Studies On FGF10 In The Cervical Loop Area Tissues Of Rat Incisors And Biological Characters Of Cervical Loop Epithelial Cells

Objective:To observe the distribution of FGFIO in cervical-loop of tooth germs by making immunohistochemistry sections in 2-3 d' s postnatal Wistar rat lower incisors.The primary cervical-loop epithelia cells were cultured by enzymolysis and tissue cultutre methods from 2d' s postnatal Wistar rat lower incisors,and methods were used to identify biotic characters of cervical-loop epithelial cells.The isolated and cultured tooth epithelial stem cells were studied in order to provide directions for establishing a foundation for transplantation of tooth epithelial stem cells.Methods:1.Experiment 1:FGF10 immunohistochemistry sections of 2-3 days postnatal Wistar rat lower incisor tooth germs were made.The postnatal Wistar rats,ages ranged from 2 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 immunohistochemistry staining methods were used to observe the distribution of FGF10 in tooth germs under light microscopy.2.Experiment 2:The tooth epithelial cells of rats were isolated and cultured to identify their biological characters. â‘ The Wistar rats 2-days-postnatal were selected and killed by cutting off the heads.The rats were disinfected with 75%alcohol for 2-3 seconds.The mandibles were clipped in asepsis by scissors to remove the unuseful tissues.Clipped mandibles were put into culture dishes contained Hanks liquid to clear blood.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 stereomicroscope and cultured in DMEM/F-12 medium contained 20%fetal bovine serum.The culturing bottles were put in incubator in condition of 100%humidity,5%CO2 and 37℃.The cervical-loop epithelial cells were purificated by differential digestion when the cultured cells overspread the culture bottle.â‘¡The growth of cervical-loop epithelial cells were observed and the cell growth curve was drew.â‘¢The cell cycle of the cervical-loop epithelial cells were detected by flow cytometer(FCM)â‘£The cell crawling slides of depurant cervical-loop epithelial cells were preparated by routine method.The primary antibody was amelogenin,the control groups were 0.1ml/L PBS fluid.The method of immunocytochemistry was according to the explanations.⑤The primary cultured cells were digested by 0.25%trypsin+0.02% EDTA or 0.24%Dispaseâ…¡to passage culture.The survival rate of digested cells were calculated and the status of passage cultured cells were observed.Results:1.FGF10 immunohistochemistry sections of 2-3 d's postnatal Wistar rat lower incisor tooth germs were made.The cervical-loop structure is seen distinctly in histological sections of tooth germs of 2 days after neonatal.Cervical-loop tisseus exhibits positive expression for FGF10 in immunohistochemistry staining.The dental papilla,basal membrane of inner enamel epithelium and preodontoblasts are positive in brown,while the control group is negtive.2.The biological characters of isolated and cultured tooth epithelial cells of rats reveal that:â‘ There are a few of cells emigrated from cervical-loop tisseus after culturing 24 hours in DMEM/F-12 medium contained 20%fetal bovine serum.The primary culture is a mixture of different cells,which are composed of two different cells:Fiber-like cells and epithelial cells. The fiber-like cells are long fusiform,the conjunction between cells is loose.The epithelial cells are polygonal and clone formation,the conjunction between cells is close.The epitheliual cells are arounded by fiber-like cells,which like of a island form.â‘¡The cells' growth curve:the logarithmic growth phase of cervical-loop epithelial cells are 3 to 7days.DT is eaqual to 42.34h。③The results of FCM:epithelial cells are 86.7%in phase G₁,2.67% in phase G₂,10.7%in phase S.â‘£Potentiality of cell differentiation:The cervical-loop epithelial cells exhibit positive expression for amelogenin in immunocytochemistry staining,while the control group is negtive.⑤Survival rate of cells digested by 0.25%trypsin contained 0.02% EDTA is 33.67%,while digested by 0.24%Dispaseâ…¡is 69.23%.Cells survived by 0.25%trypsin contained 0.02%EDTA proliferate slowly and seldom adhere to the wall.Cells survived by 0.24%Dispaseâ…¡could proliferate to clumping clones.The Activity,proliferation and metabolism of the cells are better.But cells' shape becomes abnormal at 7 day.Conclusion:1.Observation results of FGF10 immunohistochemistry sections of tooth germs are positive in cervical-loop tissue structures concluding inner enamel epithelium(ameloblast cell-lineage),dental papilla and preodontoblasts.The continuous growth of the mouse incisors,as well as their subdivision into the crown and root domains,is due to the modulating of the network of FGF10 signals.2.It is beneficial to prepare for the successive experiments by choosing purified cervical-loop epithelial cells in logarithmic growth phase:3 to 7days.3.The proportion of cells in cell cycle suggests that the growth of cervical-loop epithelial cells are similar to stem cells.4.The cervical-loop epithelial cells exhibit positive expressionfor amelogenin in immunocytochemistry staining,suggest their potentiality to be preameloblast/ameloblast.5.0.24%Dispaseâ…¡can preserve the epitheliums' adhensivation.The reason may be that Dispaseâ…¡bring less destruction to basement membranes.The reason of being anomal-shape maybe lack of extracellular matrix or growth signals like FGF10 leading to cells'growth was inhibited.