CN104833782A - Method for in-vitro screening and detection of anti-cow-endometritis medicaments - Google Patents
Method for in-vitro screening and detection of anti-cow-endometritis medicaments Download PDFInfo
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- 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 description 19
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Abstract
The invention provides a method for the in-vitro screening and detection of anti-cow-endometritis medicaments. The method is implemented through carrying out inflammatory reaction on cow endometrial epithelium cells cultured in vitro; adding a to-be-detected medicament; and determining the anti-inflammatory activity of the to-be-detected medicament by detecting the expression changes of inflammatory proteins in the cells: if the expression quantity of the inflammatory proteins are decreased, determining that the to-be-detected medicament has anti-inflammatory activity, and the number of decrease times of the expression quantity is the size of the relative anti-inflammatory activity of the to-be-detected medicament, otherwise, determining that the to-be-detected medicament has no anti-inflammatory activity, wherein the inflammatory proteins are lactoferrins. According to the method for the in-vitro screening and detection of anti-cow-endometritis medicaments disclosed by the invention, the simple and rapid in-vitro screening and detection on the anti-cow-endometritis activity of to-be-detected medicaments can be implemented. When a porous plate is used, high-throughput screening and detection can be realized, and screening and detection results have important reference values to the modern clinical application and modern development of Chinese traditional medicines.
Description
Technical field
The present invention relates to a kind of method of in-vitro screening and the anti-cow endometritis medicine of detection.
Background technology
Inflammation is very common and important pathologic process, and the initiative of anti-inflammatory drug is essential, and find anti-inflammatory drug safely and effectively need select suitable inflammatory model carry out screening and evaluation antiphlogistic effects.In recent years, along with developing rapidly of cell culture technology, inflammatory cell model is widely used in inflammation mechanism research at home and abroad, and think can be used for medicine anti-inflammatory activity screening research.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 medicine all has good antiinflammatory action.Building effective appraisal procedure medicine under development anti-inflammatory efficacy aspect is a very important strategy.
Summary of the invention
The invention provides a kind of method of in-vitro screening and the anti-cow endometritis medicine of detection.The cow uteri intimal epithelium cell utilizing in vitro culture, by detecting the anti-inflammatory activity of the expression change reflection Chinese medicine of inflammatory protein in cell, fast and easy.As adopted porous plate cultured cell, then can detect tens kinds of even hundreds of kind medicines simultaneously, thus realize the high flux screening of medicine.Wherein, inflammatory protein is lactoferrin.
Cow endometritis is the inflammatory process of uterine epithelial cell.The applicant finds after deliberation, and after milk cow generation endometritis, in endometrial tissue cell, lactoferrin (Lactoferrin) is expressed and extremely significantly raised, and the expression of anti-inflammatory drug to this albumen has significant downward effect.Therefore, by the anti-inflammatory activity of the expression change indirect reaction medicine to be checked in detection lactoferrin (Lactoferrin) in vitro epithelial cell, thus judge whether institute's testing drug has antiinflammatory action significantly, clinical front basis can be provided for treatment cow endometritis.
The invention provides a kind of method of in-vitro screening and the anti-cow endometritis medicine of detection, that the cow uteri intimal epithelium cell of in vitro culture is carried out inflammatory, add medicine to be checked again, 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 method of the present invention, medicine to be checked is preferably Chinese medicine or natural products.
Preferably, fluoroscopic examination is detected as described in.
Preferably, step is as follows:
(1) cell chulture;
(2) Cellular inflammatory is reaction induced, and making it becomes inflammatory cell;
(3) drug treating inflammatory cell to be checked: medicine to be checked is joined in inflammatory cell nutrient solution and cultivates;
(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 fixedly fix with trichloroacetic acid damping fluid.Trichloroacetic acid can make protein conformation in cell change, and exposes more hydrophobic grouping, makes it aggregate and precipitate at the bottom of porous plate hole, is a kind of desirable albuminous degeneration fixing agent efficiently.
Preferably, the formula that data described in step (7) calculate is:
, wherein n is the multiple hole count of drug treating; P is the light absorption summation of positive controls; N is blank group each hole light absorption summation, and X is the light absorption total value in the multiple hole of same drug treating, 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<1, then medicine to be checked has anti-inflammatory activity; When Y≤0.5, then medicine to be checked has remarkable anti-inflammatory activity.
