CN109298169A - Screening method, cell model and application of anti-dairy cow endometritis drugs - Google Patents
Screening method, cell model and application of anti-dairy cow endometritis drugs Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
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- G01N2201/129—Using chemometrical methods
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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Abstract
The present invention provides the method for a kind of in-vitro screening and the anti-cow endometritis drug of detection, it is that the cow uteri intimal epithelium cell of in vitro culture is induced to be inflamed after reaction, add drug to be checked, changed by inflammatory protein in detection cell relative to the expression of internal reference albumen, accurately reflect the anti-inflammatory activity of drug to be checked: if the relative expression quantity of inflammatory protein reduces, then drug to be checked has anti-inflammatory work, and it is drug to be checked with respect to anti-inflammatory activity size that relative expression quantity, which reduces multiple,；It is on the contrary then without anti-inflammatory activity；Wherein, the inflammatory protein is role of integrin-linking kinase.In-vitro screening of the invention and the method for detecting anti-cow endometritis drug may be implemented to carry out easy, quickly screening and detection to the anti-cow endometritis activity of drug to be checked in vitro.When using porous plate, high-throughput screening and detection can be realized, screening and testing result have important reference value to the modern clinic application of Chinese medicine and modernization development.
Description
Technical field
The present invention relates to the screening techniques and cell model of anti-cow endometritis drug.
Background technique
Preclinical Drug Activity determination is the significant process whether determining drug is suitable for disease treatment.Cow uteri inner membrance
Inflammation is the morbidity of milk cow postpartum height, and the initiative and screening for the anti-inflammatory drug of the disease are extremely important.To find safely and effectively
Anti-inflammatory drug generally requires to select suitable inflammatory model to screen and evaluate antiphlogistic effects.It is rapid with cell culture technology
Development, inflammatory cell model are at home and abroad widely used in inflammation mechanism research, and think to can be used for the screening of drug anti-inflammatory activity
Research.As the arrival in " antibiotic-free " epoch and the green and healthy demand of milk supply are promoted, Chinese medicine or natural products treat milk cow
The application of endometritis has received widespread attention.However, since milk cow belongs to larger animal, anti-cow endometritis drug
Preclinical Activity determination and screening remain world-famous puzzle.Although it has been reported that the cow uteri intimal epithelium of detection inflammatory
The expression variation of lactoferrin can reflect anti-inflammatory drug anti-inflammatory activity in vitro indirectly in cell, but the technology only detects
A kind of change level of the factor of lactoferrin is difficult to accurately reflect drug anti-inflammatory activity size.Because in vitro experiment, newborn
Ferritin detection level is influenced in addition to being reacted by cell pathology, is also influenced by detection cell quantity or sample size,
I.e. individually detection lactoferrin is also influenced by technical conditions.So the external work in relation to anti-cow endometritis drug
Property detection and accurate and effective appraisal procedure still without ideal research strategy.It would therefore be highly desirable to develop a kind of efficiently fast
Preclinical Activity determination and screening of the detection method of speed for anti-cow endometritis drug.
Summary of the invention
The present invention provides the method for a kind of in-vitro screening and the anti-cow endometritis drug of detection, is to utilize in vitro culture
Cow uteri intimal epithelium cell, pass through detection cell in inflammatory protein expression variation reflection Chinese medicine anti-inflammatory activity, side
Just quickly.Porous plate culture cell is such as used, then tens kinds of even several hundred kinds of drugs can be detected simultaneously, to realize the height of drug
Flux screening.Cow endometritis is the inflammatory process of uterine epithelial cell.The applicant it has been investigated that, in cow uteri
Intimal epithelium cell is inflamed after reaction, and integrin connects protein kinase (integrin-linked protein in cell
Kinase, ILK) the extremely significant raising of expression, and anti-inflammatory drug has significant downward effect to the expression quantity of the albumen,
The expression of internal reference protein actin (β-actin) does not significantly change in reason group throughout simultaneously.Therefore, pass through detection
Expression variation of the role of integrin-linking kinase in vitro in epithelial cell relative to internal reference albumen can reflect drug to be checked indirectly
Anti-inflammatory activity.Between throughout managing groups of cells, introduces internal reference protein expression level and target protein expression levels are corrected, thus more
Add and accurately judge whether institute's testing drug has significant anti-inflammatory effect, can be treatment cow endometritis drug research
Preclinical basis is provided.
