rhBMP2作用下人牙周膜成纤维细胞中cbfα1的表达研究

摘要

目的:本实验通过反转录聚合酶链反应,测定不同浓度的重组人骨形成蛋白2(rhBMP2)对人牙周膜成纤维细胞中cbfαlmRNA在不同作用时间点表达,了解rhBMP2对骨改建过程的调控.方法:原代培养人牙周膜成纤维细胞,取生长良好的第6代细胞,分别用25ng/ml、50ng/ml及100ng/ml的rhBMP2作用于细胞,于作用1、3、5、7天后收集细胞.反转录聚合酶链反应检测各组细胞4个时间作用点cbfα1 mRNA含量,PCR产物1%琼脂糖凝胶电泳后,采用Image Pr0 Plus5.0图像分析软件对电泳胶带亮度进行分析.结果:不同浓度rhBMP2对cbfα1mRNA的表达均表现为初期降低,而后明显增高,至峰值后回落.不同浓度对cbfα1 mRNA表达上调的峰值没有明显差异.100ng/ml和50ng/ml的rhBMP2浓度组能够较快地上调cbfα1mRNA表达至峰值,然后100ng/ml组表现为缓慢下降,50ng/ml组迅速回落;25ng/ml组较慢上调cbfα1mRNA的表达至峰值后,迅速回落.结论:①根据实验各组中cbfα1的表达变化,提示笔者BMP2可以上调HPDLfs中cbfα1的转录水平,并且对cbfα1的表达调控主要为启动作用,cbfα1的表达增高幅度似乎与rhBMP2浓度并不相关.②高浓度组对cbfα1的上调作用迅速而持久,低浓度组的作用则相对迟缓而短暂.提示笔者BMP2可能通过上调cbfα1的表达而促进HPDLfs向成骨细胞转化.%Objective By RT-PCR to investigating the effects of rhBMPs in different concentration on the expression of cbfα1 in human periodontal ligament fibroblasts.To clarify the molecular mechanism of BMPs' regulation to the HPDLfs,which contribute to the bone rebuilding. Methods The 6th generation well-grown primary culture fibrocyte from HPDLfs were used in the experiment. The cells were subjected to different doses of rhBMP2 (25ng/ml,50ng/ml and 100ng/ml) for 1,3,5,7 days in this experiment. mRNA expression of cbfα1 were determined by semiquantitative RT-PCR approach.electrophoresis tape brightness was analyzed by graphical analysis software Image Pro Plus5.0. Results The expression of cbfα1mRNA acted by each rhBMP2 of different concentration fell down in primary stage,then increased obviously and decreased after reaching the peak value.The peak value of expression which reacted in different concentration presented no significant difference. When the expression of cbfα1mRNA regulated rapidly to peak value in both concentration 50ng/ml and 100ng/ml,the expression decreased smoothly in 100ng/ml,but decreased sharply in 50ng/ml.The expression increased smoothly and decreased sharply in 25ng/ml at the same time.Conclusion ① Based on the different expression variety of cbfα1 in experiments,the results showed that BMPs can enhance the mRNA of cbfα1 in HPDLfs and acted mainly as startup effect on the expression regulation of cbfα1. The enhanced amplitude of cbfα1 expression seemed to be no relation with the rhBMP2 concentration. ②. The enhancive effect in high concentration on cbfα1 was quick and abiding,but it was slow and unabiding in low concentration.