中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2013年
31期
2474-2477
,共4页
熊寿良%徐宏光%王弘%张敏%俞云飞%张巍%涂成东%赵泉来%吕坤
熊壽良%徐宏光%王弘%張敏%俞雲飛%張巍%塗成東%趙泉來%呂坤
웅수량%서굉광%왕홍%장민%유운비%장외%도성동%조천래%려곤
椎间盘%颈椎%软骨细胞%基因表达
椎間盤%頸椎%軟骨細胞%基因錶達
추간반%경추%연골세포%기인표체
Intervertebral disc%Cervical vertebrae%Chondrocytes%Gene expression
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.
目的 探討自噬在人頸椎椎體終闆軟骨細胞退變模型中的作用及意義.方法 選擇2012年2至8月皖南醫學院弋磯山醫院脊柱外科住院接受頸前路椎體次全切除手術的頸椎病患者和頸椎骨摺或脫位者48例.分為對照組(17例)和頸椎病組(31例).將術中取齣的頸椎椎體終闆軟骨用酶消化法分離終闆軟骨細胞併培養,建立人頸椎椎體終闆軟骨細胞退變模型.甲苯胺藍染色,倒置相差顯微鏡及HE染色觀察細胞形態學變化,單丹磺酰戊二胺(MDC)染色觀察自噬小體,激光共聚焦顯微鏡觀察自噬標誌性蛋白微管相關蛋白1輕鏈3(LC3蛋白),運用反轉錄聚閤酶鏈反應檢測Ⅱ型膠原、蛋白多糖及免疫印跡檢測LC3蛋白的錶達.結果 成功建立人頸椎椎體終闆軟骨細胞退變模型.對照組原代終闆軟骨細胞以多角形為主,增殖速度較快,而頸椎病組原代終闆軟骨細胞以梭形為主,細胞增殖速度較對照組慢.兩組細胞中MDC染色均可見自噬小體,激光共聚焦顯微鏡下均可見LC3蛋白位于胞內和覈週.頸椎病組終闆軟骨細胞Ⅱ型膠原基因(0.628±0.254)及蛋白多糖基因(0.715 ±0.194)的錶達均低于對照組(0.845 ±0.186,0.913 ±0.254,均P＜0.05).對照組中LC3-Ⅱ/LC3-I較頸椎病組明顯上調.結論 自噬在人頸椎終闆軟骨細胞退變過程中起著重要作用.調控終闆軟骨細胞中自噬的活性可能會改善椎間盤的退變.
목적 탐토자서재인경추추체종판연골세포퇴변모형중적작용급의의.방법 선택2012년2지8월환남의학원익기산의원척주외과주원접수경전로추체차전절제수술적경추병환자화경추골절혹탈위자48례.분위대조조(17례)화경추병조(31례).장술중취출적경추추체종판연골용매소화법분리종판연골세포병배양,건립인경추추체종판연골세포퇴변모형.갑분알람염색,도치상차현미경급HE염색관찰세포형태학변화,단단광선무이알(MDC)염색관찰자서소체,격광공취초현미경관찰자서표지성단백미관상관단백1경련3(LC3단백),운용반전록취합매련반응검측Ⅱ형효원、단백다당급면역인적검측LC3단백적표체.결과 성공건립인경추추체종판연골세포퇴변모형.대조조원대종판연골세포이다각형위주,증식속도교쾌,이경추병조원대종판연골세포이사형위주,세포증식속도교대조조만.량조세포중MDC염색균가견자서소체,격광공취초현미경하균가견LC3단백위우포내화핵주.경추병조종판연골세포Ⅱ형효원기인(0.628±0.254)급단백다당기인(0.715 ±0.194)적표체균저우대조조(0.845 ±0.186,0.913 ±0.254,균P＜0.05).대조조중LC3-Ⅱ/LC3-I교경추병조명현상조.결론 자서재인경추종판연골세포퇴변과정중기착중요작용.조공종판연골세포중자서적활성가능회개선추간반적퇴변.
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 chondrocytes; 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 chondrocytes 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.