Preferably, described in-vitro screening and be detected as external high flux screening and detection, uses porous plate cultured cell simultaneously, adds different medicine to be checked wherein.Thus realize multiple medicine to be checked and detect simultaneously.
High-flux medicaments sifting is based on the screening model of external molecule or cellular level, carries out the screening of large quantization compound pharmacologically active, and the structure of the extensive sample library of diversity.High throughput screening drug can provide brand-new screening technique and means, has quick, high specific, highly sensitive feature, can greatly improve breakneck acceleration and efficiency.
Further, described porous plate is the microwell plate of printing opacity at the bottom of the black surround being applicable to fluoroscopic examination.
Most Chinese medicine preparation or natural products all itself have color, and the color of medicine itself often affects general light absorption detection method.The present invention uses fluoroscopic examination, in the dark detects, and the Color influences of medicine itself can be eliminated, and meanwhile, use fluoroscopic examination fast and easy, degree of accuracy is high.
In-vitro screening of the present invention and detect the method for anti-inflammatory drug, can be implemented in external to medicine to be checked anti-cow endometritis activity carry out easy, screen and detect fast.When using porous plate, can realize high-throughout screening and detection, screening and testing result have important reference value to the modern clinic application of Chinese medicine and modernization development.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for instructions, together with embodiments of the present invention for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the distribution plan 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 detects in the cow uteri intimal epithelium cell of in vitro culture after inflammatory reaction cell and anti-inflammatory Chinese traditional Herba Andrographitis therapeutic intervention with fluorescence immunoassay blotting, the expression change of lactoferrin in cell.
Embodiment
Following embodiment is convenient to understand the present invention better, 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 purchases available from routine biochemistry reagent shop.
embodiment 1
The method concrete steps of in-vitro screening of the present invention and the anti-cow endometritis medicine of detection are as follows:
One, cow uteri intimal epithelium cell chulture: in cow uteri intimal epithelium cell inoculated and cultured saturating 96 microwell plates at the bottom of black in growth period of taking the logarithm, at saturated humidity, 5% CO
2preculture in incubator, nutrient culture media is that DMEM/F12 nutrient solution and 10% is without endotoxic hyclone (Gibco company).Cell suspension is with 5 × 10
4the 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 nutrient solution, and join in all each hole except A1, A2, B1 and C1 tetra-holes, 100 μ l/ holes; A1, A2, B1 and C1 continue to add normal nutrient solution in tetra-holes.Tissue Culture Plate is placed in saturated humidity, 5% CO
2continue in incubator to cultivate.
Three, drug treating cell: aseptic Chinese medical extract DMEM/F12 cell culture fluid is diluted to 2g/ml and obtains medicine nutrient solution, then joins medicine nutrient solution in all drug treating cell holes with the volume in 100 μ l/ holes with multiple tracks liquid-transfering gun.Often kind of medicine at least arranges 3 multiple holes.Negative control hole A1, A2, B1, C1 and model comparison hole A3, A4, D1, E1 all add normal nutrient solution.Microwell plate is placed in saturated humidity, 5% CO afterwards
2continue in incubator to cultivate.
Four, cell and protein thereof are fixed: during drug treating cell 24h, take out Tissue Culture Plate, discard metabolism liquid, join in micropore by the trichloroacetic acid immobile liquid of 10%, every hole 100 μ l, leave standstill 2h at microwell plate being placed in 4 DEG C.
Five, close: take out microwell plate, discard immobile liquid, with TBST solution washing microwell plate 3 times, each 3min, then add 5% skim milk powder solution (BD company, TBST prepares), 100 μ l/ holes, slowly shake 1h under room temperature on shaking table.Can well complete closed with this understanding.
Six, antibody combine: get 100 μ l marked by fluorescein isothiocyanate lactoferrin antibody (Anti-Lactoferrin antibody-FITC) solution (5% skim milk powder solution preparation, lactoferrin antibody working concentration is 1-2 μ g/ml.Antibody can business be buied, as Abcam company of the U.S.) join in the middle of all micropores respectively, then keep in Dark Place in 4 DEG C, 8h or at room temperature slowly rock 2h.
