槟榔碱对Hacat及DOK细胞FHIT和SMAD4蛋白表达影响研究

Study on the expression of the FHIT and SMAD4 in Hacat and dysplastic oral keratinocyte cells under the stimulation of arecoline

目的通过研究人永生化角质形成细胞(Hacat细胞)和异常增生角质形成细胞细胞在不同浓度槟榔碱刺激下增殖活性的改变以及脆性组氨酸三聚体(FHIT)蛋白和胰腺癌缺失(SMAD4)蛋白的表达,探讨FHIT和SMAD4蛋白在口腔黏膜组织从正常到潜在恶性病变过程中的可能作用机制。方法 MTT法检测不同浓度槟榔碱(0、2.5、5、10、20、40、80、160μg/m L)、不同作用时间(24、48、72 h)条件下,Hacat细胞和异常增生角质形成细胞存活率的改变;Western Blot检测不同浓度槟榔碱(0、20、40、60、80μg/m L)作用24 h后,Hacat细胞和异常增生角质形成细胞FHIT蛋白和SMAD4蛋白的表达。结果不同浓度槟榔碱、不同时间干预后,Hacat细胞和异常增生角质形成细胞的存活率均随槟榔碱浓度的增加、干预时间的延长而降低;在槟榔碱浓度相同、干预时间相同的情况下,异常增生角质形成细胞的存活率高于Hacat细胞(P<0.05);槟榔碱浓度为0时,Hacat细胞FHIT和SMAD4蛋白表达水平高于异常增生角质形成细胞(P<0.05);不同浓度槟榔碱干预24 h后,Hacat细胞和异常增生角质形成细胞FHIT和SMAD4蛋白表达量随槟榔碱浓度的增加而下降。结论槟榔碱可导致Hacat细胞和异常增生角质形成细胞存活率降低,并呈浓度、时间依赖性;槟榔碱可诱导Hacat细胞和异常增生角质形成细胞FHIT和SMAD4蛋白表达减少;FHIT蛋白和SMAD4蛋白表达减少可能是口腔潜在恶性病变的发病机制之一。

Objective To explore the possible pathogenesis of oral potentially malignant disorders,we investigated the effect of arecoline on human normal keratinocyte Hacat cells and dysplastic oral keratinocyte with respect to cell survival rate and the expression of the FHIT and SMAD4.Methods Human keratinocyte cells （of the Hacat cell line）and dysplastic oral keratinocytes were treated with different concentrations （0,2.5,5,10,20,40,80 and 160 μg/mL）and different effecting time （24,48,72 h）of arecoline,following which cell survival rate was accessed using the MTI assay.Western blot analysis was performed to study the expression of the FHIT and SMAD4 when Hacat and dysplastic oral keratinocytes were treated with different concentrations （0,20,40,60 and 80 μg/mL）of arecoline for 24 hours.Results MTI assay showed that the survival rate of both Hacat and dysplastic oral keratinocytes were significantly decreased with the increasing concentration and the extension of the effecting time of arecoline.Furthermore,higher survival rate was found in dysplastic oral keratinocyte when compared with Hacat cells under the same stimulation （P 〈 0.05）.The initial expression of FHIT and SMAD4 was higher in Hacat cells than that in dysplastic oral keratinocyte （P 〈 0.05）.Both of these two cell lines had a decreasing expression of FHIT and SMAD4 after being treated with increasing concentration of arecoline for 24 hours.Conclusion The cell survival rate is negatively associated with the arecoline in a dose and time-dependent manner.The expression of FHIT and SMAD4 is also reduced with the increasing concentration of arecoline.Furthermore,the aberrant expression of FHIT and SMAD4 may play an important role in the modulating of cell proliferation and thus may participate in the occurrence and development of oral potentially malignant disorders.