CN102168081B - GFP (Green Fluorescent Protein) leakage test method - Google Patents
GFP (Green Fluorescent Protein) leakage test method Download PDFInfo
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- CN102168081B CN102168081B CN201010586179A CN201010586179A CN102168081B CN 102168081 B CN102168081 B CN 102168081B CN 201010586179 A CN201010586179 A CN 201010586179A CN 201010586179 A CN201010586179 A CN 201010586179A CN 102168081 B CN102168081 B CN 102168081B
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Abstract
The invention discloses a GFP (Green Fluorescent Protein) leakage test method, belonging to the technical field of toxicological detection. The method comprises the following steps of: obtaining animal cells for expressing GFP by using the transgenic technology; and then, preparing a cell suspension and a lysis solution, detecting the fluorescence value of GFP after incubation with a test substance, and calculating the leakage rate and the denaturation rate of the cell GFP. By analyzing the relationship between the leakage rate and the denaturation rate of the cell GFP and the MMAS value of the test substance, a substitute test method for judging the irritation of the test substance on eyes through GFP leakage rate and denaturation rate is established. The detection method established by the invention has the advantages of low cost, simple and quick operation, wide application test substance spectrum, high prediction accuracy and good stability and is a promising eye irritation substitute test method. Using an adenoviral vector to transducer a target gene has the advantages of high efficiency, low pathogenicity, high titer, and no integration of chromosome of a host cell in vivo, and the like.
Description
Affiliated technical field
The present invention relates to a kind of toxicology detection method, especially makeup toxicology detection method belongs to toxicology detection technique field.
Background technology
At present, the live body lagophthalmos stimulates (Draize) test to be widely used in safety evaluation of cosmetics, but because of there is subjectivity in its points-scoring system, simultaneously because a large amount of use-testing animal receives the opposition of each protection of animal tissue because of it is caused damage.Under globalization protection of animal motional effects, developed country has generally carried out to substitute, to reduce and to be optimized for " 3R " motion of core.In safety evaluation of cosmetics, external alternative experiment will progressively replace experimentation on animals.Thereby scientist comprises isolated organ model, chorioallantois film test and cells in-vitro test etc. having carried out a large amount of explorations aspect the stripped alternative method of development scientific and reliable eye irritant test both at home and abroad.In animal experiment; At least want the pathologic reaction of corneal, conjunctiva and three kinds of tissues of iris to observe and mark; Single in vitro method be difficult to simulate fully reaction in a organized way, still have no a kind of method to have sufficient test basis proof can substitute the animal eye irritation test fully at present.The more alternate test of research has the film test of chicken embryo chorioallantois, erythrocyte hemolysis test, oxyphorase denatured test, MTT test etc. at present.These methods respectively have relative merits at ease-to-operate, the suitable aspects such as thing spectrum, specificity and accuracy of being tried.
Summary of the invention
In order to overcome the above-mentioned deficiency of existing TP, the present invention provides the TP of GFP (green fluorescent protein) leakage rate in a kind of detection SIRC cell (rabbit cornea cell).
