CN109142298A - A kind of method for quantitatively determining of sugariness on a cellular level - Google Patents
A kind of method for quantitatively determining of sugariness on a cellular level Download PDFInfo
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Abstract
The present invention provides a kind of method for quantitatively determining of sugariness on a cellular level, including intracellular calcium ion fluorescent staining, laser confocal scanning, the variation of measurement microscope intracellular free calcium level.Method of the invention establishes the method using detection technique of fluorescence research sweet receptor cell intracellular free calcium level variation, quantitative people can effectively avoid the influence of human subject's factor to the perception degree of other unknown sample sweet tastes under ex vivo.Method of the invention can quick, efficient, highly sensitive detection compound sugariness, have a good application prospect.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of method for quantitatively determining of sugariness on a cellular level.
Background technique
The sense of taste that the mankind can distinguish is broadly divided into five kinds, i.e. tart flavour, sweet taste, bitter taste, delicate flavour, saline taste.It is most of to feed
Newborn class animal more prefers to sweet taste, and the food of sweet taste is usually implied with more energy, and the food of bitter taste then implies
Food may be to body nocuousness.The sweet receptor cell of mammal is mainly distributed on the taste buds cell of tongue and jaw epidermis.
In the cell of the different sense of taste of every kind of impression, taste signal transduction access is all different.
T1R gene family can encode three kinds of conservative GPCRs, i.e. T1R1, T1R2 and T1R3, T1R2 and T1R3 combination shape
At heterodimer, sweet receptor can be used as；T1R1 and T1R3 is combined and is formed heterodimer, can be used as umami receptor.
These sense of taste ligands and taste receptors interact, and activate cascade signal so as to cause neurotransmitter regulator, then in cranial nerve
Signal can be passed to sense of taste cortical centers from hypothalamus by the nerve fibre of pivot, and sense of taste cortical centers can be added the signal of collection
Work and integration, and then generate the corresponding sense of taste and feel.
Sweet cpd is varied, and including carbohydrate, artificial sweetener, thaumatin T, amino acid, sweet taste signal is main
It is by the T1R2/T1R3 dimerization receptor body impression on II type taste buds cell, sweet taste signal molecule includes simple carbon hydrate
Object (monosaccharide and polysaccharide), D- type amino acid (most of) and L-type amino acid (part), artificial sweetener etc..Correlative study proves
The involved signaling molecule of sweet taste signal conduction has α-gustducin, PLC β 2, IP3R, TRPM5 etc., and research has determined that at present
Signaling pathway molecule caused by natural sweet taste substance and artificial sweetener stimulation sweet receptor is different.
Sweet cpd rear two signal paths of primary activation in conjunction with sweet receptor.Natural sweet taste substance and sweet receptor
In conjunction with rear, activation downstream G-protein, and then it is logical to open cNMP- gate by activated adenyl cyclase (AC) hydrolysising ATP, generation cAMP
Road leads to flow of calcium ions, and intracellular free calcium level increases, and cAMP can also activate PKA, causes potassium ion on the outside of small base portion logical
Road phosphorylation, so that ion channel be made to close, it is suppressed that efflux of K+ ions causes membrane depolarization, voltage dependent calcium from
Stream in sub, calcium ion concentration increase.After artificial sweeteners' substance is in conjunction with sweet receptor, downstream G-protein is activated, activates phospholipase C
(PLC), IP3 and DAG is generated, IP3 can cause the calcium ion in endoplasmic reticulum to discharge in conjunction with the IP3R on endoplasmic reticulum, intracellular
The raising of calcium ion concentration.The activation of two accesses results in intracellular free calcium level raising.
