CN104833782B - A kind of in-vitro screening and the method detecting anti-cow endometritis medicine - Google Patents
A kind of in-vitro screening and the method detecting anti-cow endometritis medicine Download PDFInfo
- Publication number
- CN104833782B CN104833782B CN201510234048.3A CN201510234048A CN104833782B CN 104833782 B CN104833782 B CN 104833782B CN 201510234048 A CN201510234048 A CN 201510234048A CN 104833782 B CN104833782 B CN 104833782B
- Authority
- CN
- China
- Prior art keywords
- medicine
- inflammatory
- checked
- cell
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000003814 drug Substances 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000012216 screening Methods 0.000 title claims abstract description 27
- 238000000338 in vitro Methods 0.000 title claims abstract description 21
- 208000004145 Endometritis Diseases 0.000 title claims abstract description 20
- 230000003110 anti-inflammatory effect Effects 0.000 claims abstract description 29
- 230000002757 inflammatory effect Effects 0.000 claims abstract description 21
- 102000010445 Lactoferrin Human genes 0.000 claims abstract description 20
- 108010063045 Lactoferrin Proteins 0.000 claims abstract description 20
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 claims abstract description 20
- 229940078795 lactoferrin Drugs 0.000 claims abstract description 20
- 235000021242 lactoferrin Nutrition 0.000 claims abstract description 20
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 235000018102 proteins Nutrition 0.000 claims abstract description 17
- 210000000981 epithelium Anatomy 0.000 claims abstract description 8
- 210000004291 uterus Anatomy 0.000 claims abstract description 8
- 230000008859 change Effects 0.000 claims abstract description 7
- 230000009467 reduction Effects 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 36
- 229940079593 drug Drugs 0.000 claims description 15
- 210000004969 inflammatory cell Anatomy 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 230000001413 cellular effect Effects 0.000 claims description 5
- 230000004907 flux Effects 0.000 claims description 4
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 4
- 239000012930 cell culture fluid Substances 0.000 claims description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- 229930014626 natural product Natural products 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 2
- 102000036639 antigens Human genes 0.000 claims description 2
- 108091007433 antigens Proteins 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000013641 positive control Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 8
- 238000012360 testing method Methods 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 3
- 241000283690 Bos taurus Species 0.000 description 11
- 230000004054 inflammatory process Effects 0.000 description 7
- 206010061218 Inflammation Diseases 0.000 description 6
- 239000002260 anti-inflammatory agent Substances 0.000 description 6
- 229940124599 anti-inflammatory drug Drugs 0.000 description 6
- 239000012531 culture fluid Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 239000000834 fixative Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 241000746375 Andrographis Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000002357 endometrial effect Effects 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- BFVQTKQTUCQRPI-YYEZTRBPSA-N LPS with O-antigen Chemical compound O([C@@H]1[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@@H]4[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@@H]5[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O5)O)O4)O)[C@@H](O)[C@@H](CO)O3)NC(C)=O)[C@@H](O)[C@@H](CO[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)NC(C)=O)O2)NC(C)=O)[C@H](O)[C@@H](CO)OC1O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)OC([C@@H]1O)O[C@H]1[C@H](O)[C@@H]([C@@H](O)COC2[C@H]([C@@H](O)[C@H](OP(O)(O)=O)[C@@H]([C@@H](O)CO)O2)O)OC([C@H]1O)O[C@H]1[C@H](OP(O)(=O)OP(O)(=O)OCCN)[C@@H]([C@@H](O)CO)OC([C@H]1O)O[C@H]1[C@H](O[C@]2(O[C@@H]([C@@H](O)[C@H](O[C@]3(O[C@@H]([C@@H](O)[C@H](OP(O)(=O)OCCN)C3)[C@@H](O)CO)C(O)=O)C2)[C@@H](O)CO)C(O)=O)C[C@](O[C@@H]1[C@@H](O)CO)(OC[C@H]1O[C@@H](OC[C@@H]2[C@H]([C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O2)O)[C@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@H]([C@@H]1OP(O)(O)=O)OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)C(O)=O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O BFVQTKQTUCQRPI-YYEZTRBPSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000001741 anti-phlogistic effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 231100000284 endotoxic Toxicity 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Abstract
The present invention provides a kind of in-vitro screening and the method detecting anti-cow endometritis medicine, it is that the cow uteri intimal epithelium cell of In vitro culture is carried out inflammatory, add medicine to be checked, by detecting the expression change of inflammatory protein in cell, determine the anti-inflammatory activity of medicine to be checked: if inflammatory protein expression reduces, then medicine to be checked has anti-inflammatory activity, and expression reduction multiple is the relative anti-inflammatory activity size of medicine to be checked；Otherwise then without anti-inflammatory activity；Wherein, described inflammatory protein is lactoferrin.The in-vitro screening of the present invention and the method detecting anti-cow endometritis medicine, it is possible to achieve in vitro the anti-cow endometritis activity of medicine to be checked is carried out simplicity, quickly screening and detection.When using porous plate, it is possible to realize high-throughout screening and detection, screening and testing result modern clinic application and the modernization development to Chinese medicine and there is important reference value.
