The Study Of On Separation And Purification Technology Of Taste Bud Cells Of Mouse And Application

Taste,which is one of the most essential,primordial and necessary sensory functions,is used to recognize quality of food,regulate appetite and control quantity of food intake.However,the primary taste reception on the cells is the prerequisite for the taste perception,study on the cellular taste perception has been hampered because of the relatively outdated technology for separating taste bud cells.Consequently,it is crucial for the further research work in this field to establish an effective technique to obtain taste bud cells with high production,high viability,and high purity,and establish a method to culture taste bud cells.ICR mice were used as the experimental animal in our study.Taste bud cells of fungiform papillae were prepared and purified by a two-step enzymatic treatment and Percoll density gradient centrifugation,and evaluated by Avidin-bitin-peroxidase cmplex method,electron microscope, immunofluorescence technology and laser confocol scanning microscopy. Finally an effective technique for separation and purification of taste bud cells was thus established.Then on the basis of the established technique of separation and purification of taste bud cells,a short-term culturing method was developed by culturing the purified taste bud cells from our two-step enzymatic treatment and the isolated taste bud cells from the direct mechanical treatment.Finally,the thermal changes occurring during the mutual recognition between taste bud cells and sweeteners were detected by ITC,and the feasibility of thermal dynamics study of sweetener recognition by taste bud cells was discussed.The results of this study were as follows:(1)The technique for separating and purifying taste bud cells of fungiform papillae was established by optimizing the conditions for the two-step enzymatic treatment to obtain the mixed cell suspension.The mixed enzyme solution of collagenaseâ…¡(1.5mg/mL) and dispaseâ…¡(1.5mg/mL) was used to peel off lingual epithelium by incubating the excised tongue of 6-8 week-old ICR mouse for 6-8min at room temperature. Then the taste bud cell suspension was obtained by incubating the lingual epithelium for 10-20min at 37℃with the mixed solution of 0.25%trypsin and 0.02 mmol/L EDTA.Purification of taste bud cells was conducted by incubating the cell suspension for 30min with Percoll working solution and centrifuging at a speed of 1200rpm for 6min at 25℃.The purity of taste bud cells was improved from less than 10%to more than 65%after this treatment.Results from electron microscope and immunohistochemistry confirmed the feasibility of this method.(2)Short time culture method of mouse taste bud cells was established. Taste bud cells were best cultured in 5%CO₂at 37℃with Iscove's modified Dulbecco's Medium(containing 1:5 MCDB153,10%FBS,20ng/mL insulin, 100U/mL/100mg/mL penicillin/streptomycin) as culture medium,and typeâ… rat tail collagen as adhesive coating.Compared with the mechanical method,enzymatic method showed better influence for taste bud cell culturing.Results from proliferation and immunohistochemistry indicated that after two weeks of culturing,taste bud cells still exhibited the expression of the key biomarker in taste perception,such asα-gustducin. However,taste bud cells possessed only weak proliferation ability after culturing.So this method was just suitable for short-term culture.(3)Results from ITC indicated that thermal changes during the mutual recognition between taste bud cells and various sweeteners at the same concentraton were related to the sweetness of the sweeteners.The higher the sweetness was,the bigger the absolute value of thermal change was.For the low-sweetness sweetener,the interaction with taste bud cells was exothermic,while for the high-sweetness sweetener,the interaction was endothermic.In short,ITC was found to be feasible for the study on taste perception in the level of cells and lay a foundation for the following study.