人退变髓核细胞中内质网应激水平的观察研究  

Endoplasmic reticulum stress in degenerative human nucleus pulposus cells

作　　者：文天用 刘月[2] 程实 应金威[2] 阮狄克 WEN Tianyong;LIU Yue;CHENG Shi;YING Jinwei;RUAN Dike(Department of Orthopedics, Xijing Hospital, Air Force Military Medical University, Xi＇ an, Shaanxi Province, 710032;Department of Orthopedic Surgery, Navy General Hospital, Beijing, 100048, China)

机构地区：[1]空军军医大学西京医院骨科,西安710032 [2]海军总医院骨科,北京100048

出　　处：《第三军医大学学报》2018年第12期1098-1103,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81472121);海军总医院创新培育基金(CXPY201402)~~

摘　　要：目的观察探讨退变与正常髓核组织中内质网应激水平的差异。方法获取正常和退变髓核组织共9例(均为海军总医院骨科2016年1月至2017年6月期间住院手术患者),取组织及分离培养髓核细胞,通过透射电镜观察内质网形态、免疫组化法对髓核组织中葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)、C/EBP同源蛋白(C/EBP homologous protein,CHOP)的表达分布进行定性分析,RT-PCR及Western blot对髓核细胞中GRP78、CHOP进行mRNA和蛋白质水平的分析。结果透射电镜结果显示退变髓核细胞的内质网扩张、肿胀,正常片状折叠结构消失;免疫组化染色及Western blot检测结果均可见退变髓核组织GRP78、CHOP的表达升高;RT-PCR提示较之于正常组,退变组GRP78[(1.076±0.13)vs(2.471±0.53)]、CHOP[(1.09±0.21)vs(6.873±1.4)]明显升高(P<0.05)。结论退变髓核细胞中内质网应激水平较正常髓核细胞升高,提示内质网应激可能在髓核细胞退变过程中发挥作用。ObjectiveTo investigate the difference in the levels of endoplasmic reticulum （ER） stress between degenerative and normal human nucleus pulposus cells. MethodsNucleus pulposus tissues were collected from specimens of normal and degenerative intervertebral discs from 9 surgical patients （mean age 33 years） admitted in the Department of Orthopedic Surgery, Navy General Hospital between January, 2016 and June, 2017. The nucleus pulposus cells （NPCs） were isolated and cultured, and the morphology of the ER was observed using transmission electron microscopy （TEM）. The expression levels of 2 ER stress-related molecules, namely glucose-regulated protein 78 （GRP78） and C/EBP homologous protein （CHOP）, were analyzed using immunohistochemical assay, real-time PCR and Western blotting. ResultsTEM revealed obvious ER dilation and swelling in degenerative NPCs as compared with normal NPCs. Immunohistochemical assay and Western blotting showed obviously increased expressions of GRP78 and CHOP proteins in degenerative NPCs. GRP78 and CHOP mRNA expressions were also significantly increased in degenerative as compared with normal NPCs （2.471±0.53 vs 1.076±0.13, and 6.873±1.4 vs 1.09±0.21, respectively; P〈0.05）. ConclusionThe abnormal morphology of ER and increased expression of ER stress markers in degenerative NPCs suggest the involvement of ER stress in the development of NPC degeneration.

关 键 词：椎间盘退变 内质网应激 人髓核细胞 透射电镜 

分 类 号：R361.3[医药卫生—病理学] R394.3[医药卫生—基础医学]