大鼠髁突软骨细胞双向电泳图谱的比较

Study on the method of two dimensional polycrylamide gel electrophoresis on rat condylar chondrocyte

目的：探讨大鼠髁突软骨细胞的双向电泳方法,比较不同染色方法的图谱,以助于对髁突软骨细胞进行蛋白质组学研究。方法：取第3代大鼠髁突软骨细胞,提取细胞总蛋白质,进行不同pH宽度的双向电泳,对蛋白质定量、样品溶解、聚焦时间、二向平衡时间进行探讨,并比较银染（silver）和改良考马斯亮蓝染色法（coomassi blue-silver）对图谱的影响,对获得的图谱进行Pdquest（Version 7.1）图像分析。结果：采用Bio-Rad标准方法进行双向电泳获得理想的2-DE图谱。大鼠髁突软骨细胞蛋白质大多集中在pH4~7范围。在窄范围的pH4～7分离后,蛋白质点分散。考马斯亮蓝染色检测到（1203±86）个蛋白质点,银染色检测到（1769±97）个蛋白质点。与改良的考马斯亮蓝染色比较,银染能更好地显示低丰度蛋白质,但背景加深。结论：大鼠髁突软骨细胞蛋白质的双向电泳方法可靠,结果较为满意,丰富了蛋白质组学数据库,为颞下颌关节的深入研究提供了可靠的实验依据;考马斯亮蓝染色蛋白质点清晰,背景干净,更利于后续的质谱鉴定。

PURPOSE： To investigate the protein profile by two dimensional polycrylamide gel electrophoresis on the rat condylar chondrocyte in vitro.METHODS： The third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats in this study.The protein profile of the rat mandibular condylar chondrocytes was examined by two dimensional polycrylamide gel electrophoresis（2-DE-PAGE）.The 2-DE gel maps on different pH gradients were obtained.The result of modified coomassi blue-sliver staining and sliver staining was compared using Pdquest 7.1 image analysis software.RESULTS： The results showed that the good protein profile of the condylar chondrocytes was obtained by standard Bio-Rad manual.The protein was mainly in the field from pH4 to pH7.The 1203 ±86 protein points were examined on 2-DE gel map by modified coomassi blue-sliver staining,and 1769 ±97 protein points was examined by sliver staining.The silver staining map showed more distinctly but higher background than modified coomassi blue-sliver staining.CONCLUSIONS： The protein profile of the condylar chondrocytes enriches the proteomic database and gives evidence to further proteomic research.The 2-DE map obtained by modified coomassi blue-sliver staining is more suitable for MALDI-TOF mass identification.