Apoptosis Of Human Odontoblasts And Expression Of Apoptotic Protein Bcl-2, Bax With Differently Deep Caries

Objective: Apoptosis known as programmed cell death is an active ordereddeath process caused by apoptotic signals in order to maintain body’senvironmental stability. Apoptosis occurs in all life process such as growth,development, maturation and senium. It has been the hot spot in life scienceand medical research because of its important significance. In the process ofapoptosis, the bcl-2gene family is found which plays an important role. Thereis an antiapoptotic gene bcl-2and an apoptosis promoting gene bax. Studieshave shown that the effect of bcl-2gene and bax gene is achieved through thequantity of their protein in both tissues and cells, and the Bcl-2/Bax ratioregulates the apoptotic process.Apoptosis occurs in dental pulp tissue just as in other tissues in vivo.Odontoblasts are characteristic cells in pulp-dentin complex and the earliestcells to contact foreign injury stimulation. Many studies show that in theprocess of caries the dentin was exposed gradually, pulp will appear obviouspathological changes, including cell apoptosis and reparative dentin formed.But different reports are quite different, and there is no research on thepathological changes of dynamic and quantitation in caries process.This experiment explores the apoptotic mechanism in odentoblasts and thepossible correlation between apoptosis and damaging-repairing dynamicchange in caries progress through HE staining, TUNEL staining andimmunohistochemistry of Bcl-2and Bax in human tooth caries specimen.Methods:1Specimen selection and preparation: The patients are twenty to forty yearsold without systemic disease. Choose the third molar whose root developmentcompleted with the patient’s consent. Meanwhile, the tooth can be pulled outcompletely without value of treatment or keep. The teeth appear without fillings, obvious abrasion, defect or periodontal disease. Record medicalhistory, clinical symptom, pulp vitality values of dental and X-ray result of thetooth. The damages are chronic caries which happen to occlusal surface.Divide the teeth into normal tooth group, shallow caries group, moderatecaries group and deep caries group reference to chronic caries diagnosisstandard and caries damage depth classification standard. Each group has20teeth. Cut root immediately, fixed, decalcified, water washing, paraffinembedding, sectioning.2HE staining: Observe reparative dentin and the number of odentoblasts.3TUNEL staining: Observe the apoptosis in odentoblasts and calculate theapoptotic index.4Immunohistochemical staining: Using a two-step immunohistochemicalstaining to observe the expression of apoptotic related proteins Bcl-2and Baxin odentoblasts and quantify the gray value with Motic medical6.0imageanalysis system.5SPSS13.0.Results:1Results of HE staining1.1Normal tooth group: The cell layers of the pulp underneath the dentineare clear while the cell-free zone and the cell-rich zone are obvious. The tallcolumnar odontoblasts are many which have rich cytoplasm. The number ofodontoblasts is about15.42/100μm. No reparative dentine observed.1.2Shallow caries group: The cell layers of the pulp underneath the dentineare clear while the cell-free zone and the cell-rich zone are obvious. The tallcolumnar odontoblasts are many which have rich cytoplasm. The number ofodontoblasts is about15.71/100μm. No reparative dentine observed.1.3Moderate caries group: The cell layers of the pulp underneath thedentine are not clear while the cell-free zone disappears. There is reparativedentin under most of the caries parts. Under reparative dentin the odontoblastsare low columnar or cuboidal with less cytoplasm, and the number ofodontoblasts decreases to about9.91/100μm. 1.4Deep caries group: The cell layers of the pulp underneath the dentineare not clear while the cell-free zone disappears. There is thick and massreparative dentin under all of the caries parts. Under reparative dentin theodontoblasts are cuboidal or flat which have a little cytoplasm, and the numberof odontoblasts is only about6.41/100μm.No difference between natural tooth group and shallow caries group,significant difference was found between each other groups, with a P<0.05.2TUNEL staining2.1Normal tooth group: The apoptotic index of the odontoblastic zone isabout0.20.2.2Shallow caries group: The apoptotic index of the odontoblastic zone isabout0.21.2.3Moderate caries group: The apoptotic index of the odontoblastic zone isabout28.65.2.4Deep caries group: The apoptotic index of the odontoblastic zone isabout52.68.No difference between natural tooth group and shallow caries group,significant difference was found between each other groups, with a P<0.05.3Immunohistochemical staining3.1Apoptotic protein Bcl-23.1.1Normal tooth groups: The cytoplasm of odontoblasts is stained lightyellow. The gray value is about198.00.3.1.2Shallow caries groups: The cytoplasm of odontoblasts is stained lightyellow. The gray value is about197.93.3.1.3Moderate caries groups: The cytoplasm of odontoblasts is stainedyellow. The gray value is about178.98.3.1.4Deep caries groups: The cytoplasm of odontoblasts is stained darkyellow. The gray value is about151.40.No difference between natural tooth group and shallow caries group,significant difference was found between each other groups, with a P<0.05.3.2Apoptotic protein Bax 3.2.1Normal tooth group: The cytoplasm of odontoblasts is stained lightyellow. The gray value is about196.23.3.2.2Shallow caries group: The cytoplasm of odontoblasts is stained lightyellow. The gray value is about195.24.3.2.3Moderate caries group: The cytoplasm of odontoblasts is stained darkyellow. The gray value is about150.53.3.2.4Deep caries group: The cytoplasm of odontoblasts is stained brown.The gray value is about117.51.No difference between natural tooth group and shallow caries group,significant difference was found between each other groups, with a P<0.05.Conclusion:1Almost no apoptosis is observed in odontoblast zone with shallow caries.But if caries have progressed into dentin, there is obvious apoptosis.Meanwhile, the number of apoptotic cells increases with dentin caries progressgradually. The results of apoptosis in shallow caries, moderate caries and deepcaries have significant differences. It indicates possible relationship betweenodontoblast apoptosis and reparative dentine formed.2The expression levels of antiapoptotic protein Bcl-2and apoptosispromoting protein Bax are low in normal tooth group and shallow caries groupwhile increase in moderate caries group and deep caries group. The rise ofBax is much more significant, so Bcl-2/Bax ratio changes which is in line withincrease of apoptotic cells. It indicates that Bcl-2/Bax ratio is an importantmechanism of odontoblast apoptosis.