CN102323386A - A kind of experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine - Google Patents
A kind of experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine Download PDFInfo
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- CN102323386A CN102323386A CN201110157256A CN201110157256A CN102323386A CN 102323386 A CN102323386 A CN 102323386A CN 201110157256 A CN201110157256 A CN 201110157256A CN 201110157256 A CN201110157256 A CN 201110157256A CN 102323386 A CN102323386 A CN 102323386A
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Abstract
The invention discloses a kind of experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine, comprise step: (1) adopts rat basophilic HTLV cell to set up the potential sensitization in-vitro evaluation of traditional Chinese medicine model; (2) with transmission electron microscope observing, calculate the potential sensitization that one or more indexs take off in graininess index, Annexin V positive cell rate, the β-hexosaminidase release rate are judged traditional Chinese medicines.The inventive method is directly perceived, stable, reliable for the judgement of the potential sensitization of traditional Chinese medicine.
Description
Technical field
The present invention relates to drug evaluation method field, be specifically related to a kind of experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine.
Background technology
Along with the raising of modernization of Chinese medicine degree, traditional Chinese medicine has had very great development in recent years, but thing followed safety issue is also serious day by day.In the clinical preceding method for evaluating safety; The traditional Chinese medicine sensitivity test comprises initiatively systemic allergy test (ASA) and passive cutaneous anaphylaxis test (PCA); Be primarily aimed at type i allergic reaction; But most traditional Chinese medicines before new drug is clinical in the declaration material sensitivity test result all negative, have bigger difference with the high positive rate of the allergic reaction that exists in the clinical use in listing back, this species diversity points out traditional sensitivity test possibly have defective; Therefore, seeking more effective, sensitive evaluation method is the key issue that needs to be resolved hurrily at present.
Summary of the invention
The invention provides a kind of experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine, this method is directly perceived, stable, reliable for the judgement of the potential sensitization of traditional Chinese medicine.
A kind of experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine comprises step:
(1) adopt rat basophilic HTLV cell (RBL-2H3 cell) to set up the potential sensitization in-vitro evaluation of traditional Chinese medicine model;
(2) with transmission electron microscope observing, calculate the potential sensitization that one or more indexs take off in graininess index, Annexin V positive cell rate, the β-hexosaminidase release rate are judged traditional Chinese medicines.
Being configured to of described traditional Chinese medicine potential sensitization in-vitro evaluation model: rat basophilic HTLV passage is cultivated.The described cultured method that goes down to posterity adopts existing passage cultural method.
The described graininess index that takes off adopts conventional morphological method to detect.
Described Annexin V positive cell rate adopts flow cytometry to measure.
Described β-hexosaminidase (being Hex) release rate adopts spectrophotometry.
The present invention is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine and has adopted the RBL-2H3 cell to set up cell model.The RBL-2H3 cell is commercial rat basophil tumor line; There is high-affinity IgE acceptor on the surface, can take off the particle reaction after the activation, because it has the immortalization characteristic; Cultural method is simple; Growth rapidly, complex operation or drawbacks such as cell purity, lazy weight are the optimal selections of setting up the vitro detection system when having avoided from blood the gradient separations basophil.
The present invention is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine and has adopted the method for measuring Annexin V positive rate.Annexin V once was widely used in combining the phosphatidylserine on the plasma membrane in the apoptotic process.Many experiment confirm Annexin V participate in the exocytosis process.1999, the Demo reported first was come the degranulated mast cell of mark with Annexin V, and the result shows that Annexin V combines with the secretory granules of cell surface specifically, and combination degree and β-hexosaminidase are released into direct ratio.
Compared with prior art, the present invention has following advantage:
The present invention will from take off graininess index, histamine release rate, Annexin V positive rate, β-hexosaminidase release rate etc. at present common counter confirm RBL-2H3 cells in vitro evaluation model parameters, set up traditional Chinese medicine conveniently clinical before the active platform of safety evaluatio or clinical practice bad reaction prediction.
