Based On Calcium Imaging The Estabilishment And Application Of Sweetness Sense Cell Model

Sweetener was a kind of additive which can give sweet taste to foods,and are widely used in the field of food processing.The evaluation of sweetness has always depended on the sensory evaluation of human,and there is still no direct evaluation method.It exits a lot of conditionality on expression of sweetness with impersonality and true feelings because of the differences in evaluation personnel's physiology and psychology include the individual,living environment and experiences.Artificial sweeteners are in the food processing industry in the popular because it does not provide or provide very low-calorie,thus,establishing a simple objective sweetness evaluation method is extremely important.Sweet response cells are those cells that are capable of expressing sweet taste receptor proteins and are able to recognize sweetness to open downstream signaling pathways.Over the past decade,many studies have pointed out that the artificial sweeteners bind to sweet receptors,leading to intracellular sweet taste signal transduction factor Ca2+ concentration increase.In this paper,sweet response cells were used as object of study from the perspective of the sweetness and basis of sweetener taste sensation.NCI-H716 cell model was established by using the calcium imaging method.Exposure sucralose to NCI-H716 cells,and the expression of T1R2/T1R3 respectively at mRNA and protein levels was observed by Q-PCR and Western blot.The relationship between intracellular Ca2+release induced by artificial sweeteners and the expression of T1R2/T1R3 was investigated by calcium imaging technique based on the change of the expression of the sweet taste receptor.The effects of sweetness,Sweetness-stimulating intracellular Ca2+ concentration is an absolute positive correlation,so as to explore the sweetness detection method based on calcium image.The purpose of this study was to investigate the correlation between the sweetness of artificial sweeteners,the expression of T1R2/T1R3 in sweet response cells and the intracellular Ca2+ concentration of sweet-taste stimulants.The results are as follows:(1)Establishment of sweet response cell model HEK-293 cells were transiently expressed sweet receptor,qPCR was used to identify sweet receptor expression at the gene level and HEK-293T cells were successful transiently expressed.Then STC-1,NCI-H716 and HEK-293T cells were stimulated by sucralose at a sweetness of 600,and compared with the increment of intracellular Ca2+ concentration,NCI-H716 cells were the best sweet response cell model.(2)Long-term exposure to artificial sweeteners induced an increase in the amount of sweet-taste receptor proteinOn the basis of previous study of sucralose sweetness preference in this laboratory,sucralose 6 and 12 were chosen as the exposure sweetness of NCI-H716 cells.Sucralose with sweetness 12 could better promote Sweet taste receptor protein expression.Subsequently,NCI-H716 cells were exposed to sucralose with a sweetness of 12 and identified by qPCR and western blot,respectively.The mRNA and protein expression levels of the NCI-H716 cells after exposure were all significantly enhanced.It was found that the long-term exposure sucralose 12 to NCI-H716 cells could increase expression of sweet receptor protein.(3)Increased expression of sweet receptor protein facilitates an increase in intracellular Ca2+ concentration stimulated by artificial sweetenersBased on the sucralose-induced the increasing of sweet receptor expression in NCI-H716 cells,the intracellular Ca2+ concentration increased with the increase of the expression level of the sweet receptor protein under the same stimulus condition.It was found that the increase of intracellular Ca2+ concentration was positively correlated with the sweetness of artificial sweeteners,while intracellular Ca2+ concentration increments evoked by stimulating of the same sweetness of AK and sucralose to NCI-H716 cells were not consistent.(4)Sucralose(within a certain sweetness range)Sweetness can be evaluated by sweetener-induced intracellular Ca2+ concentration increment valueThe intracellular Ca2+ increment induced by unit receptor unit sweetness stimulation(AUC)of AK and sucralose,while between sweetness 200-600 of sucralose,The intracellular Ca2+ increment induced by unit receptor unit sweetness stimulation(AUC)was a fixed value.This value is taken as a constant(K),and sweetness equal to the specific value of the sweetener-stimulated intracellular Ca2+ concentration increment and the constant value.As follow:Sweetness = AUC/K(1)However,the intracellular Ca2+ increment induced by unit receptor unit sweetness of AK in the range of selected stimulus sweetness was not consistent,and the constant K value could not be obtained.In summary,the sweetness of artificial sweeteners,sweet taste receptor expression and sweetener-stimulated intracellular Ca2+concentration increment in a certain range of stimuli sweetness into absolute positive correlation,which laid the foundation for the sweetness detection method based on the research of Calcium imaging.