Calcium ion participates in as a main intracellular messenger and regulates and controls a variety of physiological activities.Cell has strict mechanism
The balance of intracellular calcium ion is controlled, one is to rely on the calcium pump and calcium channel of cytoplasma membrane, second is that by endoplasmic reticulum, mitochondria
Equal cellular calciums library.In animal and plant cells, calcium ion mainly exists in the form of three kinds: 1. and eucaryotic cell structure ingredient or macromolecular
Substance combines, and is formed and combines calcium；2. being stored in the cells cellular calcium such as endoplasmic reticulum, mitochondria library, to store calcium；3. with ion
Form exists, i.e. free calcium.Cell storage calcium intracellular and free calcium can mutually convert, under static state, in cell
Free calcium concentration is very low, and about 10-8-10-7M.When being stimulated, the free calcium in cell cytoplasm be will rise rapidly, can
Reach 10-5M。
Fluo-4AM is the acetoxymethyl ester derivative of Fluo-4, be it is a kind of can with the fluorescent dye of penetrating cell film, point
Minor is C51H50F2N2O23, molecular weight are as follows: and 1096.95, structural formula are as follows:
The fluorescence of Fluo-4AM is very weak, and fluorescence will not increase with intracellular free calcium level and be enhanced.Fluo-4AM into
It can be sheared to form Fluo-4 by intracellular esterase after entering cell, to be trapped in intracellular.Fluo-4 can with it is free
Calcium binding, in conjunction with can produce stronger fluorescence after calcium ion, maximum excitation wavelength is 494nm, and maximum emission wavelength is
516nm.The laser excitation wavelength being recommended to use in the detection of practical application laser scanning co-focusing microscope is 488nm left
The right side, launch wavelength detection range are 512-520nm.
Sugariness is to reflect the sense organ of people to food mouthfeel evaluation index, in simple terms, being of characterization in a sense
Close the level of sweetness of object or food.In daily life, people usually use sense organ, that is, tongue to judge sugariness.In section
It learns in research, mainly with various chemical reagent and analysis instrument etc. come the sugariness of test sample, but these instruments can only detect
The content of pleasantly sweet soluble solid in food, not true sample sugariness.So we are generally according to these single sweet teas
The content of taste substance and the sugariness of single sweet substance quantify the sugariness of these foods or complex compound.
The method of detection sugariness is broadly divided into two major classes at present, is analytical chemistry detection method, biological method inspection respectively
Survey method.Wherein analytical chemistry detection method mainly has high performance liquid chromatography, electronic tongues detection method, sugariness meter detection side
Method, spectrophotometer detection method；Biological method has artificial assessment etc..Analytical chemistry methods detection can be detected accurately
The content of sugar, but sugariness can not be quantified on physiological level；Although biological detection method can quantify on physiological level
Sugariness, but subjective, people the taste manually tested and assessed is variant, it can not accurate quantification sugariness.There is not one kind yet at present
The standard method of objective quantitative material sugariness on physiological level, foundation can on physiological level quantitative combination object sugariness side
Method is particularly significant for food and sweetener development.
Summary of the invention
The present invention provides one kind to detect unicellular intracellular free calcium level variation based on laser scanning co-focusing microscope
Method, and then the quantitative sugariness on physiological level.
There are two types of the cell lines that the present invention uses, respectively HEK293 cell line and HEK293-T1R2/T1R3 cell line,
It sufficiently eliminates and measures difference caused by the reaction of cell background；The HEK293-T1R2/T1R3 cell line and the cell line preparation side
Method please refers to patent 201810425704.1.
In order to achieve the above object, the present invention provides calcium ion concentrations in a kind of stable expression sweet receptor cell cytoplasm
Detection method.
A kind of method for quantitatively determining of sugariness on a cellular level, includes the following steps:
(1) the HEK293 cell of adhere-wall culture and HEK293-T1R2/T1R3 cell are suspended by normal passage step
The preparation of cell, and planted in the burnt culture dish of copolymerization respectively；
(2) when the HEK293 cell and HEK293-T1R2/T1R3 of the adhere-wall culture in the burnt culture dish of the copolymerization of step (1)
When the convergence degree of cell reaches 40%-60%, with calcium ion fluorescent solution to plantation in be copolymerized in burnt culture dish cell into
Row is incubated for；
(3) cell fluorescent images after being incubated for using laser scanning co-focusing microscope continuous observation recording step (2) are extremely
120s obtains the fluorescence at individual cells each time point in 120s of HEK293 cell and HEK293-T1R2/T1R3 cell respectively
Intensity value；Minimum fluorescent value sets in 16s to 120s maximum fluorescence value as Fm as Fo in 16s before setting；Wherein in 16s
Sweet tea sample solution to be measured is added；
(4) the fluorescence intensity change ratio of HEK293 cell and HEK293-T1R2/T1R3 cell is calculated separately with △ F/Fo
Value, wherein △ F is Fm-Fo；
(5) that HEK293-T1R2/T1R3 cell fluorescence intensity changing ratio △ F/Fo value is subtracted HEK293 cell fluorescence is strong
The value for spending changing ratio △ F/Fo is △ R；
(6) set the sugariness of sucrose solution under certain known concentration as the value of 1, F/Fo be △ Rs；The then △ that step (5) obtains
The ratio of R value and △ Rs, the relative sweetness of sweet tea sample solution as to be measured.
