| Objective:To observe the developments of tooth germs by making histological sections in postnatal Wistar rat lower incisors,and the bell period tooth germs were selected.The cervical-loop epithelia were mechanically stripped from the incisor tooth germs.The primary cells were cultured by enzymolysis and tissue cultutre methods.The immunehistochmistry methods were used to identify depurant cervical-loop epithelial cells.The isolated and cultured model of tooth epithelial stem cells was established in order to provid the seed resource of tooth epithelial stem culls and to lay a foundation for transplantation tooth epithelial stem cells.Methods:1.Making histological sections of postnatal Wistar rat lower incisor tooth germs:The postnatal Wistar rats,ages ranged from 1 day to 3 days after neonatal were selected.The rats were killed by cutting off the heads.Clipped mandibles were used to make paraffin embedded tissues sections by routine method.Gradient alcohol dehydration,xylene transparent and HE staining methods were used to observe the developments of tooth germs under light microscopy.The bell period tooth germs were used to prepare for the next experiments.2.Experiment 1:Isolating and culturing tooth epithelial stem cells of rats:①The Wistar rats after 2 days postnatal were Selected and killed by cutting off the heads.The rats were disinfected with 75%of the alcohol for 2-3 seconds.The mandibles were c(?)ipped in asepsis by scissors to remove the unuseful tissues.Clipped mandibles were put into culture dishes contained Hanks liquid to clear blood.The incisor tooth germs were mechanically stripp(?)d from cleared mandibles under stereomicroscope. Enamel organs were mechanically stripped from incisor tooth germs under stereomicroscope after enzymolysis method.The cervical-loop tisseus were mechanically stripped from enamel organs by miroforceps and cultured in culture bottle.The culture bottle was put in incubator in condition of 100%humidity,5%CO2 and 37℃.②Culturing the cervical-loop tisseus in different mediums:The isolated cervical-loop tisseus were put in different mediums(1640 medium and DMEM/F-12 medium)to observe the growth morphology of cervical-loop epithelial cells.3.Experiment 2:Purificating and identificating of the cervical-loop epithelial cells:①Purification of the cervical-loop epithelial stem cells:When the cultured cells overspread the culture bottle,the cells were digested by digested liquid in oscillating,then transfered into culture dishes coated by collagen typeⅣor nothing.The mediums were changed when cells adhered the wall.The cells were put into the incubator in condition of 100%humidity,5%CO2 and 37℃.The medium was changed every other day.After 3-5 times changing the medium,the culture dishes coated by collagenⅣor nothing were changed.It is beneficial to explore favorable method to purify cervical-loop epithelial cells which can lead to prepare for the next experiments.②Preparating cell crawling slides of depurant cervical-loop epithelial cells by routine method:Primary antibody were integrin-β1 and monoclonal antibody CK,control group was 0.1ml/L PBS fluid.The method of immunehistochmistry was according to the explanations.The cells were iditified by clone formation,staining integrin-β1 and monoclonal antibody CK.Results:1.Making histological sections of postnatal Wistar rats lower incisor tooth germs:①Histological sections of tooth germs of 1 day after neonatal:It was been seen indistinctly infant cervical-loop tisseus structure: inner enamel epithelium(ameloblast cell-lineage),stratum intermedium and outer enamel epithelium.The Aetivity,proliferation and metabolism of the cells were immature.②Histological sections of tooth germ of 2 days after neonatal:It was been seen distinctly cervical-loop tisseus structure:inner enamel epithelium(ameloblast cell-lineage),stratum intermedium and outer enamel epithelium.The Activity,proliferation and metabolism of the cells were very mature.③Histological sections of tooth germ of 3 days after neonatal:It was been seen distinctly cervical-loop tisseus structure:inner enamel epithelium(ameloblast cell-lineage),stratum intermedium and outer enamel epithelium.But the mandible of 3 days after neonatal had begun to calcificate,the cervical-loop epithelial cells were more flap than the 2 days.From the above results,it was feasible to select 2 days of postnatal Wistar rats as the experimental models.2.Experiment 1:Isolating and culturing tooth epithelial stem cells of rats:①Isolating tooth epithelial stem cells:It was easy to obtained mandibles from 2 days of postnatal Wistar rat heads becaues the heads had not calcificated fully.The total incisor tooth germs could be easily obtained under the stereomicroscope.By enzymolysis method,the cervical -loop tisseus were successfully removed.②Observing growth morphology of cervical-loop epithelial cells in different mediums:When cultured in DMEM/F-12 medium:After being cultured 24 hours,a few of cells emigrated from cervical-loop tisseus.The primary culture is a mixture of different cells,which were composed of two different cells: Fiber-like cells and epithelial cells.The fiber-like cells were long fusiform,the conjunction between cells was loose.The epithelial cells were polygonal and clone foemation,the conjunction between cells was close.The epithelial cells were arounded by fiber-like cells,which like of a island form.When cultured in 1640 medium:The growth state of cells in 1640 and in DMEM/F-12 medium was roughly the same.But the activity of the cells lived in 1640 medium was not so strong as in DMEM/F-12medium.It may be due to the difference of composition in the two different mediums.Thecells growed well in DMEM/F-12 medium.3.Experiment 2:Purification and identification of the cervicalloop epithelial cells:①Purification of cervical-loop epithelial cells:Group of culture dishes coated by collagen typeⅣ:After two to three times'purification by adhering to collagen typeⅣto remove the fiber -like cells,The cervical-loop epithelial cells could be depurated.The cells adhering to the walls were epithelial cells.Epithelial cells' volume was small and round,had plenty of plasma.The nucleus was large and has 2-3 nucleolus.The cells were polygons,froming cloning.Group of culture dishes coated nothing:The cells could not be depurated fully through two to three times to dislodge fiber-like cells. Cells adhered to the walls partly were epithelial cells,the others were fiber-like cells.②Identification of cervical-loop epithelial cells:The cervicalloop epithelial cells exhibit positive expression for integrin-β1 and monoclonal antibody CK in immunohistochemistry staining.The plasma were positive in red,while the control group was negtive. Conclusion:1.Observation results of histological sections of tooth germs after 2 days neonatal:The cervical-loop tissue structures can be obviously seen,which concluding inner enamel epithelium(ameloblast cell-lineage),stratum intermedium and outer enamel epithelium.The activity,proliferation and metabolism of the cells are very mature.It is easy to obtain mandibles from 2 days of postnatal Wistar rat heads becaues the heads had not calcificated fully.The total incisor tooth germs could be easily obtained under the stereomicroscope.By enzymolysis method,the cervical-loop tisseus were successfully removed.2.The activity of cells in 1640 medium is not so strong as in DMEM/F-12 medium.It may be due to the difference of composition in different mediums. The cells grow well in DMEM/F-12 medium.3.The quantity of cells in culture dishes coated by collagenⅣadhering to wall is larger than the group in culture dish coated nothing.lt can say that collagenⅣcan increase the epithelial cells' adhensivation.The reason may be that culture dish coated nothing was lack of extracellular matrix,lead to inhibit cells' growth.4.Identification of cervical-loop epithelial stem cells:Stem cells which derived from the ectoderm,adhered to basement membranes through expressing special integrins,such as integrin-β1.Keratins expressed in epithelial cells,which was primary structural proteins and differentiated maked products of epithelial cells.The cervical-loop epithelial cells exhibit positive expression for integrin-β1 and monoclonal antibody CK in immunohistochemistry staining.The plasma is positive in red,while the control group is negtive.It indicats that the cells cultured are cervical-loop epithelial cells riching in stem cells. |
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