山羊颞下颌关节盘细胞肌动蛋白骨架的研究

Study on the Cytoskeleton of Cultured Goat Temporomandibular Joint Disc Cells

目的:通过激光扫描共聚焦显微镜(Laser Scanning Confocal Microscope,LSCM)研究原代及传代山羊颞下颌关节盘细胞骨架(Cytoskeleton,CSK)蛋白-肌动蛋白的形态及蛋白量的改变,以说明关节盘细胞随着传代的发生,细胞骨架蛋白-肌动蛋白的差异。方法:原代(P0)山羊颞下颌关节盘细胞及传代1、2、3、4(P1、P2、P3、P4)代的关节盘细胞,分别接种在有方形盖玻片的6孔板中制作细胞爬片。对各代关节盘细胞肌动蛋白骨架进行免疫荧光染色,利用激光共聚焦显微镜获取各代关节盘细胞肌动蛋白的形态,同时利用荧光强度测定软件对各代关节盘细胞肌动蛋白荧光量进行测定。结果:丝状的肌动蛋白主要沿着细胞膜外围均匀排列,随着传代丝状结构逐渐清晰,且逐渐增粗,P4代时形成较粗的纤维束。荧光强度整体趋势是随着传代荧光量表达是递增的,P3代以内没有统计学差异,而在P4代时统计学差异明显。结论:肌动蛋白骨架随着传代过程形态学上发生了改变,在P4代时可见骨架蛋白形成较粗的纤维束,荧光量表达同其他代细胞差异明显,这为以后工程化颞下颌关节盘种子细胞的选择提供了依据。

Objective：To study the morphology and quantify the cytoskeleton（CSK）-actin in primary and passaged goat temporomandibular joint disc（TMJ）cells,and to illustrate the differences of actin protein during passaging using laser scanning confocal microscope（LCSM）.Methods：The primary,1st,2nd,3rd and 4th（P0,P1,P2,P3,P4）TMJ disc cells were inoculated into 6-well plates with square coverslips placed in each well.The CSK-actin of each generation of TMJ disc cells was stained by immunofluorescence staining and observed under the LCSM and later quantitatively analyzed by a fluorescence intensity software.Results：Filamentous actin mainly located uniformly at the outer periphery of cell membrane.The CSK-actin became thicker and clearer during cell passaging,and thick fiber bundles formed obviously in P4 generation.The fluorescence intensity also increased during passaging and was significantly higher in P4 than in P1,P2 and P3.Conclusion：The morphology of the CSK-actin changed during the passaging of TMJ disc cells.This finding might provide basis for seed cell choose in TMJ disc engineering.