Description
Technical field
The present invention relates to a kind of in-vitro screening and the method detecting anti-cow endometritis medicine.
Background technology
Inflammation is very common and important pathological process, and the initiative of anti-inflammatory drug is essential, and finds safely and effectively anti-inflammatory drug and need to select suitable inflammatory model screen and evaluate antiphlogistic effects.In recent years, along with developing rapidly of cell culture technology, inflammatory cell model is at home and abroad widely used in inflammation mechanism research, and thinks the research that can be used for the screening of medicine anti-inflammatory activity.Along with the arrival in " antibiotic-free " epoch, the antiinflammatory action of Chinese medicine receives extensive concern.Clinical and experiment confirms that a lot of Chinese medicines all have good antiinflammatory action.Building effective appraisal procedure medicine under development anti-inflammatory efficacy aspect is a very important strategy.
Summary of the invention
The present invention provides a kind of in-vitro screening and the method detecting anti-cow endometritis medicine.It is the cow uteri intimal epithelium cell utilizing In vitro culture, by detecting the anti-inflammatory activity expressing change reflection Chinese medicine of inflammatory protein, fast and easy in cell.As adopted porous plate to cultivate cell, then can detect tens kinds of even hundreds of kind medicines, thus realizing the high flux screening of medicine simultaneously.Wherein, inflammatory protein is lactoferrin.
Cow endometritis is the inflammatory process of uterine epithelial cell.The applicant it have been investigated that, after milch cow generation endometritis, in endometrial tissue cell, lactoferrin (Lactoferrin) is expressed and is extremely significantly raised, and the expression of this albumen is had significant downward effect by anti-inflammatory drug.Therefore, by detecting the anti-inflammatory activity expressing change indirect reaction medicine to be checked in lactoferrin (Lactoferrin) epithelial cell in vitro, thus judging whether institute's testing drug has antiinflammatory action significantly, it is possible to provide clinical front basis for treatment cow endometritis.
The present invention provides a kind of in-vitro screening and the method detecting anti-cow endometritis medicine, it is that the cow uteri intimal epithelium cell of In vitro culture is carried out inflammatory, add medicine to be checked, by detecting the expression change of inflammatory protein in cell, determine the anti-inflammatory activity of medicine to be checked: if inflammatory protein expression reduces, then medicine to be checked has anti-inflammatory activity, and expression reduction multiple is the relative anti-inflammatory activity size of medicine to be checked；Otherwise then without anti-inflammatory activity；Wherein, described inflammatory protein is lactoferrin.
In the method for the present invention, medicine to be checked is preferably Chinese medicine or natural product.
Preferably, described detection is fluoroscopic examination.
Preferably, step is as follows:
(1) cell is cultivated；
(2) Cellular inflammatory is reaction induced, makes inflammatory cell；
(3) drug treating inflammatory cell to be checked: medicine to be checked is joined in inflammatory cell culture fluid and cultivate；
(4) cell and protein thereof are fixed；
(5) close；
(6) antigen antibody reaction: add with fluorescein-labeled inflammatory protein antibody, react；
(7) fluorescence signal detection, data calculate and interpretation of result.
