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(50μg/L)组;③白细胞介素6(100μg/L)组;④肿瘤坏死因子α(20μg/L)组;⑤白细胞介素1β(50μg/L)+740Y-P组(PI3K激活剂);⑥白细胞介素6(100μg/L)+740Y-P组;⑦肿瘤坏死因子α(20μg/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1m RNA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αm RNA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。
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(50μg/L)group,IL-6(100μg/L)group,TNF-α(20μg/L)group,IL-1β(50μg/L)+740Y-P(a PI3K activator)group,IL-6(100μg/L)+740Y-P group,TNF-α(20μg/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-αon 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-1βand TNF-αgroups 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-αgroups 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-αin the IL-1β,IL-6 and TNF-αgroups were significantly higher than those in the control group.The expression of interleukin-1ra in the IL-1βand TNF-αgroups 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-1βand TNF-αgroups,and slightly decreased in the IL-6 group.Compared with IL-1β,IL-6 and TNF-αgroups,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-αas inflammatory factors may inhibit the expression of type II collagen in nucleus pulposus cells by inhibiting the PI3K/AKT cell proliferation pathway.
作者
梁卫东
任周梁
盛军
曹锐
盛伟斌
Liang Weidong;Ren Zhouliang;Sheng Jun;Cao Rui;Sheng Weibin(Department of Spinal Surgery,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第21期3410-3417,共8页
Chinese Journal of Tissue Engineering Research
基金
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~~
