内质网应激对吸烟诱导的髓核细胞凋亡与炎性反应的影响研究  

Influence of endoplasmic reticulum stress on smoking-induced nucleus pulposus cells apoptosis and inflammatory response

作　　者：侯孝忠 徐林飞 易威威[3] 陈艳阳[3] 沈皆亮[3] HOU Xiaozhong;XU Linfei;YI Weiwei;CHEN Yanyang;SHEN Jieliang(Department of Orthopedics,Henan Provincial Hospital,Zhengzhou Henan,450000,P.R.China;Department of Orthopedics,Henan Provincial Chest Hospital,Zhengzhou Henan,450003,P.R.China;Department of Orthopedics,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400042,P.R.China)

机构地区：[1]河南省省立医院骨科,郑州450000 [2]河南省胸科医院骨科,郑州450003 [3]重庆医科大学附属第一医院骨科,重庆400042

出　　处：《中国修复重建外科杂志》2019年第6期736-742,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：重庆市科学技术委员会基础研究与前沿探索一般项目资助(cstc2018jcyjAX0059)~~

摘　　要：目的探讨内质网应激对吸烟诱导的髓核细胞凋亡与炎性反应的影响。方法以2016年10月-2018年10月25例接受椎间盘摘除术的颈椎间盘突出症患者为研究对象,分为吸烟组(14例)和非吸烟组(11例)。两组患者年龄、性别、突出节段、Pfirrmann分级比较,差异均无统计学意义(P>0.05)。将术中获取的髓核组织,行TUNEL染色和Western blot检测,观察细胞凋亡情况。然后,采用酶序贯消化法分离培养髓核细胞并传代,取第3代细胞行ELISA检测炎性因子IL-1β和TNF-α含量,免疫荧光染色观察细胞中P65核转移情况,Western blot检测内质网应激相关蛋白GRP78和CHOP表达,透射电镜观察细胞内质网超微结构。最后,为验证内质网调控作用,采用特异性抑制剂4-PBA处理吸烟组髓核细胞后,Western blot检测GRP78、CHOP、IL-1β、TNF-α及P65蛋白相对表达量,流式细胞术检测细胞凋亡率;并以吸烟组未作处理的髓核细胞作对照。结果髓核组织观测显示,吸烟组细胞凋亡率及凋亡相关蛋白Caspase-3和PARP相对表达量均明显高于非吸烟组(P<0.05)。髓核细胞观测显示,吸烟组髓核细胞分泌IL-1β、TNF-α水平以及GRP78、CHOP蛋白相对表达量均明显高于非吸烟组(P<0.05);免疫荧光染色示吸烟组细胞P65主要表达在细胞核,而非吸烟组在细胞质;透射电镜观察显示,与非吸烟组相比,吸烟组内质网处于应激损伤状态。吸烟组髓核细胞经4-PBA处理后,GRP78、CHOP、IL-1β、TNF-α及P65蛋白相对表达量以及细胞凋亡率均较处理前明显降低(P<0.05)。结论吸烟可通过内质网应激导致髓核细胞凋亡和炎性反应加剧,抑制内质网应激有望成为延缓吸烟导致椎间盘退变的新靶点。Objective To investigate the influence of endoplasmic reticulum stress(ERS) on smoking-induced nucleus pulposus cells apoptosis and inflammatory response. Methods Between October 2016 and October 2018, 25 patients with cervical disc herniation receiving discectomy were collected and divided into smoking group(14 cases) and non-smoking group(11 cases). The baseline data of age, gender, herniated segment, and Pfirrmann grading showed no significant difference between the two groups(P>0.05). The obtained nucelus pulposus tissues were harvested to observe the cell apoptosis via detecting the apoptosis-related proteins(Caspase-3 and PRAP) by TUNEL staining and Western blot test. The nucleus pulposus cells were isolated and cultured with enzyme digestion, of which the third generation cells were used in follow-up experiments. Then, the expressions of inflammatory factors [interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α)] were detected by ELISA;the nuclear translocation of P65 was monitored by cell immunofluorescence staining. Furthermore, ERS-related proteins(GRP78 and CHOP) were detected by Western blot;and endoplasmic reticulum ultrastructure was observed under transmission electron microscope. To verify the regulatory effect of ERS, cells were pretreated by ERS specific inhibitor(4-PBA), then cell apoptosis and inflammatory response were tested.Results The nucleus pulposus tissue observation showed that the cell apoptotic rate and the expressions of apoptosisrelated proteins(Caspase-3 and PARP) were obviously higher in smoking group than in non-smoking group(P<0.05).The nucleus pulposus cells observation indicated that the expressions of the inflammatory factors(IL-1β and TNF-α) and the ERS-related proteins(GRP78 and CHOP) were also higher in smoking group than in non-smoking group(P<0.05).The results of cell immunofluorescence staining further confirmed that smoking stimulated nuclear translocation of P65 in nucleus pulposus cells. The ERS injury was much more serious in smoking group than in non-smok

关 键 词：吸烟 椎间盘退变 细胞凋亡 炎性反应 内质网应激 

分 类 号：R681.5[医药卫生—骨科学]