The technical solution adopted for the present invention to solve the technical problems is:
GFP reveals TP, comprises the following steps:
A. contain the selection of the adenovirus packaging cell of expression vector Pshuttle-CMV-GFP:
293 cell cultures are in the RPMI of 10% foetal calf serum 1640 substratum, and the SIRC cell attachment is incubated at the high sugared DMEM substratum of 10% foetal calf serum, and culture condition is 37 ℃, 5%CO
2(going down to posterity once in general 2～3 days); The vector virus rotaring redyeing 293 cell that will contain Pshuttle-CMV-GFP;
B. adenovirus prepares and titer determination:
Get 50ml 293 cells, 70% is paved with, and conventional cell cultures makes cell count reach 10
5/ mL, with 96 hole enzyme plate bed boards, every hole adds 100 μ L cell suspensions, spreads 10 rows altogether; Prepare 10 1.5mL EP pipes, every pipe adds serum-free RPMI 1640 substratum of 900 μ L, and first pipe adds 100 μ L virus liquid, mixing; The viral liquid of getting 100 μ L, first pipe joins second pipe, presses the dilution of gradient dilution method up to the 9th pipe; Every row at above-mentioned 96 hole enzyme plates adds dilution viral liquid 100 μ l, and the substratum that the 10th row adds serum-free is as negative control; 37 ℃, CO
2Incubator hatching 7-10 days, the situation of CPE appears in observation of cell; The hole count of the existing CPE of every discharge that counts, calculate cytopathy variability (like the whole pathologies of each porocyte of a certain concentration, ratio is 1, and like acellular pathology, then ratio is 0):
T＝10
1+d(S-0.5)/mL；
D=Log 10
Extent of dilution(as being 10 times of extent of dilution, d=1);
CPE cytopathy ratio sum in each concentration of S=;
Calculating the virus titer that is used for cell infection according to the KARBER formula is 10
7TCID
50/ mL;
The preparation of C.GFP positive cell suspension and cell pyrolysis liquid:
Get the GFP positive cell of 80% degree of converging, dye with the Pshuttle-CMV-GFP virus liquid inductance of confirming titre, behind the 24h with fluorescence microscope luminescence of cell situation; When cell GFP positive rate reaches 80% when above, collecting cell is prepared into 5 * 10
5/ mL cell suspension; Cell suspension is processed cell pyrolysis liquid through multigelation;
D. being tried thing handles and fluoroscopic examination:
To be made into concentration be 10%, 1% and 0.1% solution, suspension or suspension liquid with being tried PBS that thing uses the 0.1mol/L contain 1% bovine serum albumin; In volume is the EP pipe of 500 μ L, add 200 μ L cell suspension or cell pyrolysis liquids, and 200 μ L are tried thing; Control group substitutes with the PBS of the 0.1mol/L that contains 1% bovine serum albumin and is tried thing to calculate the self-dissolving luminous value; Every treatment group repeated sample number is 3; In 37 ℃ of shaking tables, hatch 10min, the treatment group that adds cell suspension is got supernatant 200 μ L and is added 96 hole enzyme plates with the centrifugal 10min of 2000r/min; The treatment group of adding cell pyrolysis liquid is directly got 200 μ L and is added 96 hole enzyme plates, on ELIASA, surveys its luminance value with 485nm and 535nm;
E.GFP spills rate, GFP sex change rate is calculated:
GFP leakage rate=[(being tried thing supernatant luminance value-contrast supernatant luminance value)/contrast luminance value] * 100%;
GFP sex change rate=[(contrast lysate luminance value-tried thing lysate luminance value)/contrast lysate luminance value] * 100%.
The criterion of present method eye irritation is: any thing that tried more than or equal to 25% in GFP sex change rate or the GFP leakage rate is judged to be eye and stimulates positively, GFP sex change rate and GFP leakage rate are judged to be eye and stimulate a feminine gender less than 25% the thing that tried simultaneously.
It is example that present method is dyed the SIRC cell with the viral liquid inductance of adenovirus expression carrier (Pshuttle-CMV-GFP); Process cell suspension and lysate after expressing GFP; Respectively with tried thing and hatched; Detect leakage rate and the sex change rate of cell GFP, analysis of cells GFP leakage rate with tried the dependency of thing MMAS value, thereby set up a kind of method (photogenic cell being handled an alternative stimulation test method) of alternate test through medicine.
GFP is not present in the higher organism cell from luminescent jellyfish, is easy to observe and detect, and therefore be biomarker albumen commonly used in the biomedical research.Present method adopts makeup raw material commonly used to handle the mammalian cell that changes the GFP gene, measures the content of the GFP that from cell, leaks.
The invention has the beneficial effects as follows that cost is low, easy and simple to handle fast, be suitable for and tried that the thing spectrum is wide, forecasting accuracy is high; Utilize adenovirus carrier transduction goal gene have high-level efficiency, low pathogenicity, high titre and in vivo unconformability go into advantages such as host cell chromosome.Present method has good stability and safety.The present invention is an example with corneal epithelial cells of rabbit (SIRC cell) only, and the method for being set up also is applicable to clones such as Hela, hepG2, C2C12.The present invention only is example with the adenovirus expression carrier, wherein also comprise can be in cell other transgenic methods of expressing green fluorescent protein, like methods such as plasmid, retrovirus, slow viruss.
