中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (21): 3410-3417.doi: 10.3969/j.issn.2095-4344.1748

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

抑制髓核细胞Ⅱ型胶原表达的炎性因子白细胞介素1β、白细胞介素6及肿瘤坏死因子α

梁卫东，任周梁，盛 军，曹 锐，盛伟斌   

  1. 新疆医科大学第一附属医院脊柱外科，新疆维吾尔自治区乌鲁木齐市 830054
  • 修回日期:2019-02-12 出版日期:2019-07-28 发布日期:2019-07-28
  • 通讯作者: 盛伟斌，博士，主任医师，新疆医科大学第一附属医院脊柱外科，新疆维吾尔自治区乌鲁木齐市 830054
  • 作者简介:梁卫东，男，1983年生，新疆维吾尔自治区阿勒泰市人，蒙古族，2009年新疆医科大学毕业，硕士，主治医师，主要从事骨科相关临床及基础研究。 并列第一作者：任周梁，男，1991年生，四川省仪陇县人，汉族，2016年新疆医科大学毕业，硕士，医师，主要从事骨科相关临床及基础研究。
  • 基金资助:

    新疆医科大学第一附属医院院内科研基金项目(20152RQN05)，项目负责人：梁卫东

Interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha inhibit expression of type II collagen in nucleus pulposus cells

Liang Weidong, Ren Zhouliang, Sheng Jun, Cao Rui, Sheng Weibin   

  1. Department of Spinal Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • Revised:2019-02-12 Online:2019-07-28 Published:2019-07-28
  • Contact: Sheng Weibin, MD, Chief physician, Department of Spinal Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • About author:Liang Weidong, Master, Attending physician, Department of Spinal Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China. Ren Zhouliang, Master, physician, Department of Spinal Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China. Liang Weidong and Ren Zhouliang contributed equally to this work.
  • Supported by:

    Institutional Research Fund Project of the First Affiliated Hospital of Xinjiang Medical University, No. 20152RQN05 (to LWD)

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摘要：

文章快速阅读：

文题释义：
髓核的组成：发育成熟的髓核是一个由软骨样细胞分散在细胞间质内，周围围绕着一个比较致密的胶原纤维网的含水球，位于椎间盘偏后位置，占椎间盘横断面积的50%-60%；由于它的含水量很高，并和软骨终板紧密接触，是椎间盘接受经软骨终板主要营养途径渗透交换营养的主要部分。
胶原蛋白Ⅱ型：胶原蛋白有不同的类型，目前为止已经发现了几十种。其中Ⅰ、Ⅲ型主要存在于皮肤血管等结缔组织中；Ⅱ型主要由软骨细胞产生，多存在于骨骼、关节、肌腱等组织；Ⅶ型主要存在于子宫胎盘中。
椎间盘退变：随着年龄的增长，髓核、纤维环及软骨终板等椎间盘各个组织的老化退变，是一系列脊柱退行性疾病的前提和病理基础，如椎间盘突出、脊柱不稳、神经根病变等。病理表现为椎间盘细胞数量减少和功能降低，导致聚合蛋白聚糖和Ⅱ型胶原蛋白进行性减少。

 

