199,suggesting that miR-199 participates in the onset of intervertebral disc degeneration(IDD)and can be used as an early diagnostic and therapeutic biomarker and therapeutic target for IDD.
作者
王伟
郭召
杨利新
乔昱皓
张利
范伟业
WANG Wei;GUO Zhao;YANG Lixin(Fourth Department of Orthopedics,Xiangjiang Branch,the Third Hospital of Hebei Medical University,Hebei,Shijiazhuang 050053,China;不详)
出处
《河北医药》
2025年第2期212-217,共6页
Hebei Medical Journal
基金
河北省自然科学基金精准医学联合基金项目(编号:H2021206139)。
关键词
椎间盘退变
微小RNA
TNF-Α
凋亡
intervertebral disc degeneration
microRNA(miRNA)
tumor necrosis factor-alpha(TNF-α)
apoptosis
