| Traditional Chinese Medicine Injection is a unique form of Chinese Medicine, an important method to handle critical symptoms. It’s based on the Traditional Chinese Pharmacology and Medicine theory, used modern technology and methodology, to abstract effective composition from Chinese medicine and nature medicine, and then made them into sterile solution or suspension. Traditional Chinese Medicine Injection is an extraordinary achievement for the modern development of Traditional Chinese Pharmacology and Medicine. It directly goes into blood instead of gastrointestinal tract, the nature of quick acting is very essential for critical symptoms. This method is not affected by digestive system and food, clinically often used for patient who suffers from coma, twitching, cramp or digestive system problem.The application of Traditional Chinese Medicine is spread widely; the report of bad reaction is also in an increasing trend, most of which are skin, respiratory system and cardiovascular system damage. An anaphylactoid reaction is also observed, different from the classic anaphylactic reaction which will reproduce a special antibody IgE after the anaphylactogen first entered the body. When the anaphylactogen stimulate the body afterward, it will be recognized and handled by the antibody, which leads the astrocyte and basophil degranulation to release massive bioactive substance to active the immunological effect.However, two of the "Chinese Pharmacopoeia"(2010) included only the guinea pigs for injection allergic reactions, active systemic anaphylaxis test (ASA) and passive cutaneous anaphylaxis test (PCA). Guinea pigs easy to allergens, was the ideal allergy test object, conventional allergy tests use of the guinea pig. But, due to the smaller guinea pig individuals, and sometimes behavior change is not typical and obvious, coupled with the behavior observed there are more subjective factors, guinea pigs prone to false positive or false negative results. Other guinea pig’s body active allergy test and passive cutaneous anaphylaxis test for the classical pathway dependent on IgE-mediated allergic reactions, a more precise detection of the effect of chemical drugs or a single component caused by classic allergic reactions, but traditional Chinese medicine injection often occurs:No IgE-mediated first contact with the adverse reactions which produce similar clinical symptoms of the classic allergic reaction, its mechanism is not yet fully clear, is called the class of allergic reactions. Against allergic reactions in this class guinea pig experiments testing the effect is not satisfactory.Therefore, to find a screening model for traditional Chinese medicine injection trigger allergic reactions is an important measure to control the adverse reaction of traditional Chinese medicine injection, this article from the in vitro cell model begin to filter out the most appropriate targeted class of allergic reactions in vitro screening model, lay the foundation to find and identify the substances trigger allergic reactions in traditional Chinese medicine injection. AIM:1. From mast cells and basophilic cell, choose one of the most suitable cell lines as a traditional Chinese medicine injection to trigger allergic reactions in vitro screening model.2. Use several clinic traditional Chinese medicine injections to evaluate whether the chosen cell lines matches the clinic trial.METHODS:1. Cell growth curve determination:The logarithmic phase of growth of three kinds of cells were seeded in96-well plates at different concentrations were measured using the MTT assay and CCK-8assay culture Od, Id,2d,3d,4d,5d, the cells vitality, at each time point, each cell, each concentration, six wells. Which the MTT group each hole by adding10μL concentration incubated for4h careful Aspirate medium5mg/mL MTT solution, dissolved DMSO solution shock micro plate reader570nm at the determination of the absorbance of each well (OD value); of CCK-8group in each hole by adding CCK-8solution, after1h incubation, micro plate reader,450nm, determination of the OD values of each hole six cells per day. The OD value of the use of the SPSS13.0calculate the mean, mean OD values obtained with the Windows Office Excel2010growth curve.2. Choose the cell lines to establish the screening of in vitro mast cell degranulation model2.1Determination of histamine releaseTake the logarithmic phase of the three types of cells seeded in24-well plates,48h adherent growth, discard the culture medium, adding Tyr screening to establish in vitro mast cell degranulation model ode fluid balance of10minutes. C48/80 stimulation group join the50μL C48/80, and set the blank control group by adding50μL of Tyrode solution. At0min,15min,30min,45min and60min to stop the reaction.2000r/min centrifuge10min at0-4℃, the supernatant spare. The Ku812directly in the tablet centrifuge same speed centrifugal supernatant spare.2.2Liquid chromatography-mass spectrometry (LC-MS) method for the determination of histamine integrationSamples after the activation of the C18solid phase extraction column, and then to0.2%ammonia3mL water, add3ml methanol to collect the methanol solution, evaporated to dryness at room temperature, take0.5mL methanol fully dissolve over a0.22μm membrane spare.Chromatographic conditions:mobile phase:acetonitrile:0.3%formic acid0.5mmol/L, ammonium acetate (60:40); flow rate:0.8mL/min; column temperature:35℃. Linear regression calculations draw the standard curve equation to calculate the histamine content of the samples to different histamine standard peak area on concentration.2.3The enzyme of the aminoglycoside net release determinationTake three types of cells in logarithmic growth phase, seeded in96-well microliter plates by adding50μL of substrate, the experimental group after the growth of48h each hole by adding50μL of C48/80of Triton Group by adding50μL of0.3%Triton X-100cell lisps, all membrane lisps, release of all amino glycosidase aminoglycoside enzyme content in the control group, the blank adding an equal amount of Tyrode solution. Reaction Omin,15min,30min,45min, and60min.By