Preferably, the burnt culture dish of copolymerization described in step (1) is suitable for carrying out oil mirror on laser confocal microscope
The Tissue Culture Dish of observation.
Preferably, the specification for being copolymerized burnt culture dish is outer diameter 35mm, internal diameter 10mm；The burnt culture dish of the copolymerization makes
With preceding needing with 25 μ g/ml Poly-L-Lysine Solutions in 37 DEG C of incubation 30min.
Preferably, calcium ion fluorescent described in step (2) is Fluo-4AM, and it is dense in Flex buffer
10 μM of degree.
Preferably, the Flex buffer includes ingredient as shown in the table:
| Reagent | Volume (mL) |
| HBSS solution | 47.5 |
| HEPES solution | 1 |
| 1M MgSO4 | 0.05 |
| 1M Na2CO3 | 0.165 |
| 1M CaCl2 | 0.065 |
| 10%BSA bovine serum albumin(BSA) | 0.5 |
| 250mM Probenecid | 0.5 |
| It amounts to | 50 |
The balanced salt solution of the HBSS solution, that is, Hanks is commercially available Thermo Products, and specific ingredient see the table below:
| Ingredient | Concentration (mg/L) | Concentration (mM) |
| CaCl2(anhydrous) | 140 | 1.261261 |
| MgCl2·6H2O | 100 | 0.492611 |
| MgSO4·7H2O | 100 | 0.406504 |
| KCl | 400 | 5.333334 |
| KH2PO4 | 60 | 0.441176 |
| NaHCO3 | 350 | 4.166667 |
| NaCl | 8000 | 137.931 |
| Na2HPO4(anhydrous) | 48 | 0.338028 |
| D-Glucose | 1000 | 5.555555 |
HEPES is n-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, and the HEPES solution is commercially available Thermo Products,
HEPES concentration is 1M, pH value 7.2-7.5；The Probenecid is probenecid.
Preferably, the pH=7.4 of the Flex buffer.
Preferably, step (2) is described is carried out with calcium ion fluorescent solution to the cell in the burnt culture dish of copolymerization is planted
The condition of incubation are as follows: be protected from light at 37 DEG C and be incubated for 45-60min, the area of attached cell covering culture dish bottom is made to reach 40%-
60%.
Preferably, the model FV1000 type of laser scanning co-focusing microscope described in step (3)；Acquisition parameters setting
Are as follows: object lens are 60 times of oil mirrors, and laser tube is ne ion laser, and excitation wavelength 488nm, launch wavelength range is 500nm-
600nm, scanning speed are 8 μ s/Pixel, and pixel is 640x 640, are carried out using XYT scanner program to cell not damaged continuous
Scanning is taken pictures, room temperature, be protected from light under the conditions of scan 4min, obtaining totalframes is no less than 30 fluorescence pictures；Then it will obtain
Continuous observation fluorescent image fluorescent quantitation is carried out to unicellular on fluorescent image with ImageJ software, obtain single in 120s
The fluorescent value of cell.
Preferably, the sucrose solution concentration that the sugariness of sucrose solution is 1 under certain known concentration described in step (6) is 150mM.