Preferably, described cell and protein thereof are fixed is fixed with trichloroacetic acid buffer.Trichloroacetic acid can make protein conformation in cell change, and exposes more hydrophobic group, so as to aggregate and precipitate is at the bottom of porous plate hole, is a kind of desirable efficient albuminous degeneration fixative.
Preferably, the formula that data described in step (7) calculate is:
, wherein n is the multiple hole count of drug treating；The light that P is positive controls absorbs summation；N is that blank group each hole light absorbs summation, and the light that X is the multiple hole of same drug treating absorbs total value, and Y is the anti-inflammatory activity ratio of medicine.
Preferably, when Y >=1, then medicine to be checked is without anti-inflammatory activity；As Y, < when 1, then medicine to be checked has anti-inflammatory activity；When Y≤0.5, then medicine to be checked has notable anti-inflammatory activity.
Preferably, described in-vitro screening and detection are external high flux screening and detection, use porous plate to cultivate cell simultaneously, add different medicine to be checked wherein.Detect thus realizing multiple medicine to be checked simultaneously.
High-flux medicaments sifting is based on the screening model of external molecule or cellular level, carries out the screening of a large amount of compound pharmacologically active and the structure of the extensive sample library of multiformity.High throughput screening drug can provide brand-new screening technique and means, has quick, high specific, highly sensitive feature, can be greatly enhanced breakneck acceleration and efficiency.
Further, described porous plate is the microwell plate of printing opacity at the bottom of the black surround suitable in fluoroscopic examination.
Most Chinese medicine preparation or natural product all itself have color, and the color of medicine itself often affects general light absorption detecting method.The present invention uses fluoroscopic examination, in the dark detects, and the Color influences of medicine itself can be eliminated, and meanwhile, uses fluoroscopic examination fast and easy, and degree of accuracy is high.
The method of the in-vitro screening of the present invention and detection anti-inflammatory drug, it is possible to achieve in vitro the anti-cow endometritis activity of medicine to be checked is carried out simplicity, quickly screening and detection.When using porous plate, it is possible to realize high-throughout screening and detection, screening and testing result modern clinic application and the modernization development to Chinese medicine and there is important reference value.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, is used for together with embodiments of the present invention explaining the present invention, is not intended that limitation of the present invention.In the accompanying drawings:
Fig. 1 is the scattergram of the various processing mode of cell.In figure except A1, A2, A3, A4, B1, D1, C1 and E1, all the other micropores are drug treating hole Y；
Fig. 2 is in the cow uteri intimal epithelium cell with fluorescence immunoassay blotting detection In vitro culture after inflammatory reaction cell and anti-inflammatory Chinese traditional Herba Andrographis therapeutic intervention, the expression change of lactoferrin in cell.
Detailed description of the invention
Below example is easy to be more fully understood that the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop.
Embodiment 1
The method of the in-vitro screening of the present invention and the anti-cow endometritis medicine of detection specifically comprises the following steps that
One, cow uteri intimal epithelium cell is cultivated: the cow uteri intimal epithelium cell inoculated and cultured of trophophase of taking the logarithm is in 96 microwell plates saturating at the bottom of black, at saturated humidity, 5%CO2Preculture in incubator, culture medium is that DMEM/F12 culture fluid and 10% is without endotoxic hyclone (Gibco company).Cell suspension is with 5 × 104The concentration inoculation of individual/ml, every hole 100 μ l.
Two, Cellular inflammatory is reaction induced: cell inoculated and cultured, after 24 hours, discards the cellular metabolism liquid in culture plate.By bacterial endotoxin LPS(O111:B4, Sigma company) concentration being configured to 1 μ g/ml is dissolved with fresh DMEM/F12 culture fluid, and join in all each hole except A1, four holes of A2, B1 and C1,100 μ l/ holes；A1, A2, B1 and C1 continuously add normal culture fluid in four holes.Tissue Culture Plate is placed in saturated humidity, 5%CO2Incubator continues cultivate.
Three, drug treating cell: aseptic Chinese medicine extract DMEM/F12 cell culture fluid is diluted to 2g/ml and obtains medicine culture fluid, then joins medicine culture fluid in all drug treating cell holes with the volume in 100 μ l/ holes with multiple tracks liquid-transfering gun.Every kind of medicine is at least provided with 3 multiple holes.Negative control hole A1, A2, B1, C1 and model comparison hole A3, A4, D1, E1 all add normal culture fluid.Afterwards microwell plate is placed in saturated humidity, 5%CO2Incubator continues cultivate.