Seven, after antibody combines, 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, setting excitation wavelength 496nm, absorbs optical wavelength 518nm, carries out two waveband and interferes the fluorescent absorption value read in each micropore.The light absorption total value (X) in multiple for same drug treating hole is substituted into the anti-inflammatory activity ratio (Y) that following formulae discovery goes out medicine.
N is the multiple hole count of drug treating; P is absorbance value summation (the i.e. OD of LPS disposal hole
a3+ OD
a4+ OD
d1+ OD
e1); N is blank group each hole absorbance value summation (i.e. OD
a1+ OD
a2+ OD
b1+ OD
c1).
Nine, result of calculation judges: when Y >=1, then judge that medicine antibiotic-free milk bovine endometritis is active; As Y<1, then judge that medicine has anti-cow endometritis active; When Y≤0.5, then judge that medicine has remarkable anti-cow endometritis active.
embodiment 2
The present embodiment fluorescence immunoassay blotting to theory of the present invention (after milk cow generation endometritis, in endometrial tissue cell, lactoferrin (Lactoferrin) is expressed and is extremely significantly raised, and the expression of anti-inflammatory drug to this albumen has significant downward effect) verify:
Cow uteri intimal epithelium cell chulture, inflammatory reaction induction and pharmaceutical intervention process are consistent with method in the present invention.After treated cell extracts total protein in conventional manner, through polyacrylamide gel electrophoresis, half-dried transfer printing, with fluorescein-labeled lactoferrin antibody (Anti-Lactoferrin antibody-FITC, purchased from American Abcam company) carry out immune-blotting method, obtain result with exposure method, result is see 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 being 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 the intervention of anti-cow endometritis Chinese medicine Herba Andrographitis, its expression significantly reduces.Illustrate that the expression of lactoferrin and Cellular inflammatory react and have high correlation, anti-cow endometritis medicine has significant downward effect to the lactoferrin of high expressed in inflammatory cell.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
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 again, 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.
2. method according to claim 1, is characterized in that: described medicine to be checked is Chinese medicine or natural products.
3. method according to claim 1 and 2, is characterized in that: described in be detected as fluoroscopic examination.
4., according to the arbitrary described method of claim 1-3, it is characterized in that: step is as follows:
(1) cell chulture;
(2) Cellular inflammatory is reaction induced, and making it becomes inflammatory cell;
(3) drug treating inflammatory cell to be checked: medicine to be checked is joined in inflammatory cell nutrient solution and cultivates;
(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.
5. method according to claim 4, is characterized in that: described cell and protein thereof are fixedly fix with trichloroacetic acid damping fluid.
6. method according to claim 4, is characterized in that: the formula that data described in step (7) calculate is:
, wherein n is the multiple hole count of drug treating; P is the light absorption summation of positive controls; N is blank group each hole light absorption summation, and X is the light absorption total value in the multiple hole of same drug treating, and Y is the anti-inflammatory activity ratio of medicine.
7. method according to claim 6, is characterized in that: when Y >=1, then medicine to be checked is without anti-inflammatory activity; As Y<1, then medicine to be checked has anti-inflammatory activity; When Y≤0.5, then medicine to be checked has remarkable anti-inflammatory activity.
8. the method according to claim 1-7, is characterized in that: described in-vitro screening and be detected as external high flux screening and detection, uses porous plate cultured cell simultaneously, adds different medicine to be checked wherein.
9. method according to claim 8, is characterized in that: described porous plate is the microwell plate of printing opacity at the bottom of the black surround being applicable to fluoroscopic examination.
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| CN109298169A (en) * | 2018-09-21 | 2019-02-01 | 中国农业科学院兰州畜牧与兽药研究所 | Screening technique, cell model and the purposes of anti-cow endometritis drug |
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| 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 |
Citations (3)
| 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 |
| 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 |
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| 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 |
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Patent Citations (3)
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|---|---|---|---|---|
| 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 |
| 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 |
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| 叶秋容 等: "天然免疫分子乳铁蛋白的抗炎机制", 《生命的化学》, vol. 33, no. 3, 31 December 2013 (2013-12-31), pages 69 - 71 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298169A (en) * | 2018-09-21 | 2019-02-01 | 中国农业科学院兰州畜牧与兽药研究所 | Screening technique, cell model and the purposes of anti-cow endometritis drug |
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