The first aspect of the invention provides:
The screening technique of anti-cow endometritis drug, includes the following steps:
The cow uteri intimal epithelium cell of in vitro culture is carried out inflammatory by step 1；
Step 2 applies drug to be detected to the epithelial cell of inflammatory；
Step 3 is changed by the expression of inflammatory protein in detection cell come to drug anti-inflammatory activity drug effect to be detected；If
Inflammatory protein expression quantity reduces, then drug to be checked has anti-inflammatory activity.
In one embodiment, epithelial cell, which carries out inflammatory, is induced using bacterial endotoxin.
In one embodiment, the inflammatory protein is that integrin connects protein kinase.
In one embodiment, the expression of inflammatory protein connects protein kinase antibody using the integrin of IRDye800 label
It is detected.
In one embodiment, detection uses laser near-infrared molecular imaging system, sets excitation wavelength 785nm, reads
Transmitting OD value at 820nm is taken, another setting excitation wavelength 680nm reads and emits OD value at 720nm.
In one embodiment, the judgement of the drug anti-inflammatory activity drug effect in step 3 is calculated using following formula:
ODM1For the OD value that role of integrin-linking kinase ILK in model hole is detected, ODT1For integrin in drug-treated hole
Connect the OD value of kinases ILK detection, ODC1The OD value detected for role of integrin-linking kinase ILK in blank control wells；
ODM2For the OD value that internal reference albumin A CTB in model hole is detected, ODT2It is detected for internal reference albumin A CTB in drug-treated hole
OD value, ODC2The OD value detected for internal reference albumin A CTB in blank control wells.E is the anti-inflammatory activity ratio of drug；When
When E >=0.5, examined drug has significant anti-inflammatory activity；As E≤0.5, then examined drug is without anti-inflammatory activity.
The processing of following steps is carried out for OD value,
A, each measurand is measured several times in the state of different illumination intensity, measurement result is put into two
It ties up in coordinate system, the abscissa of two-dimensional coordinate system is intensity of illumination, and ordinate is OD value；
B, several measured values by same measurand under identical intensity of illumination are fitted, and it is quasi- to obtain optical density
Conjunction value；
C, the optical density match value for obtaining same measurand under adjacent intensity of illumination carries out line, quasi- to optical density
Conjunction value is modified, so that line keeping parallelism of the different measurands between identical two adjacent intensities of illumination；
D, it will bring formula by modified optical density match value as standard absorbance value and be calculated.
In step B, each measured value and other sum of the distance with group measured value are calculated, the sum of selected distance result is minimum
Measured value as reference measurement values, be weighted and averaged using same group of other measured values with reference measurement values, reference measurement values
Weight ratio be 80%, be 20% with the sum of weight ratio of other measured values is organized, and with the weighting system for organizing other measured values
Number meets following relationship,
Wherein, QiFor the weighting coefficient of ith measurement value, LiIt is ith measurement value at a distance from reference measurement values, L 'iFor
With ith measurement value apart from nearest measured value at a distance from reference measurement values, k is proportionality constant.
The anti-inflammatory activity calculating of detection drug is to move egg relative to internal reference albumen flesh by inflammatory protein role of integrin-linking kinase
Difference of the white relative expression levels between different disposal group calculates.
In one embodiment, cell is cultivated simultaneously using porous plate, different drugs to be checked is added wherein, to realize
A variety of drugs to be checked detect simultaneously.
In one embodiment, the porous plate is the micropore tissue culture plate suitable for near-infrared molecular imaging system.