A kind of method for quantitatively determining of sugariness on a cellular level specifically comprises the following steps:
(1) the HEK293 cell of adhere-wall culture and HEK293-T1R2/T1R3 cell are suspended by normal passage step
The preparation of cell；The HEK293 cell of confluent cultures ware and HEK293-T1R2/T1R3 cell are specially removed into culture solution, used
5mL phosphate buffer (10mM, pH 7.4) washes twice, thin with trypsase (Trypsin) solution digestion of 1mL 0.25%
Born of the same parents 2min is added fresh culture solution and stops, is transferred in 15ml centrifuge tube, and 180g is centrifuged 5min, removes supernatant liquor；Xiang
1ml fresh medium is added in lower sediment, carries out the preparation of suspension cell, is uniformly mixed so as to obtain cell suspending liquid；
(2) a 1.5ml EP centrifuge tube separately is taken, 990 μ l fresh mediums is added in Guan Zhongxian, add 10 μ l steps
(1) cell suspending liquid obtained mixes；
(3) cell suspension obtained in 10 μ l steps (2) is taken out, is added in 25 lattice × 16 lattice blood counting chamber,
It is counted under optical microscopy mirror, determines cell concentration；Cell concentration according to measurement carries out above-mentioned steps (1) cell suspension dilute
It releases, preparation work concentration is 105The cell suspension of cells/ml；Taking 1ml concentration is about 105The cell suspending liquid of cells/ml adds
Enter to 35mm and be copolymerized in burnt culture dish, is placed in 37 DEG C of constant temperature cell incubators and is cultivated；
The present invention uses 25 lattice × 16 lattice blood counting chamber, cell concentration calculation formula are as follows: number of cells/ml=
4 block plaid total number of cells/4 × 10000 × 10000.Above-mentioned formula please refers to related document, and (all cards are bright, blood counting chamber
Use relevant calculation, " new college entrance examination (high three is physical and chemical raw) ", 4 phases in 2013,61-64)
(4) it after the cell suspending liquid of step (2) being seeded to the burnt culture dish culture for 24 hours of copolymerization, sees under an optical microscope
It examines, when the area for observing cell covering culture dish bottom reaches 40%-60%, removes the culture solution in culture dish, then
The buffer that 1mL contains Fluo-4AM (Thermo Fisher, F14202) fluorescence probe (10 μM) is added into culture dish, after
Continue and be protected from light culture 45-60min in 37 DEG C of constant temperature cell incubators, obtains the cell of Fluo-4 fluorescence probe label；
(5) with the cell of Fluo-4 fluorescence probe label in FV1000 laser scanning co-focusing microscope determination step (4)
Intracellular Fluo-4 and the fluorescence intensity emitted after calcium binding at different conditions；It is copolymerized in FV1000 scanning laser
When focusing microscope starts the 16s after scanning, sweet tea sample solution to be detected is added into culture dish, continues to sweep cell
It retouches and takes pictures, obtain fluorescence picture；The parameter setting of the laser confocal scanning microscope are as follows: object lens are 60 times of oil mirrors, laser
Pipe is ne ion laser, and excitation wavelength 488nm, launch wavelength range is 500nm-600nm, and scanning speed is 8 μ s/
Pixel, pixel are 640x 640, carry out not damaged continuous scanning to cell using XYT scanner program and take pictures, in room temperature, are protected from light
Under the conditions of scan 4min, obtain totalframes and be no less than 30 fluorescence pictures；
(6) step (5) are obtained into fluorescence picture ImageJ software and analyzes individual cells consecutive variations in preceding 2min
Fluorescence intensity；Minimum fluorescent value before choosing in 16s is set as Fo, and the maximum fluorescence value before choosing in 120s is set as Fm, single
A cell fluorescence intensity changing ratio is calculated with △ F/Fo=(Fm-Fo)/Fo formula；With calculated HEK293-T1R2/
T1R3 cell fluorescence changing ratio △ F/Fo subtracts the change in fluorescence ratio △ F/Fo intracellular of HEK293 cell, obtains HEK293-
Fluorescence intensity change ratio difference △ R intracellular between T1R2/T1R3 cell and HEK293 cell.We define under certain known concentration
The sugariness of sucrose solution is 1 (for example defining 150mM sweetness of cane sugar is 1), HEK293-T1R2/T1R3 cell and HEK293 cell
Between fluorescence intensity change ratio difference intracellular be sugariness that △ R reflects sugar juice to be detected；Change in fluorescence ratio difference △ R is got over
Greatly, then the sugariness for detecting sugar juice is bigger.