Description of drawings
Fig. 1 is the graph of a relation of Annexin V positive rate and CA concentration among the embodiment 2;
Fig. 2 is the streaming scatter diagram of control group among the embodiment 2;
Fig. 3 is 0.2mmolL for CA concentration among the embodiment 2
-1The streaming scatter diagram of drug group;
Fig. 4 is 1.0mmolL for CA concentration among the embodiment 2
-1The streaming scatter diagram of drug group;
Fig. 5 is 5.0mmolL for CA concentration among the embodiment 2
-1The streaming scatter diagram of drug group;
Fig. 6 is the transmission electron microscope picture of negative control group RBL-2H3 cell among the embodiment 4;
Fig. 7 is the transmission electron microscope picture of positive controls RBL-2H3 cell among the embodiment 4;
Fig. 8 is 0.2mmolL for CA concentration among the embodiment 4
-1The transmission electron microscope picture of drug group RBL-2H3 cell;
Fig. 9 is 1.0mmolL for CA concentration among the embodiment 4
-1The transmission electron microscope picture of drug group RBL-2H3 cell;
Figure 10 is 5.0mmolL for CA concentration among the embodiment 4
-1The transmission electron microscope picture of drug group RBL-2H3 cell.
Embodiment
The present invention combines specific embodiment to further specify as follows:
Morphological method detects takes off graininess index: rat RBL-2H3 basophil tumor line, and available from Chinese Academy of Sciences's cell bank, with containing 10% (percent by volume) NBCS, 40umL
-1The DMEM nutrient solution of gentamicin is in 37 ℃, 5% (percent by volume) CO
2With the cultivation of going down to posterity in the incubator of saturated humidity, experiment all is in exponential phase with cell.The RBL-2H3 cell in growth period of taking the logarithm, the digestion counting, the adjustment concentration of cell suspension is 5.0 * 10
6Individual mL
-1, 3 drug-treated groups are got 50 μ l cell suspensions respectively and are placed 0.5ml EP pipe, and (solvent is an incubation buffer, NaCl 140mmol/L in the incubation buffer, KCl 5mmol/L, CaCl to add 50 μ l variable concentrations chlorogenic acid test liquids
21mmol/L, MgCl
2, 0.6mmol/L, all the other are distilled water) (chlorogenic acid is hereinafter to be referred as CA), chlorogenic acid test liquid concentration is respectively 0.2mmolL
-1, 1.0mmolL
-1, 5.0mmolL
-1Negative control group is got 50 μ l cell suspensions and is placed 0.5ml EP pipe, adds 50 μ l DMEM nutrient solutions; Positive controls is got 50 μ l cell suspensions and is placed 0.5ml EP pipe, and adding 50 μ l concentration is 30 μ gmL
-1The incubation buffer that receives reagent thing C48/80.3 drug-treated groups, negative control group and positive controls hatch 30,60 at 37 ℃ respectively, 120min, ice bath 10min cessation reaction, and (every solencyte final concentration is 1.0 * 10 to add 150 μ l A Li Xinlan dye liquors then respectively
6Individual mL
-1), dyeing 10min mixes the back microscopically and is counting cytochrome, calculates according to formula and takes off graininess index (DI), and take the photograph sheet with MOTIC biology microscope IMAQ analytic system.
Take off graininess index (DI)=[(negative control group cytochrome-drug-treated group and cytochrome)/negative control group cytochrome] * 100%.
The RBL-2H3 cell is the monolayer adherence growth, under inverted phase contrast microscope, can see heterogeneous particles a large amount of in the endochylema.This experiment is observed cell for circular with A Li Xinlan dyeing RBL-2H3 cell, examines not paintedly, and equally distributed light blue particulate matter is arranged in the cell.Behind the RBL-2H3 cell degranulation, distortion in various degree takes place in cell, and born of the same parents' endoparticle is sparse, even formation of vacuoles, the endochylema fade.The observer unifies observation caliber, behind the adjustment cell concentration, is counting taking off granular cell within sweep of the eye, and is calculating according to formula and to take off graininess index (DI), and the result sees table 1.
