Characteristic Expression Of Three ThermoTRPV Channel Proteins In Human Odontoblast-like Cells By Immunoelectron Microscopy

Objective Dentine hypersensitivity(DH)causes short,sharp dental pain and is associated with dentinal exposure to various stimuli(e.g.mechanical,thermal,p H,and osmotic condition).Three hypotheses(i.e.,the neural theory,the odontoblastic transduction theory,and the hydrodynamic theory)have been proposed to explain the tooth pain mechanism.Amongst the three hypotheses,the hydrodynamic theory has been widely accepted,which assumes that dentinal fluid flow induced by noxious stimuli may activate mechanoreceptors resulting in pain sensation.Recent years,"Odontoblast hydrodynamic receptor theory" has been presented that odontoblasts as the outermost cells of dental pulp express transient receptor potential(TRP)channels and play important roles as sensory receptors in perceiving external noxious stimuli.TRP channels have been demonstrated to be activated by various external stimuli including temperature,acid,osmotic pressure,mechanical stimuli and chemical stimuli.Transient receptor potential vanilloid-1(TRPV1)channel is also named as capsaicine receptor and has been most studied.TRPV1,transient receptor potential vanilloid-2(TRPV2)and transient receptor potential vanilloid-3(TRPV3)are members of the TRP family and directly related to the cellular mechanism of temperature-sensing in human.They can be activated by adjacent range of heat temperature [TRPV1(>43°C),TRPV2(>52°C)and TRPV3(33-39°C)] as homotetramers.These three TRPV channels have been proved to be expressed in HODs-like cells.However,the different distribution of three thermo TRPV channels at subcellular level in HODs-like cells is still unclear.Methods The frozen HODs-like cells obtained from human dental pulp stem cells(HDPCs)were induced in vitro with the method of S.Gronthos in the early stage of the study.After the cells were resuscitated,3to 4 passages of HOD-like cells were selected.The HODs-like cells were identified by reverse transcription polymerase chain reaction(RT-PCR)and immunofluorescence(IF)firstly to conform whether the characteristic proteins expressed in HODs-like cells,dentin sialophosphoproteins(DSPP)and nestin.A portion of cells were lysed and the gene expressions of TRPV1,TRPV2 and TRPV3 channels were detected by RT-PCR.The same batch of cells was selected for fixation.The expression of TRPV1,TRPV2 and TRPV3 channel proteins were detected by IF.Then the HOD-like cells samples were made into ultrathin sections,mounted on nickel grids,floated of three TRPV antibodies conjugated with 10 nm colloidal gold particles and observed under immunoelectron microscopy(IEM)at60,000 magnifications.The relative intracellular distributions of these three channels were analyzed quantitatively on IEM images using a robust sampling,stereological estimation and statistical evaluation method based on labelling density(LD)and relative labeling index(RLI)mentioned by Mayhew.Results1.The result of RT-PCR and IF showed that DSPP and nestin are expressed in HODs-like cells at gene and protein level.IF showed that HODs-like cells are tall,columnar,and polarized cells with long processes.These results comfirmed the HODs-like cells cultured in vitro have the characteristics of HODs.At the same time,the result of RT-PCR and IF demonstrated the gene and protein expression of TRPV1,TRPV2 and TRPV3 channels in HOD-like cells respectively.2.The result of IEM demonstrated the expression of TRPV1,TRPV2 and TRPV3 channel proteins in HOD-like cells at subcellular level,such as nucleus,cytoplasm,mitochondria,endoplasmic reticulum(ER)membrane,primary cilia,and odontoblast processes.3.The quantitative IEM analysis revealed that the RLI of colloidal gold particles conjugated with TRPV1,TRPV2,and TRPV3 proteins in nucleus,cytoplasm and primary cilia was less than1,the RLI of colloidal gold particles conjugated with TRPV1,TRPV2 and TRPV3 proteins in mitochondria,ER and odontoblast processes was more than 1and ?2 value was more than 10% of total ?2.TRPV1,TRPV2 and TRPV3 proteins are preferentially labeled in HODs-like cells.Conclusions This experiment demonstrated that HODs-like cells cultured in vitro have HODs characteristics.The result of IF and RT-PCR demonstrated that expression of TRPV1,TRPV2 and TRPV3 channel proteins and gene in HOD-like cells.The quantitative IEM analysis revealed that the TRPV1,TRPV2 and TRPV3 channel proteins are preferentially labeled in mitochondria,ER membrane and odontoblast processes.HODs could play an important role in mediating pulp thermo-sensation due to the expression of these three TRPV channels.The difference of relative intracellular distributions of three channels suggests that special structures such as processes may have an important role to sensing of the outer stimuli firstly.