The second aspect of the invention provides:
It is by the cow uteri intimal epithelium of in vitro culture for screening the cell model of anti-cow endometritis drug
Cell carries out inflammatory and constructs to obtain.
The third aspect of the invention provides:
Above-mentioned cell model is for screening the purposes in anti-cow endometritis drug.
Beneficial effect
The method of in-vitro screening and detection anti-inflammatory drug of the invention may be implemented in vitro to drug to be checked anti-milk cow
The scorching activity of Endometrium carries out easy, quickly screening and detection.When using porous plate, high-throughput screening and inspection can be realized
It surveys, screening and testing result have important reference value to the modern clinic application of Chinese medicine and modernization development.
Most of Chinese materia medica preparation or natural products all itself have color, and the color of drug itself often influences general
Light absorption detection method.The present invention is detected using laser near infrared spectrum, while avoiding visible light interference, thus not only
The equipment substance such as interference of the drug intrinsic colour to detection in visible range is eliminated, and significantly reduces culture plate
Autofluorescence.
The technical method detects target protein and internal reference albumen simultaneously, by calculating target protein expression levels and internal reference albumen table
Up to horizontal relative changing value, so that the target protein expression levels after being corrected, more accurately reflect drug anti-inflammatory activity
True horizon.
Detailed description of the invention
Fig. 1 is the distribution map of the various processing modes of cell.In figure in addition to A1, A2, A3, A4, B1, D1, C1 and E1, remaining
Micropore is drug-treated hole T；
Fig. 2 is inflammation of the cow uteri intimal epithelium cell in varying strength that in vitro culture is detected with fluorescence immunoassay blotting
Property reaction condition under integrin connection protein kinase and internal reference albumen β actin expression variation.LPS dosage is bigger in figure,
Inflammatory reaction intensity is bigger, and integrin connection kinase level is higher, and the expression of β actin becomes between each group
Change not significant.
Fig. 3 is that the cow uteri intimal epithelium cell of in vitro culture is detected using Laser Near infrared imaging system different strong
Integrin connection kinase variation under the conditions of the inflammatory reaction of degree.LPS dosage is bigger in figure, and inflammatory reaction intensity is got over
Greatly, the integrin connection protein kinase transmitting optical density detected is bigger, and the expression of β actin changes between each group
It is not significant.
Fig. 4 is anti-in inflammatory using the cow uteri intimal epithelium cell of Laser Near infrared imaging system detection in vitro culture
Under the conditions of answering and after anti-inflammatory Chinese traditional cordate houttuynia therapeutic intervention, the expression variation of integrin connection protein kinase in cell.Fish raw meat
After grass intervenes Cellular inflammatory reaction, the OD value of integrin connection protein kinase is significantly reduced in cell；The table of β actin
Change between each group up to level not significant.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.
The present invention provides the method for a kind of in-vitro screening and the anti-cow endometritis drug of detection, is induction in vitro culture
Cow uteri intimal epithelium cell be inflamed after reaction, add drug to be checked, pass through detection cell in inflammatory protein phase
Expression variation for internal reference albumen, to determine the anti-inflammatory activity of drug to be checked: if inflammatory protein expression quantity reduces, medicine to be checked
Object has anti-inflammatory work, and it is drug to be checked with respect to anti-inflammatory activity size that expression quantity, which reduces multiple,；It is on the contrary then without anti-inflammatory activity；Its
In, the inflammatory protein is role of integrin-linking kinase (integrin-linked kinase, ILK), and internal reference albumen is that β flesh moves egg
White (β-actin, ACTB).In-vitro screening of the invention and the method for detecting anti-cow endometritis drug, may be implemented
Easy, quickly screening and detection is carried out to the anti-cow endometritis activity of drug to be checked in vitro.When using porous plate, energy
Enough to realize high-throughput screening and detection, screening and testing result have weight to the modern clinic application of Chinese medicine and modernization development
The reference value wanted.