Table 1 chlorogenic acid is to the influence (
n=5) of RBL-2H3 cell degranulation index
Compare * P＜0.05, * * P＜0.01, t-test. with control group
The result can tentatively point out medicine whether can cause that the RBL-2H3 cell part takes place takes off particle, and can show certain correlativity from time and concentration.
Embodiment 2
Cells were tested by flow cytometry Annexin V positive cell rate: the cultural method cultivation of going down to posterity by among the embodiment 1 obtains being in exponential phase RBL-2H3 cell, gets to be in exponential phase RBL-2H3 cell, and adjustment concentration of cell suspension in digestion back is 5 * 10
6Individual/ml, add respectively in the 0.5mlEppendorf pipe; 3 drug group are that (solvent is an incubation buffer, NaCl 140mmol/L in the incubation buffer, KCl 5mmol/L, CaCl for every pipe 100ul cell suspension and 100ul variable concentrations chlorogenic acid test liquid
21mmol/L, MgCl
2, 0.6mmol/L, all the other are distilled water), chlorogenic acid test liquid concentration is respectively 0.2mmolL
-1, 1.0mmolL
-1, 5.0mmolL
-1Control group is that 100ul cell suspension and 100ul contain 10% (percent by volume) NBCS, 40umL
-1The DMEM nutrient solution of gentamicin; 3 drug group and control group 37 ℃ hatch 1h after collecting cell, 2000rpm * 5min is centrifugal, presses the experiment flow operation of Annexin V kit suggestion, with the binding buffer liquid re-suspended cell that Annexin V kit carries, the adjustment concentration of cell suspension is 1 * 10
6Individual/ml, 100ul adjustment back cell suspension adds the Annexin V working fluid 5ul that Annexin V kit carries, and incubated at room 15min adds binding buffer liquid that 400ulAnnexin V kit carries again and mixes the back and go up machine testing.
The specific secretory granules film with activation mast cell surface of Annexin V combines.Adopt flow cytometry that degranulated mast cell RBL-2H3 is carried out Annexin V blip counting, quantitative test mast cell RBL-2H3 receives variable concentrations chlorogenic acid (CA) to stimulate the degranulated degree in back.The result shown in table 2 and Fig. 1-5,0.2mmolL
-1, 1.0mmolL
-1, 5.0mmolL
-1The CA of concentration can make RBL-2H3 cell Annexin V stained positive cell rate obviously increase, and is the significant concentration dependence, and prompting CA can promote RBL-2H3 cell phosphatidylserine to turn up, and causes the cell degranulation reaction.
Table 2 chlorogenic acid is induced the RBL-2H3 cell phosphatidylserine rate (
n=3) of turning up
| Group | Chlorogenic acid concentration (mmolL -1) | Annexin V positive cell rate (%) |
| Control group | - | 3.91±2.1 |
| Drug group | 0.2 | 15.20±1.0** |
| Drug group | 1.0 | 23.20±4.0** |
| Drug group | 5.0 | 46.60±4.6** |
* and control group compare, P＜0.01, t-test.
Annexin V positive rate (being Annexin V positive cell rate) indirect reaction cell degranulation rate.
