自噬在人颈椎椎体终板软骨细胞退变模型中的表达变化及其意义  

Autophagy expression and its significance in chondrocytes from a degenerate model of human cervical endplate

作　　者：熊寿良[1] 徐宏光[1] 王弘[1] 张敏[1] 俞云飞[1] 张巍[1] 涂成东[1] 赵泉来[1] 吕坤[2] 

机构地区：[1]241001安徽芜湖,皖南医学院附属弋矶山医院脊柱外科 [2]241001安徽芜湖,皖南医学院附属弋矶山医院中心实验室

出　　处：《中华医学杂志》2013年第31期2474-2477,共4页National Medical Journal of China

基　　金：国家自然科学基金（30973025、81272048）;卫生部公益性行业专项基金（201002018）;安徽省自然科学基金（1308085MH152）

摘　　要：目的 探讨自噬在人颈椎椎体终板软骨细胞退变模型中的作用及意义.方法 选择2012年2至8月皖南医学院弋矶山医院脊柱外科住院接受颈前路椎体次全切除手术的颈椎病患者和颈椎骨折或脱位者48例.分为对照组（17例）和颈椎病组（31例）.将术中取出的颈椎椎体终板软骨用酶消化法分离终板软骨细胞并培养,建立人颈椎椎体终板软骨细胞退变模型.甲苯胺蓝染色,倒置相差显微镜及HE染色观察细胞形态学变化,单丹磺酰戊二胺（MDC）染色观察自噬小体,激光共聚焦显微镜观察自噬标志性蛋白微管相关蛋白1轻链3（LC3蛋白）,运用反转录聚合酶链反应检测Ⅱ型胶原、蛋白多糖及免疫印迹检测LC3蛋白的表达.结果 成功建立人颈椎椎体终板软骨细胞退变模型.对照组原代终板软骨细胞以多角形为主,增殖速度较快,而颈椎病组原代终板软骨细胞以梭形为主,细胞增殖速度较对照组慢.两组细胞中MDC染色均可见自噬小体,激光共聚焦显微镜下均可见LC3蛋白位于胞内和核周.颈椎病组终板软骨细胞Ⅱ型胶原基因（0.628±0.254）及蛋白多糖基因（0.715 ±0.194）的表达均低于对照组（0.845 ±0.186,0.913 ±0.254,均P＜0.05）.对照组中LC3-Ⅱ/LC3-I较颈椎病组明显上调.结论 自噬在人颈椎终板软骨细胞退变过程中起着重要作用.调控终板软骨细胞中自噬的活性可能会改善椎间盘的退变.Objective To explore the autophagy expression and examine its significance in chondrocytes in a degenerate model of human cervical vertebrae endplate. Methods Cartilage endplates were obtained from 48 hospitalized patients with cervical vertebral fracture or dislocation at our hospital between February 2012 to August 2012. They were divided into cervical spondylosis group with cervical spondylotic myelopathy （n = 31 ） and control group （n = 17 ）. Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro. The cells were stained with toluidine blue and hematoxylin and eosin; laser scanning confocal microscope and monodansyl cadaverine （MDC） were used to observe autophagy in endplate chondroeytes; reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the mRNA expression of type Ⅱ collagen and aggrecan and Western blot for the protein of LC3. Results A degenerative cell model of human cervical endplate ehondrocytes was established successfully in vitro. Compared with the common group, the cellular morphologies of degenerative group showed spindle changes. Autophagic body was stained with MDC. Intracellular and perinuclear LC3 protein was detected by laser confocal microscopy. Compared with the control group, the mRNA expressions of aggrecan （0. 715 ± 0. 194） and type Ⅱ collagen （0. 628 ± 0. 254 ） markedly decreased （ 0. 845 ± 0. 186,0. 913 ± 0. 254, P 〈0. 05） and LC3-Ⅱ/LC3-I declined in cervical spondylosis group. Conclusion Autophagy plays an important pathogenic role in the process of human cervical disc degeneration. And regulating its expression may improve disc degeneration in endplate cartilage cells.

关 键 词：椎间盘 颈椎 软骨细胞 基因表达 

分 类 号：R681.5[医药卫生—骨科学]