High-flux medicaments sifting is to carry out a large amount of compounds based on the screening model of external molecule or cellular level
The screening of pharmacological activity and the building of the extensive sample library of diversity.High throughput screening drug can provide completely new
Screening technique and means have the characteristics that quick, high specific, high sensitivity, can greatly improve breakneck acceleration and efficiency.
In a typical embodiment, using following method:
Preferably, steps are as follows:
(1) cell culture；
(2) Cellular inflammatory is reaction induced, makes inflammatory cell；
(3) drug-treated inflammatory cell to be checked: drug to be checked is added in inflammatory cell culture solution and is cultivated；
(4) it closes；
(5) it antigen-antibody reaction: is added and has fluorescein-labeled inflammatory protein antibody, reacted；
(6) detection of laser near-infrared molecular imaging system, data calculating and interpretation of result.
Preferably, the fluorescein-labeled inflammatory protein antibody is that the anti-integrin connection albumen of IRDye800 label swashs
Enzyme antibody (anti-ILK-IRDye800).
Preferably, the formula that data described in step (6) calculate are as follows:
ODM1For the OD value that role of integrin-linking kinase ILK in model hole is detected, ODT1For integrin in drug-treated hole
Connect the OD value of kinases ILK detection, ODC1The OD value detected for role of integrin-linking kinase ILK in blank control wells；
ODM2For the OD value that internal reference albumin A CTB in model hole is detected, ODT2It is detected for internal reference albumin A CTB in drug-treated hole
OD value, ODC2The OD value detected for internal reference albumin A CTB in blank control wells.E is the anti-inflammatory activity ratio of drug；When
When E >=0.5, examined drug has significant anti-inflammatory activity；As E≤0.5, then examined drug is without anti-inflammatory activity.
The processing of following steps is carried out for OD value,
A, each measurand is measured several times in the state of different illumination intensity, measurement result is put into two
It ties up in coordinate system, the abscissa of two-dimensional coordinate system is intensity of illumination, and ordinate is OD value；
B, several measured values by same measurand under identical intensity of illumination are fitted, and it is quasi- to obtain optical density
Conjunction value；
C, the optical density match value for obtaining same measurand under adjacent intensity of illumination carries out line, quasi- to optical density
Conjunction value is modified, so that line keeping parallelism of the different measurands between identical two adjacent intensities of illumination；
D, it will bring formula by modified optical density match value as standard absorbance value and be calculated.
In step B, each measured value and other sum of the distance with group measured value are calculated, the sum of selected distance result is minimum
Measured value as reference measurement values, be weighted and averaged using same group of other measured values with reference measurement values, reference measurement values
Weight ratio be 80%, be 20% with the sum of weight ratio of other measured values is organized, and with the weighting system for organizing other measured values
Number meets following relationship,
Wherein, QiFor the weighting coefficient of ith measurement value, LiIt is ith measurement value at a distance from reference measurement values, L 'iFor
With ith measurement value apart from nearest measured value at a distance from reference measurement values, k is proportionality constant.
By above-mentioned processing, the accuracy of measurement of OD value can effectively improve, reduce detection error.
Embodiment 1
Specific step is as follows for the method for in-vitro screening and the anti-cow endometritis drug of detection of the invention:
One, cow uteri intimal epithelium cell culture: the cow uteri intimal epithelium cell inoculation training of logarithmic growth phase
It supports in 96 microwell plates, in saturated humidity, 5%CO2Preculture in incubator, culture medium are DMEM/F12 culture solution and 10% nothing
Endotoxic fetal calf serum (Gibco company).Cell suspension is with 5 × 104The concentration of a/ml is inoculated with, every 100 μ l of hole.
Two, Cellular inflammatory is reaction induced: after cell inoculation culture 24 hours, discarding the cell metabolism liquid in culture plate.It will
Bacterial endotoxin LPS (O111:B4, Sigma company) dissolves the concentration for being configured to 10 μ g/ml with fresh DMEM/F12 culture solution,
And it is added in all holes in addition to tetra- holes A1, A2, B1 and C1,100 holes μ l/；Tetra- hole relayings of A1, A2, B1 and C1
It is continuous that the normal culture solution (Fig. 1) without LPS is added.Tissue culture plate is placed in saturated humidity, 5%CO2Continue to cultivate in incubator.
