RESUMO
OBJECTIVE: Ameloblastin is an enamel matrix protein expressed in several tissues. Many potential mechanisms have been identified by which ameloblastin functions as an extracellular matrix protein. However, the biological effects of ameloblastin on gingival epithelial cells remain unclear. In the present study, we established a novel system to purify recombinant human ameloblastin and clarified its biological functions in epithelial cells in vitro. DESIGN: Recombinant human ameloblastin was isolated from COS-7 cells overexpressing HaloTag-fused human ameloblastin by the HaloTag system and then purified further by reverse-phase high-performance liquid chromatography. SCC-25 cells, derived from human oral squamous cell carcinoma, were treated with recombinant ameloblastin and then cell survival was assessed by a WST-1 assay. Cell cycle analysis was performed by flow cytometry. RESULTS: The novel purification system allowed effective recovery of the recombinant ameloblastin proteins at a high purity. Recombinant ameloblastin protein was found to suppress the proliferation of SCC-25 cells. Flow cytometric analysis showed that ameloblastin treatment induced cell cycle arrest G1 phase. CONCLUSIONS: We developed a procedure for production of highly purified recombinant human ameloblastin. Biological analyses suggest that ameloblastin induces cell cycle arrest in epithelial cells and regulates the progression of periodontitis.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Proteínas do Esmalte Dentário/farmacologia , Células Epiteliais/metabolismo , Neoplasias Bucais/tratamento farmacológico , Proteínas Recombinantes/farmacologia , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Citometria de Fluxo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (k(cat)/K(m)) and enhanced tolerance against DNase 1digestion. To be pointed out, intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides responsible for nuclear cytoplasmic transport of proteins.