大鼠髁突软骨细胞发育中相关信号通路的初步筛查

目的:通过建立的大鼠髁突软骨细胞发育过程中蛋白表达差异谱,分析可能参与的信号通路,为进一步认识髁突软骨生理性及病理性生长改建的信号调控机制提供相关信息。方法:收获出生后1、7、14、28 d共4组SD大鼠髁突软骨细胞,甲苯胺蓝染色、Ⅱ型胶原免疫组化染色鉴定软骨细胞。提取各组软骨细胞总蛋白,采用i TRAQ标记定量蛋白,2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(MALDI-TOF-TOF),获取出生后大鼠髁突软骨细胞发育过程中差异蛋白的表达谱,所得数据用MASCOT软件处理,筛选样本之间有意义的差异蛋白,运用GO法及David软件进行KEGG信号通路分析。结果 :共鉴定137种具有可信度表达的蛋白,有44种蛋白参与至少27条KEGG信号通路,其中ECM-受体相互作用、焦点粘连、actin骨架调节、Ca2+信号通路、血管平滑肌收缩、Gn RH信号通路、肌醇三磷酸代谢、磷脂酰肌醇信号系统以及核糖体等信号通路具有统计学意义。结论:在大鼠髁突软骨发育中,各信号通路蛋白在时间、空间上差异性表达,共同形成复杂的信号传递网络。

Study of Related-signal Pathways Regulate Chondrocyte Maturation and Differentiation in the Mandibular Condyle of the Rat

Objective： To analyze related-signal pathways regulating proliferation and differentiation of rat mandibular condylar chondrocytes. Methods：The 1-, 7-, 14-, and 28-day-old SD rats′condylar chondrocytes were harvested in vitro, identified by toluidine blue stain and type II collagen immunohistochemistry reaction. After extracting from rat condyle in each group, total proteins were labeled with iTRAQ reagents and resolved using 2D nano-high performance liquid chro-matography （HPLC）. Protein spots were then identified by mass spectrometry. Using matrix-assisted laser desorption ioniza-tion-time-of-flight/time-of-flight technology （MALDI-TOF/TOF） to quantitative analysis the differential protein expression of rat condylar chondrocytes during their development. All data was normalized by MASCOT software to search useful differ-entially expressed proteins, then analyzed by Gene Ontology and David software, KEGG pathway database. Results： 137 differentially reliability proteins were identified, of which 44 proteins were found to be involved in at least 27 signalling subpathways. There were statistically significant differences in ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, calcium signaling pathway, vascular smooth muscle contraction, GnRH signal pathway, inositol phosphate metabolism, phosphatidylinositol signaling system and ribosome. Conclusion： This study indicated that during rat condylar development, differential proteins involved in signal transduction pathways expressed spatially and temporally.