Embodiment
Below in conjunction with embodiment the present invention is further specified.
Utilize adenovirus expression carrier Pshuttle-CMV-GFP virus liquid inductance to dye the SIRC cell, treat to process cell suspension and lysate after it expresses GFP, hatch with the various solution (or suspension) that tried the thing different concns respectively; Utilize ELIASA to detect leakage rate and the sex change rate of cell GFP at last, and analysis of cells GFP leakage rate with tried the dependency of thing MMAS value, thereby set up a kind of alternative zooperal method, promptly detect the TP of GFP leakage rate in the SIRC cell.Being tried thing comprises: tensio-active agent, acid, alkali, alcohol, ester, ketone, amine, inorganic salt and organic salt, table 1 have provided 18 kinds and have been tried that name is claimed and the MMAS value.
Embodiment one:
Being tried thing is lactic acid:
In the RPMI of 10% foetal calf serum 1640 substratum, the SIRC cell cultures is in the high sugared DMEM substratum of 10% foetal calf serum with 293 cell cultures, and culture condition is 37 ℃, 5%CO
2Went down to posterity once in general 2～3 days; Get the GFP positive cell of 80% degree of converging, use T=10
7TCID
50The Pshuttle-CMV-GFP of/mL virus liquid inductance dyes, behind the 24h with fluorescence microscope luminescence of cell situation; When cell GFP positive rate reaches 80% when above, collecting cell is prepared into 5 * 10
5/ mL cell suspension; Get the part cell suspension through multigelation, process cell pyrolysis liquid; Using the PBS of the 0.1mol/L contain 1% bovine serum albumin to be made into concentration lactic acid is 10%, 1% and 0.1% solution, in volume is the EP pipe of 500 μ L, adds 200 μ L cell suspension or cell pyrolysis liquids, and 200 μ L lactic acid; Control group substitutes lactic acid with the PBS of the 0.1mol/L that contains 1% bovine serum albumin; Every treatment group repeated sample number is 3; In 37 ℃ of shaking tables, hatch 10min,, get supernatant 200 μ L and add 96 hole enzyme plates with the centrifugal 10min of 2000r/min; On ELIASA, survey lactic acid treatment group supernatant luminance value, control group supernatant luminance value and control group luminance value with 485nm and 535nm and be respectively 6626.67,6565.33 and 20194, calculating GFP, to spill rate be 0.3%; On ELIASA, be respectively 30730 and 317 with 485nm and 535nm survey control group lysate luminance value, lactic acid group lysate luminance value, calculating GFP sex change rate is 98.97%.Lactic acid GFP sex change positive (sex change rate >=25%) is described, delimiting lactic acid is that eye stimulates the positive.
Embodiment two:
Being tried thing is Virahol:
In the RPMI of 10% foetal calf serum 1640 substratum, the SIRC cell cultures is in the high sugared DMEM substratum of 10% foetal calf serum with 293 cell cultures, and culture condition is 37 ℃, 5%CO
2Went down to posterity once in general 2～3 days; Get the GFP positive cell of 80% degree of converging, use T=10
7TCID
50The Pshuttle-CMV-GFP of/mL virus liquid inductance dyes, behind the 24h with fluorescence microscope luminescence of cell situation; When cell GFP positive rate reaches 80% when above, collecting cell is prepared into 5 * 10
5/ mL cell suspension; Get the part cell suspension through multigelation, process cell pyrolysis liquid; Using the PBS of the 0.1mol/L contain 1% bovine serum albumin to be made into concentration Virahol is 10%, 1% and 0.1% solution, in volume is the EP pipe of 500 μ L, adds 200 μ L cell suspension or cell pyrolysis liquids, and 200 μ L Virahols; Control group substitutes Virahol with the PBS of the 0.1mol/L that contains 1% bovine serum albumin; Every treatment group repeated sample number is 3; In 37 ℃ of shaking tables, hatch 10min,, get supernatant 200 μ L and add 96 hole enzyme plates with the centrifugal 10min of 2000r/min; On ELIASA, survey Virahol treatment group supernatant luminance value, control group supernatant luminance value and control group luminance value with 485nm and 535nm and be respectively 21378.67,3253 and 27214, calculating GFP, to spill rate be 66.6%; On ELIASA, be respectively 38882 and 12935.5 with 485nm and 535nm survey control group lysate luminance value, Virahol group lysate luminance value, calculating GFP sex change rate is 66.73%.Virahol treatment group GFP sex change positive (sex change rate >=25%) is described, delimiting Virahol is that eye stimulates the positive.
