The effect on dentin-pulp complex by FIP-2 isolated from rat wounded pulp

牙髓创伤愈合后的牙洞准备可能涉及与牙洞位置有关的反应性或修复性牙本质形成；然而，对分子反应知之甚少。我们的目标是分离和分析损伤牙髓中诱导或抑制的基因，以确定参与牙髓损伤反应的分子过程。以健康大鼠牙髓和损伤大鼠牙髓组织为材料，通过基因消减技术，获得23条cDNA片段。通过文库筛选，我们筛选到了与人FIP-2同源的大鼠14.7K相互作用蛋白(RFIP)-2A和B基因，参与调控膜转运和细胞形态发生。RT-PCR分析表明，在损伤的牙髓中只有rFIP-2B的诱导。原位杂交分析显示rFIP-2s在成年大鼠和胚胎组织中有独特的表达。RFIP-2A和B的转录受rFIP-2基因启动子交替使用的调控。将rFIP-2A或B-pAcGFP1-高尔基体导入正常大鼠肾细胞(NRK-52E)，发现rFIP-2B定位于高尔基体，而缺失rFIP-2B N端250个氨基酸的截短蛋白rFIP-2A普遍存在于细胞质中。在大鼠牙髓成纤维细胞中，肿瘤坏死因子-α可显著诱导rFIP-2B的表达，其诱导作用依赖于c-jun氨基末端激酶通路。RFIP-2B定位于细胞质，并在细胞死亡刺激下移位到细胞核。结果提示，rFIP-2的表达受选择性启动子调控，rFIP-2B是由肿瘤坏死因子-α介导的重要分子，可能参与了牙髓炎症过程中的细胞死亡途径。

Pulpal wound healing followed by cavity preparation may involve reactionary or reparative dentinogenesis in relation to the cavity position; however, little is known about the molecular responses. We aimed to isolate and analyze genes induced or suppressed in the wounded pulp to identify molecular processes involved in the pulp responses to injury. Twenty-three cDNAs were isolated by cDNA subtraction between healthy and wounded pulp of rats. By library screening, we identified rat 14.7K-interacting protein (rFIP)-2A and B genes homologous to human FIP-2, being involved in regulating membrane trafficking and cellular morphogenesis. RT-PCR analysis showed induction for only rFIP-2B in the wounded pulp. In situ hybridization analysis revealed unique expression of rFIP-2s in adult and embryonic tissues of rats. Transcription of rFIP-2A and B was regulated by alternative use of promoters at rFIP-2 locus. When the rFIP-2A or B-pAcGFP1-Golgi construct was transfected into normal rat kidney (NRK-52E) cells, rFIP-2B was localized in Golgi of whereas rFIP-2A, which is a truncated protein lacking the N-terminal 250 amino acids of rFIP-2B, existed ubiquitously in the cytoplasm. In rat pulp fibroblasts (RPC-C2A) cells, rFIP-2B was significantly induced by tumor necrosis factor (TNF)-α, and the induction was dependent on c-jun N-terminal kinase (JNK) pathway. rFIP-2B was localized in the cytoplasm, and translocated into the nucleus by cell death stimuli. The results suggest that rFIP-2 expression is regulated by the alternative promoter site, and rFIP-2B is a crucial molecule mediated by TNF-a, may be involved in cell death pathway during pulp inflammation.