Four, cell and protein thereof are fixed: during drug treating cell 24h, take out Tissue Culture Plate, discard metabolism liquid, and the trichloroacetic acid fixative by 10% joins in micropore, every hole 100 μ l, are placed in by microwell plate at 4 DEG C and stand 2h.
Five, close: take out microwell plate, discard fixative, with TBST solution washing microwell plate 3 times, each 3min, be subsequently adding 5% defatted milk powder solution (BD company, TBST prepares), 100 μ l/ holes, under room temperature, on shaking table, slowly shake 1h.Can well complete with this understanding to close.
Six, antibodies: (5% defatted milk powder solution preparation, lactoferrin antibody working concentration is 1-2 μ g/ml to take lactoferrin antibody (Anti-Lactoferrinantibody-FITC) solution of 100 μ l marked by fluorescein isothiocyanate.Antibody is commercially available, such as Abcam company of the U.S.) it is added separately in the middle of all micropores, then keep in Dark Place in 4 DEG C, 8h or at room temperature slowly rock 2h.
Seven, after antibodies, available multiple tracks liquid-transfering gun reclaims antibody, or discards antibody-solutions, then uses TBST buffer solution microwell plate 3 times, 3min/ time.
Eight, reading and calculating: microwell plate is put into all band microplate reader, sets excitation wavelength 496nm, absorbing light wavelength 518nm, carries out two waveband and interferes the fluorescent absorption value read in each micropore.Light absorption total value (X) in multiple for same drug treating hole is substituted into below equation and calculates the anti-inflammatory activity ratio (Y) of medicine.
N is the multiple hole count of drug treating；P is absorbance value summation (the i.e. OD of LPS disposal holeA3+ODA4+ODD1+ODE1)；N is each hole absorbance value summation (the i.e. OD of blank groupA1+ODA2+ODB1+ODC1).
Nine, result of calculation judges: when Y >=1, then judge medicine antibiotic-free milk cattle endometritis activity；When Y < when 1, then judges that medicine has anti-cow endometritis activity；When Y≤0.5, then judge that medicine has notable anti-cow endometritis activity.
Embodiment 2
The present embodiment with fluorescence immunoassay blotting to the theory of the present invention (after milch cow generation endometritis, in endometrial tissue cell, lactoferrin (Lactoferrin) is expressed and is extremely significantly raised, and the expression of this albumen is had significant downward effect by anti-inflammatory drug) it is verified:
Cow uteri intimal epithelium cell is cultivated, inflammatory reaction is induced and pharmaceutical intervention process is consistent with method in the present invention.After treated cell extracts total protein in conventional manner, through polyacrylamide gel electrophoresis, half-dried transfer, with fluorescein-labeled lactoferrin antibody (Anti-Lactoferrinantibody-FITC, purchased from American Abcam company) carry out immune-blotting method, obtaining result with exposure method, result is referring to Fig. 2.The expression of lactoferrin in 1 cell when being the reaction of normal NIP in figure, the expression of lactoferrin in 3 cells when be inflammatory reaction, 2 is the expression of lactoferrin in cell after anti-inflammatory drug intervention.
As seen from Figure 2, cell lactoferrin expression when inflammatory reaction extremely significantly increases, and after anti-cow endometritis Chinese medicine Herba Andrographis intervention, its expression significantly reduces.Illustrating that the expression of lactoferrin reacts with Cellular inflammatory and has high correlation, the lactoferrin of high expressed in inflammatory cell is had significant downward effect by anti-cow endometritis medicine.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature carries out equivalent replacement.All within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (10)
1. the method for an in-vitro screening and the anti-cow endometritis medicine of detection, it is characterized in that: be that the cow uteri intimal epithelium cell of In vitro culture is carried out inflammatory, add medicine to be checked, by detecting the expression change of inflammatory protein in cell, determine the anti-inflammatory activity of medicine to be checked: if inflammatory protein expression reduces, then medicine to be checked has anti-inflammatory activity, and expression reduction multiple is the relative anti-inflammatory activity size of medicine to be checked；Otherwise then without anti-inflammatory activity；Wherein, described inflammatory protein is lactoferrin；Specifically comprise the following steps that
(1) cell is cultivated；
(2) Cellular inflammatory is reaction induced, makes inflammatory cell；
(3) drug treating inflammatory cell to be checked: medicine to be checked is joined in inflammatory cell culture fluid and cultivate；
(4) cell and protein thereof are fixed；
(5) close；
(6) antigen antibody reaction: add with fluorescein-labeled inflammatory protein antibody, react；
(7) fluorescence signal detection, data calculate and interpretation of result.
