马蹄内翻足大鼠模型踝部骨骼、组织及脊髓蛋白质组学分析

目的应用全反式维甲酸(all-trans retinoic acid,ATRA)诱发Wistar大鼠马蹄内翻足模型为实验材料,应用蛋白质学技术探讨先天性马蹄内翻足发病机理。方法按135mg/kg体重经口给予孕10天Wistar大鼠ATRA,孕21天剖检胎鼠,提取畸形鼠和对照鼠脊髓、踝部组织(去除皮肤)及踝部骨骼总蛋白,双向电泳分离蛋白,考马斯亮蓝染色,PDQuest软件、质谱及生物信息学分析差异蛋白。应用半定量逆转录PCR检测马蹄内翻足畸形鼠和对照鼠脊髓、踝部骨骼和胫骨后肌群组织xiap、col2α1、tnnt1和ngfr基因表达。应用免疫组化染色检测Xiap蛋白表达。应用TUNEL法进行脊髓、踝部骨骼细胞凋亡检测。结果经双向电泳发现马蹄内翻足模型胎鼠多种蛋白质表达存在差异,质谱及生物信息学进一步分析发现Ⅱ型胶原蛋白等16种蛋白表达发生改变。逆转录PCR检测进一步证实xiap、col2α1在脊髓、踝部骨骼和胫骨后肌群组织表达下调,tnnt1在踝部骨骼和胫骨后肌群组织表达下调,ngfr在以上组织表达无异常。免疫组化染色进一步证实Xiap蛋白表达下调。神经、骨骼细胞凋亡发生率是对照组细胞凋亡发生率的5.4和10倍。结论马蹄内翻足大鼠模型踝部组织(去除皮肤)、踝部骨骼及脊髓多种蛋白质存在表达差异。Xiap、Col2α1和sTnT与马蹄内翻足相关,Ngfr与马蹄内翻足无关。ATRA可以诱导脊髓、骨骼凋亡发生率升高,与Xiap表达下调有关,在马蹄内翻足发生过程中发挥重要作用。

Proteomic analysis of the ankle joint bone, ankle joint tissue and spinal cord of clubfoot-like deformity in rat fetuses

OBJECTIVE: To explore the etiology of idiopathic talipes equinovarus (ITEV) in all-trans retinoic acid (ATRA) induced clubfoot-like deformity in rat fetuses with two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). METHODS: Clubfoot-like deformity model in rat fetuses was induced with ATRA (135 mg/kg) in gestation day (GD10) pregnant Wistar rats. 2-DE was applied to separate the total proteins of ankle joint tissue, ankle joint bone and spinal cord of the animal models. The Coomassie Brilliant Blue staining gels were analyzed by 2-DE software PDQuest 7.1.0. Selected differential protein spots were identified with peptide mass fingerprinting based on matrix-assisted laser adsorption/ionization time-of-flight mass spectrometry and database searching. xiap, tnnt1 and col2 alpha 1, three genes of the differential proteins, were identified furthermore. Apoptosis study was made in terminal deoxynucleotidyl transferase nick end labeling. RESULTS: There were many differential expressed proteins in the clubfoot-like deformity model. Out of the differentially expressed proteins,16 protein spots were identified to be differentially expressed in the clubfoot-like deformity model with MS. Three of the 16 protein spots, xiap, tnnt1 and col2 alpha 1 were confirmed to be significantly down-regulated by the RT-PCR, and Xiap was further confirmed to be significantly down-regulated with immunohistochemistry. Another randomly selected gene, ngfr, did not express differently in ATRA-induced clubfoot-like deformity in rat fetuses. The rates of the apoptosis in the spinal, bone of the clubfoot-like deformity fetuses was 5.4 and 10 times of those of the normal fetuses respectively. CONCLUSION: The results suggest that there are certain differently expressed proteins in ankle joint tissue, ankle joint bone and spinal cord of the ATRA-induced clubfoot-like deformity in rat fetuses, and Xiap, sTnT, and Col2 alpha 1 show a significant correlation with ITEV. Ngfr is not correlation with ITEV. Apoptosis plays a key role in the development of ITEV and related to the decreased expression of the Xiap.