CAJ | 학술논문

目的探讨骨性关节炎软骨下骨成骨细胞的分离、培养、鉴定方法及生长特性.方法复制改良Hulth兔膝关节不稳模型；采用Ⅰ型胶原酶消化联合组织块贴附法获得软骨下骨成骨细胞；应用倒置显微镜、Ⅰ型胶原免疫化学染色以及瑞氏-姬姆萨染色来进行形态学观察和生物学鉴定；利用软骨细胞与滑膜细胞分别于软骨下骨成骨细胞共培养,应用四甲基偶氮唑蓝检测软骨下骨成骨细胞的增殖活性；检测细胞的Ⅰ型胶原在基因水平的表达.结果 Ⅰ型胶原酶消化后在组织块贴附第11天有细胞开始从组织块周围爬出；Ⅰ型胶原酶免疫化学染色后可见胞浆内出现黄褐色颗粒,为阳性反应.瑞氏-姬姆萨染色显示细胞染成蓝紫色,胞核饱满,核仁清晰；四甲基偶氮唑蓝检测显示在3d时,滑膜细胞和软骨细胞对于软骨下骨成骨细胞的增殖有着明显的促进作用,在第6、10、14天时这种促进作用变得不明显,而软骨下骨成骨细胞的增殖活性超过2个共培养组.Ⅰ型胶原表达水平检测显示在第10天时,软骨细胞对于软骨下骨成骨细胞分泌Ⅰ型胶原的促进作用最强,而滑膜细胞对其的促进作用不明显.结论酶消化联合组织块贴附法可获得理想的软骨下骨成骨细胞；利用Ⅰ型胶原酶免疫化学染色鉴别软骨下骨成骨细胞方法简便易行；利用软骨下骨成骨细胞与软骨细胞和滑膜细胞共培养的体系适用于骨性关节炎(0A)微环境的研究,且该体系可以模拟类OA软骨下骨成骨细胞、滑膜细胞和软骨细胞相互影响作用的进程.
목적 탐토골성관절염연골하골성골세포적분리、배양、감정방법급생장특성.방법 복제개량Hulth토슬관절불은모형；채용Ⅰ형효원매소화연합조직괴첩부법획득연골하골성골세포；응용도치현미경、Ⅰ형효원면역화학염색이급서씨-희모살염색래진행형태학관찰화생물학감정；이용연골세포여활막세포분별우연골하골성골세포공배양,응용사갑기우담서람검측연골하골성골세포적증식활성；검측세포적Ⅰ형효원재기인수평적표체.결과 Ⅰ형효원매소화후재조직괴첩부제11천유세포개시종조직괴주위파출；Ⅰ형효원매면역화학염색후가견포장내출현황갈색과립,위양성반응.서씨-희모살염색현시세포염성람자색,포핵포만,핵인청석；사갑기우담서람검측현시재3d시,활막세포화연골세포대우연골하골성골세포적증식유착명현적촉진작용,재제6、10、14천시저충촉진작용변득불명현,이연골하골성골세포적증식활성초과2개공배양조.Ⅰ형효원표체수평검측현시재제10천시,연골세포대우연골하골성골세포분비Ⅰ형효원적촉진작용최강,이활막세포대기적촉진작용불명현.결론 매소화연합조직괴첩부법가획득이상적연골하골성골세포；이용Ⅰ형효원매면역화학염색감별연골하골성골세포방법간편역행；이용연골하골성골세포여연골세포화활막세포공배양적체계괄용우골성관절염(0A)미배경적연구,차해체계가이모의류OA연골하골성골세포、활막세포화연골세포상호영향작용적진정.
Objective To study the method of cell isolation,primary culture and identification of subchondral bone cell of osteoarthritis(OA) rabbits.Methods The rabbit instable knee joint models were made by modified Hulth modeling method.The osteoblasts were harvested from the subchondral bone of rabbits by collagenase and tissue explants attachment.The morphology observation and biological identification were performed by inverted microscope and immunocytochemistry staining,respectively.The proliferative activity of cells were detected by MTT and the expression of Ⅰ-collagen at gene level was detected.Results The cells started to appeared on the 11th day after the attachment.The cells form were fusiformis and triangle,the nucleolus were clear.The cultured cells had typical osteoblast morphological characteristics.The cells obtained from subchondral bone of rabbits were identified to be osteoblast by immunocytochemistry staining.The proliferative activity of cells were equably proliferation which detected by MTT.Conclusion The modified method provides better way to obtain ideal subchondral osteoblast and the co-culture method is suitable for the study of OA microenvironment,which can simulate interactions of the subchondral osteoblast,synovial cells and chondrocyte.