Embodiment three:
Being tried thing is glycerine:
In the RPMI of 10% foetal calf serum 1640 substratum, the SIRC cell cultures is in the high sugared DMEM substratum of 10% foetal calf serum with 293 cell cultures, and culture condition is 37 ℃, 5%CO
2Went down to posterity once in general 2～3 days; Get the GFP positive cell of 80% degree of converging, use T=10
7TCID
50The Pshuttle-CMV-GFP of/mL virus liquid inductance dyes, behind the 24h with fluorescence microscope luminescence of cell situation; When cell GFP positive rate reaches 80% when above, collecting cell is prepared into 5 * 10
5/ mL cell suspension; Get the part cell suspension through multigelation, process cell pyrolysis liquid; Using the PBS of the 0.1mol/L contain 1% bovine serum albumin to be made into concentration glycerine is 10%, 1% and 0.1% solution, in volume is the EP pipe of 500 μ L, adds 200 μ L cell suspension or cell pyrolysis liquids, and 200 μ L glycerine; Control group substitutes glycerine with the PBS of the 0.1mol/L that contains 1% bovine serum albumin; Every treatment group repeated sample number is 3; In 37 ℃ of shaking tables, hatch 10min,, get supernatant 200 μ L and add 96 hole enzyme plates with the centrifugal 10min of 2000r/min; On ELIASA, survey glycerin treatment group supernatant luminance value, control group supernatant luminance value and control group luminance value with 485nm and 535nm and be respectively 8207,8122.67 and 20344.7, calculating GFP, to spill rate be 0.41%; On ELIASA, be respectively 30336 and 30971.5 with 485nm and 535nm survey control group lysate luminance value, glycerine group lysate luminance value, calculating GFP sex change rate is 0.02%.Glycerin treatment group GFP sex change negative (sex change rate＜25%) is described, and the GFP leakage rate is lower than 25%, delimiting glycerine be that eye stimulates a feminine gender.
Because live body lagophthalmos irritant test (Draize Test) is a classical way of judging the compound eye irritation, therefore selects for use 18 kinds of MMAS value and cell GFP leakage rates of being tried thing to analyze.Acid and alkalescence is tried thing and is caused the GFP sex change, and other effect that is tried thing pair cell GFP leakage is relevant with MMAS value and the concentration of being tried thing.Concentration is 5% o'clock, and the thing that tried of most of MMAS values＞15 has obvious effect to the leakage of the GFP of SIRC cell; Cause cell GFP leakage rate＞25% and when 0.5% and 0.05% concentration, only there is minority to be tried thing (mainly being tensio-active agent).Choose the known thing that tried of MMAS value of GFP sex change negative (sex change rate＜25%) and 100% concentration; It is 5% o'clock SIRC cell GFP leakage rate and the dependency of being tried thing MMAS value that analysis is tried substrate concentration; Relation conefficient reaches 0.888, therefore, the thing that tried of GFP sex change rate positive (>=25%) is judged to be and has eye irritation; For the thing that tried of GFP sex change negative (sex change rate＜25%), GFP leakage rate 25% is decided to be " value of defining " judges the eye irritation that is tried thing.Tried of the influence of substrate concentration difference in order to get rid of in the Draize test to correlation analysis; GFP leakage rate during being tried thing MMAS value and this test and obtain 5% concentration when selecting the concentration 100% of bibliographical information for use is carried out correlation analysis; Have 11 kinds and tried thing (sequence number 3-8 in the table 1; 13,15-18) satisfy analysis condition.The result shows: the relation conefficient of the GFP leakage rate when these 11 kinds MMAS and these when being tried thing 100% concentration are tested 5% concentration is 0.888 (p＜0.001).In the GFP LOT, if GFP leakage rate 25% is decided to be " value of defining ", 11 kinds are tried thing all by correct division, and wherein 7 kinds of eyes stimulate the positive, and 4 kinds of eyes stimulate negative.