Embodiment 3
β-hexosaminidase release rate assay method: cultivate by the cultural method that goes down to posterity among the embodiment 1 and to obtain being in exponential phase RBL-2H3 cell, get and be in exponential phase RBL-2H3 cell inoculation nutrient culture media in 24 porocyte culture plates and (contain 10% (percent by volume) NBCS, 40umL
-1The DMEM nutrient solution of gentamicin) conventional overnight incubation, every porocyte number is 2 * 10
5Individual, inhale next day and remove nutrient solution and with incubation buffer (NaCl 140mmol/L in the incubation buffer, KCl 5mmol/L, CaCl
21mmol/L, MgCl
2, 0.6mmol/L, all the other are distilled water) and drip washing 3 times, every again hole adds 37 ℃ of preincubate 10min of 100 μ l incubation buffer, obtains Incubating Solution; (solvent is an incubation buffer to 3 drug group, NaCl 140mmol/L in the incubation buffer, KCl 5mmol/L, CaCl in order in the Incubating Solution of every hole, to add 100 μ l variable concentrations chlorogenic acid test liquids respectively
21mmol/L, MgCl
2, 0.6mmol/L, all the other are distilled water), chlorogenic acid concentration is respectively 0.2mmolL
-1, 1.0mmolL
-1, 5.0mmolL
-1Negative control group is in the Incubating Solution of every hole, to add 100 μ l incubation buffer; Positive controls is in the Incubating Solution of every hole, to add 100 μ l C48/80; 3 drug-treated groups, negative control group and positive controls are hatched 30min, 60min, 120min at 37 ℃ respectively; Hatch and finish back ice bath 10min cessation reaction; Get each hole supernatant 50 μ l and change in 96 well culture plates, (substrate is 1mmolL to add 50 μ l substrates simultaneously
-14-nitrobenzene-N-acetyl-β-D-glucosamine glycosides (the citric acid-sodium citrate buffer soln of p-nitrophenyl-N-acety-β-D-glucosaminide), pH 4.5) is hatched 60min for 37 ℃, adds 0.1mol/L (M) Na at last
2CO
3/ NaHCO
3The WS (pH=10) 150 μ l cessation reactions are measured each hole reactant liquor absorbance of 405nm.
Remove each hole residue supernatant; Incubation buffer is cleaned 2 times, and every then hole adds 200 μ l TritonX-100 (1%, mass percent) cell lysis; Lysate 3000rpm * 1min is centrifugal; Get supernatant 50 μ l and change in 96 well culture plates, the absorbance of the same time-and-motion study cell pyrolysis liquid is calculated different disposal group β-hexosaminidase burst size according to formula.
β-hexosaminidase burst size (%)=[supernatant absorbance/(supernatant absorbance+cell pyrolysis liquid absorbance)] * 100%
Hex is a kind of enzyme that is present in the mast cell granule, is discharged into the extracellular with taking off particle, and research shows that its release rate is parallel with the histamine release rate.Shown in table 3 result; 0.2mmolL-1 hatching interior its β of back 120min-hexoside enzyme release rate with the RBL-2H3 cell, concentration chlorogenic acid (CA) do not see obvious increase; But 1.0mmolL-1 concentration chlorogenic acid can obviously improve RBL-2H3 cell Hex release rate after hatching 60min; 5.0mmolL-1 the concentration chlorogenic acid can obviously improve its Hex release rate after hatching 30min; This release rate presents tangible correlativity with chlorogenic acid concentration and incubation time, and the prompting chlorogenic acid can promote the RBL-2H3 cell to discharge Hex, and the side light chlorogenic acid can bring out the RBL-2H3 cell degranulation.
Table 3 chlorogenic acid is to the influence (
n=6) of Hex release rate in the RBL-2H3 cell
Compare * * P＜0.01, t-test with control group.
Embodiment 4
Collect negative control, variable concentrations chlorogenic acid test liquid, concentration is respectively 0.2,1.0,5.0mmolL
-1And positive control C48/80 handles the RBL-2H3 cell 1 * 10 of 1h
7Individual, after precooling PBS damping fluid is washed, the centrifugal 15min of 2000rpm; 2.5% (mass percent) glutaraldehyde water solution of 1ml precooling is added in the centrifuge tube of centrifugal back formation cell mass as immobile liquid, and 4 ℃ are spent the night, and 4 ℃ of 1% (mass percent) osmic acid WS are fixing; Conventional preparing electron microscopy specimen program dehydration, infiltration; Ultra-thin section after the Epon812 embedding, normal dyeing is observed under the H-600A type transmission electron microscope and is taken pictures.
Transmission electron microscope observing cause after the effect of RBL-2H3 cell soup that degranulated form changes.The result sees Fig. 6-10 (* 4800), shows, negative control group RBL-2H3 entoblast is obvious, and after birth has more microvillus, and the granular that differs in size in kytoplasm balloon-shaped structure, cell contain fine and close granule (Fig. 6); And CA0.2,1.0,5.0mmolL
-1And after the positive drug C48/80 effect, the nuclear chromatin cohesion, endoplasmic reticulum has expansion; Particularly the cell endoparticle reduces, and cell cytosol is sparse, and vesica increases; A large amount of cavity spline structures occur, and the visible particle vesica is moved to plasma membrane and is merged with it and typical cases such as exocytosis take off the particle response feature.Along with increasing of CA concentration, the cavity spline structure increases in the visible cell, and degranulation has the trend of enhancing.