adding150μL of Stop Solution to terminate the reaction, the micro plate determination of the OD value of405nm hole reaction solution, and results excluding the blank control to the experimental group that the C48/80stimulate group aminoglycoside group of enzyme release with Triton lisps All membrane aminoglycosides release of the ratio of the total measure of the experimental group C48/80stimulated the net release of amino glycosidase.2.4Degranulation percentageTake three of the logarithmic growth phase cells planted in24-well plates.48h stickers wall growth, discard the culture medium by adding Tyrode fluid balance of10minutes. The experimental group joins C48/80stimulation, and set the blank control group to join the Tyrode solution. At Omin,15min,30min,45min and60min to stop the reaction of neutral red staining, random count of100cell degranulation in cells number calculation sell off particles percentage.The results above used SPSS13.0analyze software’s One Way Anova method. Histamine determination of peak area on the concentration of the linear regression calculation used Office Excel20103. Several common traditional Chinese medicine injections’ affection to P815cell.3.1The IC50of detection of several drugs commonly used in the injection of P815cellsTraditional Chinese medicine injection sample preparation liquid mother liquor, and10times the volume per diluted to a concentration, were set up together with the mother liquor, including six concentration, the experiment followed by a blank control, six concentrations of Chinese medicine injection group, each group of six complex holes. Take the logarithmic phase growth P815cells were seeded in96-well plates for12h, the blank control group by adding50μL medium, traditional Chinese medicine injection group by adding50μL of different concentrations of traditional Chinese medicine injection, cultured for36h, added to each well at room temperature balancing MTT solution and incubated4h under the same conditions, Aspirate medium and adding to the DMSO solution shocks dissolved at the determination of the OD of the hole and then the growth inhibition rate (%). Each TCM injections repeated measurements three times and averaged.3.2Several common traditional Chinese medicine injections’affection to P815cellThe establishment of in vitro mast cell degranulation model to filter the same way to detect a variety of traditional Chinese medicine injection60min after histamine release of P815cells to stimulate the enzyme aminoglycoside IC50of net release and cell degranulation. The experiment set C48/80to stimulate the positive control group and Tyrode stimulate the negative control group. The results above used SPSS13.0analyze software’s One Way Anova method.Results:1. Determination of cell growth curveMTT assay and the determination of CCK-8cell growth curve trend is basically the same, and come to the RBL-2H3cells in0.8×105/mL-1.0×105/mL within the P815cells at0.5×105/mL-0.8×105/mL range, Ku812cell growth curve sleep obvious exponential growth phase and plateau in0.8×105/mL-1.0×105/mL range, there is no significant latency2. Choose the cell lines to establish the screening of in vitro mast cell degranulation modelAfter stimulation with0-60min, three cell lines allergic active substance release were significantly increased in the same stimulus conditions, the histamine release in the P815cell degranulation and the hexamine enzyme net release to the sudden increase of the three indicators are significantly higher than the other two cell lines occurred early, the sudden increase in effect.3. Several common traditional Chinese medicine injections’affection to P815cellFilter out the P815cell sensitization of three methods of detection of several commonly used commercial traditional Chinese medicine injection; three methods of measuring results are in line. Which Xue Sai Tong injection, Qing Kai Ling Injection, Shuang Huang Lian powder for injection and water injection, the Xiangdan injection the Zhongjiefeng injection of matrine injection three indicators with the negative control group were significant differences, namely (P<0.01), prompted more than Chinese medicine injection of P815cells have a strong allergenic. Danshen injection three indexes with the control group compared to the significant difference, P<0.05, suggesting that Danshen injection sensitization of P815cells exist. Gall wood injection net degranulation aminoglycoside enzyme release indicator of degree two with blank control group was significantly different, P<0.05, suggesting that the injection of the gall bladder wood can also be partly the result of the removal of the P815cell particle reactions. Bupleurum injection fluid, Ginkgo biloba extract injection, Shengmai Injection in the three indexes with the control group no significant difference, suggesting that the three P815cells is safer in terms of sensitization.Conclusion:Among three kinds of mast cell or basophil cell line, the P815cell line is most suitable to trigger allergic reactions in vitro screening model which caused by traditional Chinese medicine injections. Under the stimulation of C48/80, P815demonstrate the obvious degranulation reaction, and the results are easily detected with good reproducibility. Under the stimulation of several common traditional Chinese medicine injection, determined by the experimental design of the detection methods from this paper, the result matches most of the official announcement and the State Food and Drug Administration and Adverse Drug Reactions Bulletin described in traditional Chinese medicine injection adverse reaction. Under the stimulation of high allergic traditional Chinese medicine, we observed apparently different result of P815cell and the nagetive comparation; under the non-allergic traditional Chinese medicine’s stimulation, we observed apparently different result of P815and the positive comparation. The result shows P815may be a new screening of in vitro mast cell degranulation model when testing allergic reaction of traditional Chinese medicine. |
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