Table 1:
Claims (1)
1. a GFP reveals TP, it is characterized in that, comprises the following steps:
A. contain the selection of the adenovirus packaging cell of expression vector Pshuttle-CMV-GFP:
293 cell cultures are in the RPMI1640 of 10% foetal calf serum substratum, and the SIRC cell attachment is incubated at the high sugared DMEM substratum of 10% foetal calf serum, and culture condition is 37 ℃, 5%CO
2The vector virus rotaring redyeing 293 cell that will contain Pshuttle-CMV-GFP;
B. adenovirus prepares and titer determination:
Get 50ml 293 cells, 70% is paved with, and conventional cell cultures makes cell count reach 10
5/ mL, with 96 hole enzyme plate bed boards, every hole adds 100 μ L cell suspensions, spreads 10 rows altogether; Prepare 10 1.5mL EP pipes, every pipe adds serum-free RPMI 1640 substratum of 900 μ L, and first pipe adds 100 μ L virus liquid, mixing; The viral liquid of getting 100 μ L, first pipe joins second pipe, presses the dilution of gradient dilution method up to the 9th pipe; Every row at above-mentioned 96 hole enzyme plates adds dilution viral liquid 100 μ l, and the substratum that the 10th row adds serum-free is as negative control; 37 ℃, CO
2Incubator hatching 7-10 days, the situation of CPE appears in observation of cell; The hole count of the existing CPE of every discharge that counts, calculate the cytopathy variability:
T＝10
1+d(S-0.5)/mL；
D=Log10
Extent of dilution
CPE cytopathy ratio sum in each concentration of S=;
Calculating the virus titer that is used for cell infection according to the KARBER formula is 10
7TCID
50/ mL;
The preparation of C.GFP positive cell suspension and cell pyrolysis liquid:
Get the GFP positive cell of 80% degree of converging, dye with the Pshuttle-CMV-GFP virus liquid inductance of confirming titre, behind the 24h with fluorescence microscope luminescence of cell situation; When cell GFP positive rate reaches 80% when above, collecting cell is prepared into 5 * 10
5/ mL cell suspension; Cell suspension is processed cell pyrolysis liquid through multigelation;
D. being tried thing handles and fluoroscopic examination:
To be made into concentration be 10%, 1% and 0.1% solution, suspension or suspension liquid with being tried PBS that thing uses the 0.1mol/L contain 1% bovine serum albumin; In volume is the EP pipe of 500 μ L, add 200 μ L cell suspension or cell pyrolysis liquids, and 200 μ L are tried thing; Control group substitutes with the PBS of the 0.1mol/L that contains 1% bovine serum albumin and is tried thing to calculate the self-dissolving luminous value; Every treatment group repeated sample number is 3; In 37 ℃ of shaking tables, hatch 10min, the treatment group that adds cell suspension is got supernatant 200 μ L and is added 96 hole enzyme plates with the centrifugal 10min of 2000r/min; The treatment group of adding cell pyrolysis liquid is directly got 200 μ L and is added 96 hole enzyme plates, on ELIASA, surveys its luminance value with 485nm and 535nm;
E.GFP spills rate, GFP sex change rate is calculated:
GFP leakage rate=[(being tried thing supernatant luminance value-contrast supernatant luminance value)/contrast luminance value] * 100%;
GFP sex change rate=[(contrast lysate luminance value-tried thing lysate luminance value)/contrast lysate luminance value] * 100%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002070703A2 (en) * | 2001-03-02 | 2002-09-12 | Florigene Ltd | Cell visual characteristic-modifying sequences |
| CN101688858A (en) * | 2007-05-02 | 2010-03-31 | 新加坡科技研究局 | Method of monitoring retinopathy |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002070703A2 (en) * | 2001-03-02 | 2002-09-12 | Florigene Ltd | Cell visual characteristic-modifying sequences |
| CN101688858A (en) * | 2007-05-02 | 2010-03-31 | 新加坡科技研究局 | Method of monitoring retinopathy |
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