兔颞下颌关节盘纤维软骨细胞分离培养的实验研究  

Experimental Study of Isolation and Cultivation of Rabbit Temporomandibular Joint Disc Fibrochondrocytes

作　　者：匡世军[1] 张志光[1] 何一青[1] 廖明庭[1] 苏凯[1] 张娟[1] 

机构地区：[1]中山大学光华口腔医学院附属口腔医院口腔颌面外科,广东广州510055

出　　处：《中山大学学报（医学科学版）》2006年第3期318-321,共4页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金资助项目(20010754)

摘　　要：目的探讨颞下颌关节盘纤维软骨细胞的分离及体外扩增的条件,观察关节盘纤维软骨细胞体外培养的生物学特性。方法在无菌条件下,切取2周龄新西兰白兔颞下颌关节盘,剪至1.0m3的碎块,0.5g/mL胰酶消化30min、胶原酶消化4h,200目筛网过滤,获得关节盘纤维软骨细胞。在倒置显微镜下连续观察细胞的形态变化及贴壁率,甲苯胺蓝染色、、胶原免疫组化染色鉴定,测定其生长曲线。透射电镜观察细胞超微结构。结果原代培养的关节盘纤维软骨细胞12h可观察到贴壁细胞,48h贴壁细胞逐渐增多,大部分细胞为短梭形,小部分为多角形,第4天可见多个细胞克隆形成,第8天时细胞彼此相连,铺满平底,细胞以梭形为主,部分多角形。传代后12h贴壁率达90%,大部分为多角形,4～5d即可张满瓶底。甲苯胺蓝染色可见异染颗粒,、胶原免疫组化染色胞浆内可见棕黄色颗粒。细胞内线粒体、内质网发达、高尔基体发达。结论体外分离培养的兔颞下颌关节盘纤维软骨细胞具有较强的增殖能力。[Objective] This study were to explore a practical method of the separation and culture of fibrochondrecytes from rabbit temperomandibular joint （TMJ） disc, and to observe the biological characteristics of cultured fibrechondrecytes. [Methods] The small pieces （1.0 mm^3 in size） of isolated and chopped Rabbit TMJ disc, which was dissected from a 2-week-old New Zealand white rabbits under sterile conditions, were digested with 0.5% trypsin for 30 minutes followed by a four-hour digestion with collagenase. The solution was filtered （with 200 mesh fiher） and collected. Morphological changes and the rate of adherence were constantly observed with phase-contrast microscope. Immunohistochemical staining for type Ⅰ and type Ⅱ collagen as well as glycosaminoglycans were performed in order to plot the growth curve.Ultrastructures of fibrechondrocytes were observed under transmission electron microscope.[Result] At 12th hour of incubation, primary fibrochondrocytes began to be adhesive. At 48th hour of incubation, the increased number of adhesive fibrochondrecytes was noticed. Most of them presented in short spindle-shaped while the rest were polygon-shaped. On day 4, the formation of colonies could be seen. Then most of the cells were spindle-shaped. On day 8, the perliferating fibrochondrocytes, which most of them were spindle-shaped and some were polygon-shaped, started to meet （or： contact） each other to form a monolayer of fibrechondrecytes covering the bottom of the incubation disc. At 12th hour after passage, the rate of adherence of the fibrochondrocytes was 90%. Most of them were polygon-shaped. At 5th day after passage, these adhesive cells filled with the bottom of the incubation disc. Immunohistochemical staining of type I and I1 collagen as well as glycosaminoglycans exhibited positive results.The fibrechondrocytes cytoplasms were rich in mictochondria, endoplasm reticulum and Golgi apparatus. [Conclusion] The fibrechondreytes of isolated rabbit TMJ disc presented proliferous ability in c

关 键 词：纤维软骨 细胞培养 组织工程 关节盘 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]