Three, drug-treated cell: sterile Chinese medical extract is diluted to 2g/ml with DMEM/F12 cell culture fluid and is obtained
Then drug culture solution is added to all drug-treated cells with the volume in 100 holes μ l/ with multiple tracks liquid-transfering gun by drug culture solution
3 multiple holes are at least arranged in Kong Zhong, every kind of drug.In addition, negative control hole A1, A2, B1, C1 and inflammatory model hole A3, A4, D1,
The normal culture solution (Fig. 1) for being free of any drug is all added in E1.Microwell plate is placed in saturated humidity, 5%CO later2Incubator
In continue to cultivate.
Four, cell and its protein are fixed: when drug-treated cell 12h, being taken out tissue culture plate, are discarded metabolism liquid, use
TBST solution washs microwell plate 3 times, each 3min, and 5% skimmed milk power solution (BD company, TBST are prepared), 100 μ are then added
The hole l/, slowly shakes 1h on shaking table at room temperature.It can complete the closing of unspecific binding sites well with this condition.
Five, antibody combines: the integrin connection protein kinase antibody (Anti-ILK for taking 100 μ l IRDye800 to mark
Antibody-IRDye800) β-actin antibody (anti-β-actin the antibody- of solution and/or ALexa680 label
ALexa680) (5% skimmed milk power solution is prepared, and antibody working concentration is 1-2 μ g/ml.Antibody is commercially available, such as the U.S.
Abcam company) it is added separately in all micropores, it is then protected from light and slowly shakes 1h at room temperature.
Six, after antibody combines, antibody can be recycled with multiple tracks liquid-transfering gun, or discard antibody-solutions, then uses TBST
Buffer washs microwell plate 3 times, 3min/ times.
Seven, microwell plate detection and calculating: is put into laser near-infrared molecular imaging system, detection inflammatory protein setting excitation
Optical wavelength 785nm reads and emits OD value in each micropore at 820nm, while detecting internal reference albumen setting exciting light
680nm reads the transmitting OD value at 720nm.The transmitting OD value of same drug-treated multiple holes is substituted into following formula
Calculate the anti-inflammatory activity ratio (E) of drug.
ODM1For the OD value that role of integrin-linking kinase ILK in model hole is detected, ODT1For integrin in drug-treated hole
Connect the OD value of kinases ILK detection, ODC1The OD value detected for role of integrin-linking kinase ILK in blank control wells；
ODM2For the OD value that internal reference albumin A CTB in model hole is detected, ODT2It is detected for internal reference albumin A CTB in drug-treated hole
OD value, ODC2The OD value detected for internal reference albumin A CTB in blank control wells.E is the anti-inflammatory activity ratio of drug.
Eight, calculated result judges: as E >=0.5, then judging that examined drug has significant anti-cow endometritis activity；When E≤
When 0.5, then judge that examined drug does not have anti-cow endometritis activity.
Embodiment 2
The present embodiment Western blot and laser near-infrared molecular imaging system are to theory of the invention (in cow uteri
Intimal epithelium cell in inflammatory reaction integrin connection protein kinase (integrin-linked protein kinase,
ILK extremely significant raising) is expressed) it is verified:
The cell culture of cow uteri intimal epithelium, inflammatory reaction Induction Process and detection method are consistent with 1 method of embodiment.
