Expression Of Pannexin3in Dental Pulp And Its Function In Dentin Hypersensitivity

Part1:The expression profile of Pannexin3in human dental pulp and odontoblastsObjective: To investigate the expression profile of Pannexin3at gene and protein level in the human dental pulp and odontoblast like cell, we use RT-PCR and Western blot.Methods: Dental pulps were harvested from the freshly extracted human (15-32years of age) tooth..Explant culture method was used to isolate dental pulp stem cells, which followed be induced into odontoblast-liked cells. The epression of Pannexin3in human dental pulps and odontoblasts at gene and protein level were identified by PCR and Western blot. Inaddion, the distribution of Pannexin3in human dental pulps and odontoblasts were identified by Immunofluorescence co-localization analysis.Results: The result of Polymerase chain reaction (PCR) and Western blot have revealed the presence of Pannexin3gene in human dental pulp cells. Consistent with the Western blot result, pannexin3was localized in the endoplasmic reticulum and plasmalemma, where are the contact sites between adjacent cells.What’s more; the immunofluorescence clearly demonstrates the overlap between pannexin3and DSPP-expressing cells. In dental pulp, both DSPP and Pannexin3were abundantly expressed in the dentinal tubule and odontoblast layer, particularly at the pulp-dentin junction areas.Conclusion:The pannexin3is expressed in the the dentinal tubule and odontoblast layer of human dental pulp tissue. Furthmore, the pannexin3mainly localizes in the endoplasmic reticulum and plasmalemma. Part2:The function of Pannexin3in the release of ATP into extracellular space.Objective: The purpose of this study is to investigate the function of Pannexin3in the release of ATP into extracellular space, when odontoblasts exposed to external stimulus.Methods: We constructed PLL3.7\CMV-PANNEXIN3\CMV-RFP lentivirus vectors to obtain overexpressed pannexin3odontoblasts. siRNA and shRNA were used to knockdown pannexin expression. The expression of the target gene was detected by PCR and Western blot to evaluate the overexpression and interference effects.The experiment seted five groups: control group, Pnnexin3over-expression group, siRNA interference group, shRNA interference group and carbenoxolone (CHX) blocking group. Each group of cells was stimulated by hot and cold stimulus, meanwhile the ATP release profile was recored by Quinacrine staining. Image Pro-plus was used to process statistic analysis of fluorescence variance. The extracellular ATP levels were measured by the bioluminescence assay.Results: The overexpression and knockdown transfection efficiency was successfully constructed by PLL3.7\CMV-PANNEXIN3\CMV-RFP lentivirus vectors, siRNA, shRNA techniques, which were confirmed by PCR, and Western blot. And fluorescence photographs showed that the pannexin3enpression level can be achieved Based on the fluorescence monitoring, ATP rapidly released into the extracellular space when exposed to hot and cold stimulus. The pannnexin3overexpressed cells release ATP most quickly among the groups and siRNA, shRNA and CHX could obviously inhibite the progress of ATP released into the extracellular.Conclusion: Hot and cold stimulus could open Pannexin3hemichannel and influence ATP release into the extracellular space. Part3: The role of ATP as a signal molecule of dental pulp sensory nerves in the pain transmissionObjective: To investigate the effects of ATP on pulp nociception mediated by P2X3receptor activation and the role in Ca ions wave in trigeminal neurons.Methods: Immunofluorescence was used to detect the expression of P2X3receptor in the mice trigeminal ganglion tissue.Trigeminal ganglion neurons were obtained through enzymatic digestion methods,which was identified by morphology observation and peripheral protein immunofluorescence detection.The whole-cell patch-clamp recording technique was utilitied to record the flow of Ca ions, when neuron was stimuliated by ATP, α, βme-ATP and A-317491. Male rats were divided into dentin sensitivity group and control group,The dentin of the experiment rats mandibular first molars were exposed with a bur powered by a electric rotary hand piece.All the rats were injected with PBS, ATP, α, βme-ATP and A-317491. Next, all mandibular first molars of the rats were stimulated by hot or cold water, and the times of licking or lifting a paw, flinches or scratch their head were recored.Results: Trigeminal ganglion neurons were isolated through enzymatic digestion methods; the cytoskeleton of cultured neurons was revealed a mesh, linear arrangement. P2X3receptor was distributed in the peripheral portion of the trigeminal ganglion. The result of whole-cell patch-clamp verified the calcium influx when added ATP and α, βme-ATP, and this influx was inhibited by A-317491. Cold and hot stimulus can significantly increased the paw lift or flinches or scratch times in ATP and α, βPme-ATP injuection groups, however the times decreased in A-317491injection group-Conclusion: ATP could activate P2X3receptors on the trigeminal ganglion, and influence the calcium influx, participate in pain transmission.