Wherein, step (3) the Fluo-4AM fluorescence probe solution contains 10 μM of Flex buffer dilution, the Flex
The composition of buffer are as follows:
| Reagent | Volume (mL) |
| HBSS solution | 47.5 |
| HEPES solution | 1 |
| 1M MgSO4 | 0.05 |
| 1M Na2CO3 | 0.165 |
| 1M CaCl2 | 0.065 |
| 10%BSA bovine serum albumin(BSA) | 0.5 |
| 250mM Probenecid | 0.5 |
| It amounts to | 50 |
The balanced salt solution of the HBSS solution, that is, Hanks is commercially available Thermo Products (Cat
No.14025092), specific ingredient see the table below:
| Ingredient | Concentration (mg/L) | Concentration (mM) |
| CaCl2(anhydrous) | 140 | 1.261261 |
| MgCl2·6H2O | 100 | 0.492611 |
| MgSO4·7H2O | 100 | 0.406504 |
| KCl | 400 | 5.333334 |
| KH2PO4 | 60 | 0.441176 |
| NaHCO3 | 350 | 4.166667 |
| NaCl | 8000 | 137.931 |
| Na2HPO4(anhydrous) | 48 | 0.338028 |
| D-Glucose | 1000 | 5.555555 |
HEPES indicates n-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, and the HEPES solution is the production of commercially available Thermo company
Product (Cat No.15630080), HEPES solution concentration are 1M, pH value 7.2-7.5；The Probenecid is probenecid.
Measuring method of the invention is compared to the advantage of existing detection sweet taste technology:
1, the present invention stablizes calcium ion concentration in the living cells of expression source of people sweet receptor by detection and changes come evaluation
The sugariness for closing object, can react sugariness on physiological level.Rather than as existing analytical chemistry detection method and biology side
Method detection method detects the content of sweet taste soluble solid in food, is then determined according to the content of these single sweet substances
Measure the sugariness of these foods or complex compound.The sugariness of the not true compound of these measurements.
2, measuring method of the invention simulates source of people sweet receptor to the perception degree of sweet substance on a cellular level, from
Source of people sweet receptor being quantified on cellular level, degree being perceived to sweet substance, people can be quantified under ex vivo to other unknown samples
The perception degree of product sweet taste.The present invention establishes a stable sugariness detection platform in vitro, it is possible to prevente effectively from human subject
The influence of factor.Method of the invention can quick, efficient, highly sensitive detection compound sugariness, have a good application prospect.
Specific embodiment
Embodiment 1
Under 150mM sucrose solution, HEK293 cell and the variation of HEK293-T1R2/T1R3 cell intracellular free calcium level:
(1) it is copolymerized repopulating cell in burnt culture dish:
Burnt special culture dish (outer diameter 35mm, internal diameter 10mm) will be copolymerized with 1ml poly-D-lysine (25 μ g/ml) 37 DEG C of incubations
30min three times with phosphate buffer (10mM, pH 7.4) cleaning, then cell is passed in proportion and is copolymerized in burnt culture dish.To
Cell adherent growth -48h for 24 hours in culture dish observes that the area of cell covering culture dish bottom reaches under an optical microscope
40%-60%.
(2) Flex buffer (pH=7.4) is configured
| Reagent | Volume (mL) |
| HBSS solution | 47.5 |
| HEPES solution | 1 |
| 1M MgSO4 | 0.05 |
| 1M Na2CO3 | 0.165 |
| 1M CaCl2 | 0.065 |
| 10%BSA bovine serum albumin(BSA) | 0.5 |
| 250mM Probenecid | 0.5 |
| It amounts to | 50 |
The balanced salt solution of the HBSS solution, that is, Hanks is commercially available Thermo Products, and specific ingredient see the table below:
| Ingredient | Concentration (mg/L) | Concentration (mM) |
| CaCl2(anhydrous) | 140 | 1.261261 |
| MgCl2·6H2O | 100 | 0.492611 |
| MgSO4·7H2O | 100 | 0.406504 |
| KCl | 400 | 5.333334 |
| KH2PO4 | 60 | 0.441176 |
| NaHCO3 | 350 | 4.166667 |
| NaCl | 8000 | 137.931 |
| Na2HPO4(anhydrous) | 48 | 0.338028 |
| D-Glucose | 1000 | 5.555555 |
HEPES indicates n-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid；The HEPES solution is the production of commercially available Thermo company
Product (Cat No.15630080), concentration 1M, pH value 7.2-7.5；The Probenecid is probenecid.
(3) fluorescence probe marks cell:
Configured Flex buffer is placed on warm bath 30min in 37 DEG C of water-baths.The Fluo- for being 10mM by 10 μ l concentration
4AM is diluted to 10ml Flex buffer, obtains Fluo-4AM (10 μM) dilution, is protected from light.Remove the training being copolymerized in burnt culture dish
Fluo-4AM (10 μM) dilution of 1ml is added in nutrient solution, every ware, and 37 DEG C are protected from light culture 45-60min.After culture, remove
Fluo-4AM dilution (10 μM) is added 1ml Flex buffer solution for cleaning one time.1ml Flex buffer is added in every ware, in case after
Continuous FV1000 laser scanning co-focusing microscope detection.
