The Application Of Popliteal Lymph Node Assay To Evaluate The Allergenic Of Traditional Chinese Medicine Injections

The integration of the modern science and the Traditional Chinese medicine theory producted Traditional Chinese medicine Injections. They are composed of low molecular weight compands mainly. As TCMIs are applied to Clinical popularly, especially intravenous injection is increasing. Their adverse reactions report have also been increasing year by year, mainly are immune mediated hypersensitivity reactions. Although according to requirement, different allergy tests have been done, still can’t fully reflect and predict the happen of them. Constantly allergic shows that their safety evaluation still have some problems, especially the immune toxicological. Therefore, the safety evaluation of TCMI Clinical research techniques and principles explicitly pointed out that encouraged the use of new technologies, new methods, but need to provide the feasible basis, as the foundation of the assessment.The popliteal lymph node assay is a mature experimental method to predict the allergenic of the LMWCs. It has advantages in reliable, fast and repeatable. It focuses on the ability of drugs causing immune response rather than the symptoms related to animals. Presented and recognition process, lymphocytes activation, proliferation and differentiation, the secretion of cell factors and specific antibodies are studied with local draining node to test the allergenic of the drugs.According to the characteristics of TCMIs, This study wants to establish a sensitivity method to evaluate the allergenic of TCMIs by analysis of multiple popliteal lymph node immune cells surface molecules by flow cytometry.1. To establish experimental method Depending on PLNA, The BALB/c female mice were injected different concentration of positive drugs50ul to the toes, after three days, five days, seven days, Animals were sacrificed. Comprehensively analysed the morphological and surface molecular changes of PLN, the result showed that five days after injection PLN cell had significantly change. we determined the best time for detection on the fifth day.Then, Using of flow cytometry analysis a series of surface moleculars change, such as antigen presenting cell (dendritic cells, and macrophages) and T, B lymphocytes to make sure sensitive, stable allergenic index which can reflect the strength of the immune response caused by the exogenou small molecules. In all, T, B and antigen presenting cells proportions and early active molecule CD69, MHCII molecule and collaborative stimulation of molecules CD80、CD86、CD40、CD154, effective T cells surface molecular CD44can be comprehensive analysis to evaluate allergenic of drugs and provide important reference and basis. This experimental method can be used to evaluate allergenic nature.2. To evaluate the allergenic of29TCMIs by the established experimental methodThe established experimental method was used to test the allergenic of29kinds of TCMIs, the results show that different TCMIs caused distinct changes of the surface molecular. They were consisted with existing report. It indicated that this method can provide important basis to evaluate allergenic of TCMIs.3. To evaluate the allergenic of TCMIs form different manufacturer, batch number and forms by the established experimental methodThe study on injections from different formulations and manufacturer had a certain difference. Xue sai tong injection from different formulations and batches, Tan re qing and Zheng qing feng tong ning injections from different batches showed that their allergenic had certain differences.4. To discuss the adjuvant whether can be used in the established experimental methodAjuvant and Alum adjuvants were commonly used in animal experiments. So we explored if they can be applied to the established experimental method. The results showed that CFA was not appropriate for PLNA, but Alum should deserves further investigation. The established experimental method that comprehensive analysis the change of surface molecules by flow cytometry can be used to evaluate the allergenic potential;The change for different kinds of TCMIs are diverse; The established experimental method can provide important basis for allergenic evaluation. TCMIs from different manufacturer, batch number and forms caused distinctive change, the allergenic potential are dissimilar.Because the TCMIs are complex and specific, it is necessary to adopt various methods and comprehensive evaluate the allergenic potential of them. That we can have the accurate results.