摘要
背景：炎性因子在椎间盘退变中起着重要的作用，其抑制Ⅱ型胶原表达机制尚未完全明确。
目的：探讨炎性因子白细胞介素1β、白细胞介素6、肿瘤坏死因子α对髓核细胞Ⅱ型胶原表达的影响及作用机制。  
方法：成年雄性SD大鼠由新疆医科大学动物实验中心提供，实验方案经新疆医科大学第一附属医院动物实验伦理委员会批准(批准号为IACUC20151203-09)。分离、培养及鉴定SD大鼠髓核细胞，将其随机分为7组：①对照组；②白细胞介素1β(50 μg/L)组；③白细胞介素6(100 μg/L)组；④肿瘤坏死因子α(20 μg/L)组；⑤白细胞介素1β(50 μg/L)+740Y-P组(PI3K激活剂)；⑥白细胞介素6(100 μg/L)+740Y-P组；⑦肿瘤坏死因子α (20 μg/L)+740Y-P组；并进行相应干预。48 h后收集髓核细胞进行RT-PCR，Western Blot检测及免疫荧光染色，分别检测白细胞介素1β、白细胞介素6和肿瘤坏死因子α对髓核细胞中基质金属蛋白/金属蛋白酶组织抑制因子，炎性因子表达对Ⅰ型、Ⅱ型胶原蛋白，聚集蛋白聚糖以及PI3K/AKT信号通路蛋白表达的影响。
结果与结论：①白细胞介素1β组和肿瘤坏死因子α组中金属蛋白酶组织抑制因子1 mRNA表达量明显低于对照组(P < 0.05)；白细胞介素1β、白细胞介素6和肿瘤坏死因子α组中基质金属蛋白酶9和基质金属蛋白酶13 mRNA表达量明显高于对照组(P < 0.05)；所有实验干预组中白细胞介素6 mRNA表达量明显高于对照组(P < 0.05)；白细胞介素1β、白细胞介素6和肿瘤坏死因子α组中白细胞介素1β和肿瘤坏死因子α mRNA表达量明显高于对照组；白细胞介素1β和肿瘤坏死因子α组中白细胞介素1ra表达量明显低于对照组(P < 0.05)；②与对照组相比，白细胞介素1β和肿瘤坏死因子α组的Ⅱ型胶原和聚集蛋白聚糖表达明显降低，而白细胞介素6组的则略有降低；与白细胞介素1β、白细胞介素6和肿瘤坏死因子α组相比，对应的给予PI3K激活剂组的表达则明显升高；③结果表明：炎性因子白细胞介素1β、白细胞介素6、肿瘤坏死因子α可能通过抑制PI3K/AKT细胞增殖通路来抑制髓核细胞Ⅱ型胶原的表达。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-2532-2335(梁卫东)；0000-0002-3342-2504(任周梁)

关键词: 白细胞介素1β, 白细胞介素6, 肿瘤坏死因子α, 髓核细胞, Ⅱ型胶原, PI3K/AKT

Abstract:

BACKGROUND: The role of inflammatory factors in the degeneration of intervertebral discs has received increasing attention. The mechanism by which inflammatory factors inhibit the expression of type II collagen has not been fully elucidated.
OBJECTIVE: To observe the expression of type II collagen in nucleus pulposus cells after intervention with inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and the mechanism of action.
METHODS: Adult male Sprague-Dawley rats were provided by the Animal Experimental Center of Xinjiang Medical University, and the study protocol was approved by the Animal Experimental Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval No. IACUC20151203-09). Nucleus pulposus cells from rats were isolated, cultured, identified and randomly divided into seven groups: control group, IL-1β (50 μg/L) group, IL-6 (100 μg/L) group, TNF-α (20 μg/L) group, IL-1β (50 μg/L)+740Y-P (a PI3K activator) group, IL-6 (100 μg/L)+ 740Y-P group, TNF-α (20 μg/L)+740Y-P group. After 48 hours of intervention, nucleus pulposus cells were collected. RT-PCR, western blot and immunofluorescence staining were used to detect the effects of IL-1β, IL-6 and TNF-α on expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase, type I and type II collagens, Aggrecan, and PI3K/AKT signaling pathway proteins in nucleus pulposus cells.
RESULTS AND CONCLUSION: (1) The expression of tissue inhibitor of metalloproteinase-1 mRNA in IL-1β and TNF-α groups was significantly lower than that in the control group (P < 0.05). The mRNA levels of matrix metalloproteinases-9 and -13 in the IL-1β, IL-6 and TNF-α groups were significantly higher than those in the control group (P < 0.05). The expression of IL-6 mRNA in all experimental intervention groups was significantly higher than that in the control group (P < 0.05). The mRNA levels of IL-1β, IL-6 and TNF-α in the IL-1β, IL-6 and TNF-α groups were significantly higher than those in the control group. The expression of interleukin-1ra in the IL-1β and TNF-α groups was significantly lower than that in the control group (P < 0.05). (2) Immunofluorescence results showed that compared with the control group, the expression of type II collagen and Aggrecan significantly decreased in the IL-1β and TNF-α groups, and slightly decreased in the IL-6 group. Compared with IL-1β, IL-6 and TNF-α groups, the expression of type II collagen and Aggrecan was significantly increased in the three corresponding PI3K activator groups. These findings indicate that IL-1β, IL-6 and TNF-α as inflammatory factors may inhibit the expression of type II collagen in nucleus pulposus cells by inhibiting the PI3K/AKT cell proliferation pathway.

Key words: interleukin-1β, interleukin-6, tumor necrosis factor-α, nucleus pulposus cells, type II collagen, PI3K/AKT

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