It is whole to resist respectively through polyacrylamide gel electrophoresis, half-dried transfer after treated cell extracts total protein in conventional manner
Close element connection protein kinase antibody (Anti-ILK antibody) and anti-actin antibody (anti-ACTB antibody) into
Row immune-blotting method, with exposure method obtain as a result, result referring to fig. 2.In addition, using laser near-infrared molecular imaging skill
Art, which has detected, cultivates cell in the connection kinase variation of inflammatory reaction integrin in microwell plate.Different LPS dosage (0,
1,5,10,100,200 μ g/ml) it is different to the inflammatory reaction of cell induction, LPS dosage is bigger, and Cellular inflammatory response intensity is got over
Greatly.From figure 2 it can be seen that LPS dosage is bigger, inflammatory reaction intensity is bigger, and integrin connection kinase level is got over
Height, internal reference protein expression level change not significant between each group.From figure 3, it can be seen that LPS dosage is bigger, inflammatory reaction is strong
Degree is bigger, and the integrin connection protein kinase transmitting optical density detected is bigger, and internal reference protein expression level becomes between each group
Change not significant.
Embodiment 3
(in milk cow endometritis occurs for the present embodiment to theory of the invention with laser near-infrared Molecular imaging techniques
Afterwards, integrin connects the extremely significant raising of kinase in endometrial epithelial cell, and anti-inflammatory drug is to the table of the albumen
There is significant downward to act on up to measurer) it is verified:
The cell culture of cow uteri intimal epithelium, inflammatory reaction induction and pharmaceutical intervention process and method one in the present invention
It causes.Treated cell connects protein kinase antibody (Anti-ILK respectively with the integrin of IRDye800 label
Antibody-IRDye800) and ALexa680 label internal reference albumen β-actin antibody (anti-β-actin antibody-
ALexa680) carry out laser near-infrared molecular imaging detection, with laser scanning acquisition as a result, result referring to fig. 4.It can be with from figure
Find out, integrin connects the transmitting OD value of protein kinase in (no LPS with drug-treated) cell when normal no inflammation is reacted
Very low, the transmitting OD value of integrin connection protein kinase is very high in the cell of the LPS processing of 10 μ g/ml, and cordate houttuynia body
Integrin connects the transmitting OD value of protein kinase significantly lower than inflammatory model in the cell that extract is jointly processed by with LPS
Group；The transmitting OD value of internal reference albumen β-actin is uniform in each processing group.After obtaining each hole transmitting OD value, by numerical value
The calculation formula in the present invention is substituted into, the results show that the anti-inflammatory activity ratio of cordate houttuynia is E=0.772, table after technology detection
The bright drug has significant anti-inflammatory activity.
As seen from Figure 4, the expression quantity of cell integrin connection protein kinase in inflammatory reaction is extremely significant increases, and
Its expression quantity significantly reduces after anti-cow endometritis Chinese Medicinal Houttuynia Cordata Thunb is intervened.Illustrate the expression of integrin connection protein kinase
Amount is reacted with Cellular inflammatory with high correlation, and anti-cow endometritis drug is to integrin highly expressed in inflammatory cell
Connecting protein kinase has significant downward effect.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (10)
1. the screening technique of anti-cow endometritis drug, which comprises the steps of:
The cow uteri intimal epithelium cell of in vitro culture is carried out inflammatory by step 1；
Step 2 applies drug to be detected to the epithelial cell of inflammatory；
Step 3 reflects drug anti-inflammatory activity drug effect to be detected by the expression variation of inflammatory protein in detection cell；If scorching
Disease expressing quantity reduces, then drug to be checked has anti-inflammatory activity.
2. the screening technique of anti-cow endometritis drug according to claim 1, which is characterized in that epithelial cell into
Row inflammatory is induced using bacterial endotoxin.
3. the screening technique of anti-cow endometritis drug according to claim 1, which is characterized in that the inflammation egg
White to connect protein kinase for integrin, the expression of inflammatory protein connects protein kinase antibody using the integrin of IRDye800 label
It is detected.
4. the screening technique of anti-cow endometritis drug according to claim 1, which is characterized in that detection is using sharp
Light near-infrared molecular imaging system sets excitation wavelength 785nm, reads and emits OD value at 820nm.