Claims (5)
1. an experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine is characterized in that, comprises step:
(1) adopt rat basophilic HTLV cell to set up the potential sensitization in-vitro evaluation of traditional Chinese medicine model;
(2) with transmission electron microscope observing, calculate the potential sensitization that one or more indexs take off in graininess index, Annexin V positive cell rate, the β-hexosaminidase release rate are judged traditional Chinese medicines.
2. the experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine according to claim 1 is characterized in that, being configured to of described traditional Chinese medicine potential sensitization in-vitro evaluation model: rat basophilic HTLV passage is cultivated.
3. the experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine according to claim 1 is characterized in that, the described graininess index that takes off adopts conventional morphological method to detect.
4. the experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine according to claim 1 is characterized in that, described Annexin V positive cell rate adopts flow cytometry to measure.
5. the experimental technique that is used for the potential sensitization of in-vitro evaluation traditional Chinese medicine according to claim 1 is characterized in that, described β-hexosaminidase release rate adopts spectrophotometry.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590492A (en) * | 2012-02-22 | 2012-07-18 | 江苏苏中药业集团股份有限公司 | Method for detecting anaphylactoid reaction of Shengmai liquid preparation |
| CN103257237A (en) * | 2013-05-09 | 2013-08-21 | 中国农业大学 | In-vitro detection method of allergen in food |
| CN105004705A (en) * | 2015-08-07 | 2015-10-28 | 天津中医药大学 | Detection method of medicine injection anaphylactic and anaphylactoid reactions and new application of adopted dye |
| CN113061116A (en) * | 2020-01-02 | 2021-07-02 | 上海医药工业研究院 | Aminopyrimidine compound, preparation method, composition and application thereof |
-
2011
- 2011-06-13 CN CN201110157256A patent/CN102323386A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| KENNETH M.JOHNSON、MELISSA PHILLIPS、CHENGWANG、GOLDA A.KEVETTER: "Chronic Phencyclidine Induces Behavioral Sensitization and Apoptotic Cell Death in the Olfactory and Piriform Cortex", 《JOURNAL OF NEUROSCIENCE RESEARCH》 * |
| 李钦、赵吟、郑晓亮、张信岳: "药用注射辅料聚山梨酯80诱发类过敏反应的细胞研究", 《中国临床药理学与治疗学》 * |
| 郑晓亮、李钦、赵吟、颜冬梅、屠凌岚、张信岳: "清开灵注射液诱导嗜碱性粒细胞脱颗粒致类过敏反应作用研究", 《中国中药杂质》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590492A (en) * | 2012-02-22 | 2012-07-18 | 江苏苏中药业集团股份有限公司 | Method for detecting anaphylactoid reaction of Shengmai liquid preparation |
| CN103257237A (en) * | 2013-05-09 | 2013-08-21 | 中国农业大学 | In-vitro detection method of allergen in food |
| CN103257237B (en) * | 2013-05-09 | 2015-05-20 | 中国农业大学 | In-vitro detection method of allergen in food |
| CN105004705A (en) * | 2015-08-07 | 2015-10-28 | 天津中医药大学 | Detection method of medicine injection anaphylactic and anaphylactoid reactions and new application of adopted dye |
| CN105004705B (en) * | 2015-08-07 | 2018-05-01 | 天津中医药大学 | The detection method of drug injection allergy anaphylactoid reaction and dyestuff new application used |
| CN113061116A (en) * | 2020-01-02 | 2021-07-02 | 上海医药工业研究院 | Aminopyrimidine compound, preparation method, composition and application thereof |
| CN113061116B (en) * | 2020-01-02 | 2023-07-25 | 上海医药工业研究院 | Aminopyrimidine compound, preparation method, composition and application thereof |
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Application publication date: 20120118 |