2. method according to claim 1, it is characterised in that: described medicine to be checked is Chinese medicine or natural product.
3. method according to claim 1 and 2, it is characterised in that: described detection is fluoroscopic examination.
4. method according to claim 1, it is characterised in that: it is fixed with trichloroacetic acid buffer that described cell and protein thereof are fixed.
5. method according to claim 1, it is characterised in that: the formula that data described in step (7) calculate is:
, wherein n is the multiple hole count of drug treating；The light that P is positive controls absorbs summation；N is that blank group each hole light absorbs summation, and the light that X is the multiple hole of same drug treating absorbs total value, and Y is the anti-inflammatory activity ratio of medicine.
6. method according to claim 5, it is characterised in that: when Y >=1, then medicine to be checked is without anti-inflammatory activity；As Y, < when 1, then medicine to be checked has anti-inflammatory activity；When Y≤0.5, then medicine to be checked has notable anti-inflammatory activity.
7. the method according to claim 1,2,4,5 or 6, it is characterised in that: described in-vitro screening and detection are external high flux screening and detection, use porous plate to cultivate cell simultaneously, add different medicine to be checked wherein.
8. method according to claim 3, it is characterised in that: described in-vitro screening and detection are external high flux screening and detection, use porous plate to cultivate cell simultaneously, add different medicine to be checked wherein.
9. method according to claim 7, it is characterised in that: described porous plate is the microwell plate of printing opacity at the bottom of the black surround suitable in fluoroscopic examination.
10. method according to claim 8, it is characterised in that: described porous plate is the microwell plate of printing opacity at the bottom of the black surround suitable in fluoroscopic examination.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510234048.3A CN104833782B (en) | 2015-05-08 | 2015-05-08 | A kind of in-vitro screening and the method detecting anti-cow endometritis medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510234048.3A CN104833782B (en) | 2015-05-08 | 2015-05-08 | A kind of in-vitro screening and the method detecting anti-cow endometritis medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104833782A CN104833782A (en) | 2015-08-12 |
| CN104833782B true CN104833782B (en) | 2016-07-06 |
Family
ID=53811795
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510234048.3A Expired - Fee Related CN104833782B (en) | 2015-05-08 | 2015-05-08 | A kind of in-vitro screening and the method detecting anti-cow endometritis medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104833782B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2642251C1 (en) * | 2016-11-03 | 2018-01-24 | Министерство сельского хозяйства Российской Федерации Федеральное государственное бюджетное образовательное учреждение высшего образования "Вятская государственная сельскохозяйственная академия" (ФГБОУ ВО ВГСХА) | Method for endometritis treatment in cows |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298169B (en) * | 2018-09-21 | 2022-04-15 | 中国农业科学院兰州畜牧与兽药研究所 | Screening method, cell model and application of anti-cow endometritis drug |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006129867A2 (en) * | 2005-06-01 | 2006-12-07 | Senju Pharmaceutical Co., Ltd. | ENHANCED EXPRESSION OF LACTOFERRIN mRNA BY LACRITIN |
| CN101463343A (en) * | 2008-12-30 | 2009-06-24 | 同济大学 | Cell model for screening antiphlogistic medicament and method for screening medicament by using the same |
| CN103983770A (en) * | 2014-05-12 | 2014-08-13 | 中国科学院上海巴斯德研究所 | Screening method of small molecular anti-inflammatory drug |
| CN104017882A (en) * | 2014-06-18 | 2014-09-03 | 中国农业科学院兰州畜牧与兽药研究所 | Fluorescent quantization PCR kit and detection method for detecting cow endometrial cell inflammatory reaction and application of kit |
-
2015