(4) laser confocal scanning microscope obtains XYT axis fluorescence picture
It is micro- that laser scanning co-focusing is opened in order, is opened 488nm and is excited light laser；
Software is opened, adjusting scanning speed is 8 μ s/Pixel, and pixel is 640x 640, and opening time axis scan pattern swashs
It shines as 488nm, receiving optical channel is 500nm-600nm；
Object lens are adjusted to 60 times of mirrors, a drop cedar oil is added dropwise on camera lens, culture dish is fixed on above objective table；
Camera lens is risen, cedar oil and culture dish following table face contact are made, then adjusts thin quasi- burnt spiral and finds cell, until
Under eyepiece can clear view to cellular morphology；
It is good to find cellular morphology, the finely dispersed visual field (facilitating subsequent fluorescent quantitation)；
Quickly scanning fluorescent image, by adjust excitating light strength, HV (enhancing picture signal), Gain (enhancing signal),
Offset (background correction) and focal plane obtain clearly scanning calcium ion fluorescent image in cell；
The Flex buffer in culture dish is removed with liquid-transfering gun, adjusts focal plane again；
Start to scan fluorescent image, when scanning a length of 4min, totalframes 60；
When starting the 16s after scanning, (cut in the 150mM sucrose solution that 200 μ l are vacantly added dropwise in testing goal cell peripheral
Culture dish is not touched, prevents the visual field mobile)；
It waits sweep time to terminate, saves image data, subsequent progress data collection and processing.
(5) quantitative unicellular intracellular free calcium level variation
Image file is opened in Image J software, randomly selects three in the fluorescence picture shot in (4) step
There is 2-3 cell in the visual field in each visual field, extracts cell respectively in the fluorescence values of each frame image.
The minimum fluorescent value of 16s was Fo in the past, and the maximum fluorescence value of former 2min is Fm, was obtained according to following formula single thin
The change in fluorescence ratio of born of the same parents: △ F/Fo=(Fm-Fo)/Fo；It, will to the change in fluorescence average of relatives of cells multiple in image file
HEK293-T1R2/T1R3 cell fluorescence intensity changing ratio △ F/Fo value subtracts HEK293 cell fluorescence intensity changing ratio △
The value of F/Fo obtains change in fluorescence ratio △ Rs, value 0.284.
Embodiment 2
Under the stimulation of 50mM sucrose, HEK293 cell and the variation of HEK293-T1R2/T1R3 cell intracellular free calcium level, step
Rapid and condition is the same as embodiment 1.
Obtaining change in fluorescence ratio △ R is 0.105.The sugariness of the 150mM sucrose solution of embodiment 1 is set as 1, then △ R/
△ Rs value is 0.370, then the sucrose solution relative sweetness of the present embodiment 50mM is 0.370.
Embodiment 3
Under the stimulation of 250mM sucrose, HEK293 cell and the variation of HEK293-T1R2/T1R3 cell intracellular free calcium level, step
Rapid and condition is the same as embodiment 1.
Obtaining change in fluorescence ratio △ R is 0.487.The sugariness of the 150mM sucrose solution of embodiment 1 is set as 1, then △ R/
△ Rs value is 1.71, then the relative sweetness of the sucrose solution of the present embodiment 250mM is 1.71.
Embodiment 4
Under the stimulation of 150mM glucose, HEK293 cell and the variation of HEK293-T1R2/T1R3 cell intracellular free calcium level,
Step and condition are the same as embodiment 1.
Obtaining change in fluorescence ratio △ R is 0.224.The sugariness of the 150mM sucrose solution of embodiment 1 is set as 1, then △ R/
△ Rs value is 0.789, then the relative sweetness of the present embodiment 150mM glucose solution is 0.789.
Embodiment 5
Under the stimulation of 50mM Sucralose, HEK293 cell and HEK293-T1R2/T1R3 cell intracellular free calcium level become
Change, step and condition are the same as embodiment 1.