5. the screening technique of anti-cow endometritis drug according to claim 4, which is characterized in that in step 3
The judgement of drug anti-inflammatory activity drug effect is calculated using following formula:
ODM1For the OD value that role of integrin-linking kinase ILK in model hole is detected, ODT1It is connected for integrin in drug-treated hole
The OD value of kinases ILK detection, ODC1The OD value detected for role of integrin-linking kinase ILK in blank control wells；ODM2For
The OD value that internal reference albumin A CTB is detected in model hole, ODT2The optical density detected for internal reference albumin A CTB in drug-treated hole
Value, ODC2For the OD value that internal reference albumin A CTB in blank control wells is detected, E is the anti-inflammatory activity ratio of drug；When E >=0.5
When, examined drug has significant anti-inflammatory activity；As E≤0.5, then examined drug is without anti-inflammatory activity.
6. the screening technique of anti-cow endometritis drug according to claim 5, which is characterized in that for optical density
Value carries out the processing of following steps,
A, each measurand is measured several times in the state of different illumination intensity, measurement result is put into two-dimentional seat
In mark system, the abscissa of two-dimensional coordinate system is intensity of illumination, and ordinate is OD value；
B, several measured values by same measurand under identical intensity of illumination are fitted, and obtain optical density fitting
Value；
C, the optical density match value for obtaining same measurand under adjacent intensity of illumination carries out line, to optical density match value
It is modified, so that line keeping parallelism of the different measurands between identical two adjacent intensities of illumination；
D, it will bring formula by modified optical density match value as standard absorbance value and be calculated.
7. the screening technique of anti-cow endometritis drug according to claim 6, which is characterized in that in step B, meter
Each measured value and other sum of the distance with group measured value are calculated, the smallest measured value of the sum of selected distance result is used as reference to survey
Magnitude is weighted and averaged using same group of other measured values with reference measurement values, and the weight ratio of reference measurement values is 80%, together
The sum of the weight ratio of other measured values of group is 20%, and meets following relationship with the weighting coefficient for organizing other measured values,
Wherein, QiFor the weighting coefficient of ith measurement value, LiIt is ith measurement value at a distance from reference measurement values, L 'iFor with
For i measured value apart from nearest measured value at a distance from reference measurement values, k is proportionality constant.
8. the screening technique of anti-cow endometritis drug according to claim 1, which is characterized in that use porous plate
Cell is cultivated simultaneously, different drugs to be checked are added wherein, to realize a variety of drugs to be checked while detect；The porous plate is
Micropore tissue culture plate suitable for near-infrared molecular imaging system.
9. the cell model for screening anti-cow endometritis drug, which is characterized in that be by milk cow of in vitro culture
Endometrium epithelial cell carries out inflammatory and constructs to obtain.
10. cell model as claimed in claim 9 is for screening the purposes in anti-cow endometritis drug.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005083437A1 (en) * | 2004-02-26 | 2005-09-09 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with integrin-linked kinase 2 (ilk2) |
| CN104080786A (en) * | 2011-11-08 | 2014-10-01 | 因特利凯有限责任公司 | Treatment regimens using multiple agents |
| CN104833782A (en) * | 2015-05-08 | 2015-08-12 | 中国农业科学院兰州畜牧与兽药研究所 | Method for in-vitro screening and detection of anti-cow-endometritis medicaments |
| US20180074045A1 (en) * | 2015-05-27 | 2018-03-15 | Cannabics Pharmaceuticals Inc | System and method for high throughput screening of cancer cells |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005083437A1 (en) * | 2004-02-26 | 2005-09-09 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with integrin-linked kinase 2 (ilk2) |
| CN104080786A (en) * | 2011-11-08 | 2014-10-01 | 因特利凯有限责任公司 | Treatment regimens using multiple agents |
| CN104833782A (en) * | 2015-05-08 | 2015-08-12 | 中国农业科学院兰州畜牧与兽药研究所 | Method for in-vitro screening and detection of anti-cow-endometritis medicaments |
| US20180074045A1 (en) * | 2015-05-27 | 2018-03-15 | Cannabics Pharmaceuticals Inc | System and method for high throughput screening of cancer cells |
Non-Patent Citations (9)
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