Studies On Hypersensibility Of Traditional Chinese Medicine Injections With The Activation Markers On Murine Basophils

The Chinese medicine is one of component elements in China traditional culture. Traditional preparation is include pill, power, plaster and elixir, nowadays some new preparation appearance on the basis of these. However, because of easy to absorb and effect sharp, the injection quickly hold the market and achieved good clinical efficacy. But as a result of increased use the traditional Chinese medicine injection (TCMI), the adverse reaction become great quantity, the phenomenon is also given widely attention in-group.Most of the TCMI adverse reactions are allergic reactions or anaphylactoid reactions, It means that it is lead to the mast cell and basophil degranulation, and release inflammatory mediators. Basophils are one of the most important effection cells in immunity reaction, take part in natural immune response and acquired immune response. Basophils play a important role in starting and continuing phase of immunity reaction. Utilize flow cytometry, basophil activation test (BAT) was considered as a effective means to analyze allergen exist allergenicity. Murine basophils still don’t have specificity surface marker, but combination markers can identify the basophil in peripheral blood. Recently studies said that CD200R surface expression as a marker of murine basophil activation. According to the literature and the characteristic of basophil we choose three surface markers include CD200R,CD107a and CD69. Analyze the feasibility of these as the activation markers of basophils. At last, in this study we intend to establish a murine BAT to observe the diversification of the basophils surface activation molecule expression. Analysis of the TCMI whether exist the allergenic potential.1Experimental Study1.1Identification of the murine basophils in peripheral bloodAcquire the BALB/c murine peripheral blood, Cells were stained with anti-IgE/anti-CD4/anti-CD45R B220, acquisition and analysis with flow cytometry (FCM). Measuring the fluorescence intensity of IgE+/CD4-/CD45R B220-, The rate of the basophils is0.7%in leukocyte. This method can identification of the murine basophils in peripheral blood, and utilize it in the BAT with Flow cytometry.1.2Activation of the murine basophilsAnti-IgE as the basophils’activator, add in murine peripheral blood and incubation in some time for FCM. Measuring the fluorescence intensity of CD200R, CD107a and CD69. The CD200R, CD107a and CD69all significantly increased after the anti-IgE stimulate. So the3molecular could be as markers in murine basophil activation.1.3Optimize the condition of the anti-IgE induce murine basophils activationAccording to the rules and characteristics of the surface markers expression, in order to select the optimum activation conditions, we choose3densities and3incubation times of anti-IgE for detecting the expression of the CD200R, CD107a and CD69.Finally, we determine the time-concentration of the anti-IgE are CD200R:30min-125ng/mL; CD107a:5min-125ng/mL;CD69:60min-125ng/mL.1.4Ionomycin, PMA and Ionomycin+PMA stimulate the murine basophils activationAcquire the murine peripheral blood, Ionomycin, PMA and Ionomycin+PMA as stimulator to affect the basophils. Observe the expression of the activated markers in different stimulate times.The result show that, after ionomycin stimulate the activation markers expression increase are not obvious. PMA and Ionomycin+PMA both make the murine basophils surface CD200R, CD107a and CD69expression increase.1.5Basophils activation of the sensitized mouseSensitization of mouse with ovalbumin(OVA), then acquire the murine peripheral blood, add in PBS, IgE and OVA in vitro. Measuring the fluorescence intensity of CD200R, CD107a and CD69on the surface of basophils.The result show that, Sensitization of mouse, stimulation with anti-IgE and OVA can both increase the expression of CD200R, CD107a and CD69.1.6TCMI stimulat murine basophils, the activation markers variations situationAcquire the murine peripheral blood, add in PBS, anti-IgE or ionomycin+PMA and TCMI separately in vitro. Detection and analysis with FCM, to measure the fluorescence intensity of CD200R, CD107a and CD69on the surface of basophils. We found that, anti-IgE or ionomycin+PMA can increase the expression of CD200R, CD107a and CD69. In the TCMI groups, XueSaiTong injection make the expression of CD200R increase, and the CD69is tended to increase. 1.7Allergic experiments of TCMI to detection the activation markers variations situationIntraperitoneal injection of BALB/c mouse with physiological saline, OVA and TCMI twice in two weeks. A week later, Acquire the murine peripheral blood, add in physiological saline, OVA and TCMI separately in vitro. To Measure the fluorescence intensity of CD200R, CD107a and CD69with FCM. The result is that Shuang HuangLian injection make the expression of the CD200R increased. QingKaiLing injection make the expression of CD200R, CD107a and CD69are tended to increase.2ConclusionsThe antibody groups IgE-FITC+/CD4-PE/cy7-/CD45R B220-PE/cy7-can identify the murine basophils in the peripheral blood. CD200R, CD107a and CD69surface expression as activation markers of murine basophil. Anti-IgE as a stimulator to effect the murine basophils. The optimum conditions are CD200R:30min-125ng/mL; CD107a:5min-125ng/mL;CD69:60min-125ng/mL. Non-IgE dependent activation of the murine basophils can make the expression of CD200R, CD107a and CD69increased. CD200R, CD107a and CD69can be the both Non-specific immune and specific immune activation markers in the murine basophils. Stimulate the basophils in vitro, the expression of CD200R, CD107a and CD69in positive group all increased obviously. The expression of CD200R and CD69in XueSaiTong group are tended to increase. It is better to deeply study to avid the anaphylactoid reaction. Analyze8TCMI allergic reactions by FCM, anti-IgE can make the expression of CD200R, CD107a and CD69all increased obviously after sensibilization. Shuanghuanglian injection exist the phenomenon that it can make the expression of CD200R increased after specificity stimulation. The expression of CD200R and CD69in QingKaiLing group are tended to increase. It is better to pay great attention to the allergenicity of Shuanghuanglian and QingKaiLing injection, avoid the allergy reaction.