Obtaining change in fluorescence ratio △ R is 5.9.The sugariness of the 150mM sucrose solution of embodiment 1 is set as 1, then △ R/ △
Rs value is 20.7, then the relative sweetness of the present embodiment 50mM Sucralose is 20.7.
Claims (9)
1. a kind of method for quantitatively determining of sugariness on a cellular level, which comprises the steps of:
(1) the HEK293 cell of adhere-wall culture and HEK293-T1R2/T1R3 cell are subjected to suspension cell by normal passage step
Preparation, and planted respectively in being copolymerized in burnt culture dish；
(2) when the HEK293 cell and HEK293-T1R2/T1R3 cell of the adhere-wall culture in the burnt culture dish of the copolymerization of step (1)
Convergence degree when reaching 40%-60%, plantation is incubated in being copolymerized cell in burnt culture dish with calcium ion fluorescent solution
It educates；
(3) cell fluorescent images after being incubated for using laser scanning co-focusing microscope continuous observation recording step (2) to 120s,
The fluorescence intensity at individual cells each time point in 120s of HEK293 cell and HEK293-T1R2/T1R3 cell is obtained respectively
Value；Minimum fluorescent value sets in 16s to 120s maximum fluorescence value as Fm as Fo in 16s before setting；Wherein it is added in 16s
Sweet tea sample solution to be measured；
(4) the fluorescence intensity change rate value of HEK293 cell and HEK293-T1R2/T1R3 cell is calculated separately with △ F/Fo,
Wherein △ F is Fm-Fo；
(5) HEK293-T1R2/T1R3 cell fluorescence intensity changing ratio △ F/Fo value HEK293 cell fluorescence intensity is subtracted to become
The value for changing ratio △ F/Fo is △ R；
(6) set the sugariness of sucrose solution under certain known concentration as the value of 1, F/Fo be △ Rs；The then △ R value that step (5) obtains
With the ratio of △ Rs, the relative sweetness of sweet tea sample solution as to be measured.
2. measuring method as described in claim 1, which is characterized in that the burnt culture dish of copolymerization described in step (1) be suitable for
The Tissue Culture Dish of oil mirror observation is carried out on laser confocal microscope.
3. measuring method as claimed in claim 2, which is characterized in that the specification for being copolymerized burnt culture dish is outer diameter 35mm,
Internal diameter 10mm；The burnt culture dish of the copolymerization is needed with 25 μ g/ml Poly-L-Lysine Solutions before use in 37 DEG C of incubation 30min.
4. measuring method as described in claim 1, which is characterized in that calcium ion fluorescent described in step (2) is
Fluo-4AM, and its concentration in Flex buffer is 10 μM.
5. measuring method as claimed in claim 4, which is characterized in that the Flex buffer include it is as shown in the table at
Point:
The balanced salt solution of the HBSS solution, that is, Hanks, specific ingredient see the table below:
HEPES is n-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, and the HEPES solution concentration is 1M, pH value 7.2-7.5；Institute
Stating Probenecid is probenecid.
6. measuring method as claimed in claim 5, which is characterized in that the pH=7.4 of the Flex buffer.
7. measuring method as described in claim 1, which is characterized in that step (2) is described to use calcium ion fluorescent solution pair
Plant the condition that cell is incubated in the burnt culture dish of copolymerization are as follows: be protected from light at 37 DEG C and be incubated for 45-60min, cover attached cell
The area of lid culture dish bottom reaches 40%-60%.
8. measuring method as described in claim 1, which is characterized in that laser scanning co-focusing microscope described in step (3)
Model FV1000 type；Acquisition parameters setting are as follows: object lens are 60 times of oil mirrors, and laser tube is ne ion laser, excitation wavelength
For 488nm, launch wavelength range is 500nm-600nm, and scanning speed is 8 μ s/Pixel, and pixel is 640x 640, using XYT
Scanner program carries out not damaged continuous scanning to cell and takes pictures, room temperature, be protected from light under the conditions of scan 4min, obtaining totalframes is not
Less than 30 fluorescence pictures；Then by the continuous observation fluorescent image of acquisition with ImageJ software to unicellular on fluorescent image
Fluorescent quantitation is carried out, the fluorescent value of individual cells in 120s is obtained.
9. measuring method as described in claim 1, which is characterized in that sucrose solution under certain known concentration described in step (6)
The sucrose solution concentration that sugariness is 1 is 150mM.
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