| Traditional Chinese Medicine Injection (TCMI), a new preparation, is originally created in our country. TCMI has been widely used in the emergence treatement of severe diseases and in the curation of infectious diseases, cardiovascular diseases and cancer, thus, it has great market demand. With the extensive application of Traditional Chinese Medicine Injection, the adverse drug reactions (ADRs) happens frequently, especially anaphylactic shock and death. As a result, ADRs of Traditional Chinese Medicine Injection have attracted widespread attention of social public. Qinkailing Injection (QKLI) and Gegensu Injection (GGSI) are two kinds of Traditional Chinese Medicine Injection that have high incidents of anaphylactic reactions.The traditional allergen detection model is not sensitive for the detection of Traditional Chinese Medicine Injection because of the composition of Traditional Chinese Medicine Injection being complicated and of low content. Thus, more sensitive method is needed to overcome the difficulties of Traditional Chinese Medicine Injection safety evaluation.In this study, the feasibility of in vitro allergen detection model was investigated using RBL-2H3 cells with the Dinitrophenylated bovine serum albumin (DNP-BSA) and Ovalbumin (OVA) worked as allergens. The in vitro model was developed by optimizing the experimental protocols from buffer solution, detection indicators, detection time, anti-serum preparation, sensitization kinetics, and so on. Then we made use of the in vitro model in the detection of anaphylaxis induced by GGSI and QKLI in order to provide experimental basis for the pre-clinical evaluation of anaphylaxis induced by Traditional Chinese Medicine Injection.1. The improvement of RBL-2H3 cell model1.1 The improvement of RBL-2H3 cell model by DNP-BSAAim:To optimize the factors affecting the cell model, develop a sensitive detection system and apply in the detection of anaphylaxis induced by DNP-BSA. Methods:To develop a sensitive in vitro model, first, we investigated the detection protocols by choosing degranulation buffer solution and detection indicators. Then, we studied the rules of antibody and antigen concentration on RBL-2H3 cell degranulation. Finally, we applied the improved RBL-2H3 cell degranulation model in the detection of anaphylaxis induced by DNP-BSA and compared the detection sensitivity with Wistar and BN rats Passive Cutaneous Anaphylaxis (PCA) assay. Results:DMEM cell culture was more suitable for cell degranulation assay than Tyrode A buffer solution.β-hexosaminidase, being used as RBL-2H3 degranulation biomarkers, could detect lower concentrations of anti-dinitrophenol monoclonal IgE antibody (anti-DNP IgE) than histamine and LTC4. The rule of antibody concentration on cell degranulation was a "bell" curve, while the antigen concentration was a logarithmic curve. The cell model could detect lower concentrations of anti-DNP IgE compared to rats PCA model. Conclusion:The optimized RBL-2H3 cell model had a higher sensitivity compared to rats PCA assay, and was suitable to be applied in the detection of anaphylaxis induced by TCMI.1.2 The improvement of of RBL-2H3 cell model by OVAAim:To optimize the factors affecting the anti-serum preparation and the process of cell sensitization, develop a sensitive detection system and apply in the detection of anaphylaxis induced by OVA. Methods:BN rat, which was a high IgE responder rat strain, were used as sensitized animals. They were sensitized on days 1,3,5,7,9 through subcutaneous injection. In the study, the effect of adjuvant, the serum collecting time and sensitization kinetics were investigated in order to improve the in vitro model. Finally, we applied the improved RBL-2H3 cell degranulation model in the detection of anaphylaxis induced by OVA and compared the detection sensitivity with Wistar and BN rats PCA assay. Results:Antibody level in the serum would reach peak on day 21 after the first sensitization when BN rats were sensitized by the combination of OVA and aluminum hydroxide adjuvant. The optimized serum collecting time would elevate the detection senstitivity. In the sensitization kinetics study, we found that the longer the cells incubated with anti-serum, the stronger the cells responded to antigen. The cell model could detect higher titers of anti-OVA serum compared to rats PCA model.Conclusion:The optimized RBL-2H3 cell model had a higher sensitivity, and was suitable to be applied in the detection of anaphylaxis induced by protein of minute quantity.2. The verification of RBL-2H3 cell modelAim:To verify the detection accuracy and sensitivity of RBL-2H3 cell model using Bovine Serum Albumin (BSA), an allergen with weak allergenicity. Methods: The allergenicity of BSA was detected by the modified RBL-2H3 cell model according to the DNP-BSA and OVA assay, and the result was compared with the Wistar and BN rats PCA assay. Results:The highest anti-BSA antibody titers detected by Wistar and BN rats PCA assay was 32, respectively, while the modified RBL-2H3 cell assay was 128.Conclusion:It had been demonstrated that RBL-2H3 cell model had good accuracy and the detection sensitivity of RBL-2H3 cell assay was higher than that of rats PCA assay.3. The detection of anaphylaxis induced by GGSI and QKLI using RBL-2H3 cell model and the study of related mechanismAim:To detect the anaphylaxis induced by GGSI and QKLI and study the related mechanism. Methods:In the study, we applied the improved RBL-2H3 cell degranulation model in the detection of anaphylaxis induced by GGSI and QKLI and compared the detection sensitivity with BN rats PCA assay. The changes in rat blood cells were detected by the blood routine test. The type of antibody in the sensitized rat serum was also detected by ELISA method and PCA assay. Results:It was demonstrated that GGSI and QKLI could induce anaphylaxis reaction by both rat PCA assay and RBL-2H3 assay. After sensitization by GGSI and QKLI, the leukocyte and the total IgE,IgG content in the rats serum were increased compared to the normal rats and OVA sensitized rats. Conclusion:RBL-2H3 cell model was suitable to be applied in the detection of anaphylaxis induced by GGSI and QKLI. It was indicated from the results of blood routine test and serum antidody ELISA kits that GGSI and QKLI could induce the typeⅠallergic reaction. |
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