- 2015-05-08 CN CN201510234048.3A patent/CN104833782B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006129867A2 (en) * | 2005-06-01 | 2006-12-07 | Senju Pharmaceutical Co., Ltd. | ENHANCED EXPRESSION OF LACTOFERRIN mRNA BY LACRITIN |
| CN101463343A (en) * | 2008-12-30 | 2009-06-24 | 同济大学 | Cell model for screening antiphlogistic medicament and method for screening medicament by using the same |
| CN103983770A (en) * | 2014-05-12 | 2014-08-13 | 中国科学院上海巴斯德研究所 | Screening method of small molecular anti-inflammatory drug |
| CN104017882A (en) * | 2014-06-18 | 2014-09-03 | 中国农业科学院兰州畜牧与兽药研究所 | Fluorescent quantization PCR kit and detection method for detecting cow endometrial cell inflammatory reaction and application of kit |
Non-Patent Citations (1)
| Title |
|---|
| 天然免疫分子乳铁蛋白的抗炎机制;叶秋容 等;《生命的化学》;20131231;第33卷(第3期);第69、71页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2642251C1 (en) * | 2016-11-03 | 2018-01-24 | Министерство сельского хозяйства Российской Федерации Федеральное государственное бюджетное образовательное учреждение высшего образования "Вятская государственная сельскохозяйственная академия" (ФГБОУ ВО ВГСХА) | Method for endometritis treatment in cows |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104833782A (en) | 2015-08-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Preparation of anti-tetracycline antibodies and development of an indirect heterologous competitive enzyme-linked immunosorbent assay to detect residues of tetracycline in milk | |
| CN104792894B (en) | Children's activity tuberculosis protein specificity spectrum and construction method thereof | |
| Ben-Bassat et al. | Changes in the structural organization of the surface membrane in malignant cell transformation | |
| Patramool et al. | Proteomic analysis of an Aedes albopictus cell line infected with Dengue serotypes 1 and 3 viruses | |
| George et al. | Rapid and sensitive detection of respiratory virus infections for directed antiviral treatment using R-Mix cultures | |
| US20230273218A1 (en) | Kit for quantitative detection using fluorescent microarray | |
| CN104833782B (en) | A kind of in-vitro screening and the method detecting anti-cow endometritis medicine | |
| Lu et al. | Sepsis-induced impairment of neutrophil chemotaxis on a microfluidic chip | |
| CN114720684A (en) | Interleukin cytokine multi-flow type fluorescence detection kit, detection method and application | |
| CN102023207B (en) | Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof | |
| CN106191286A (en) | Brucellar detection method, test kit and application thereof | |
| CN105241986B (en) | Protein characteristic spectrum for distinguishing latent tuberculosis children infectors and active tuberculosis children patients | |
| Labelle et al. | Effects of ophthalmic disease on concentrations of plasma fibrinogen and serum amyloid A in the horse | |
| CN104330564B (en) | Test kit for NPC early diagnosis | |
| Sharifian et al. | Urinary adrenomedullin level in children with acute pyelonephritis before and after treatment | |
| Liu et al. | Development of a colloidal gold Immunochromatographic assay for duck enteritis virus detection using monoclonal antibodies | |
| EP2016416A2 (en) | In vitro method for the identification and early identification and for the concomitant monitoring of the therapy of drug- and addictive substance-induced liver damage | |
| CN102967666A (en) | Quantitative detection method for content of hog cholera virus E2 proteins | |
| CN113960302A (en) | Kidney toxicity detection method based on high content technology and application thereof | |
| Njaanake et al. | Urinary cytokines in Schistosoma haematobium-infected schoolchildren from Tana Delta District of Kenya | |
| CN113419023A (en) | Thin layer chromatography detection method of exogenous mixture II | |
| CN111012786B (en) | Small molecular compound for activating inflammatory bodies and application thereof | |
| Tao et al. | Metabolic profiles in Madin–Darby canine kidney cell lines infected with H3N2 canine influenza viruses | |
| CN109298169B (en) | Screening method, cell model and application of anti-cow endometritis drug | |
| RU2137132C1 (en) | Method of allergy diagnosis of hemorrhagic fever with renal syndrome as index of neutrophil damage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160706 |