| To study the preferred plan for the detection of Thl and Th2 cell of BN rats by using flow cytometry.Isolated the peripheral blood mononuclear cell(PBMC)from the peripheral blood of BN rats,combined different concentration of stimulants which contains Phorbol-12-myristate-13-acetate(PMA)and Ionomycin(Ion)with different concentration of blocker which contains Brefedlin A(BFA)to cultivate the PBMC.The four-color scheme of flow cytometry was used to detect Thl and Th2 cells.When the concentration of PM A was 75 ng/mL,the concentration of Ion was 1.5?g/mL,the concentration of BFA was 7.5 ng/mL,the result of Thl/Th2 was the 0.56±0.04)%/(1.11±0.04)%,which was the highest among the five groups.The result of both the lower dose groups and the higher dose groups decreased.Lower concentration of stimulants and blockers cannot reach the purpose of enhance the activation of T cell and increase the detection efficiency.And higher concentration of stimulants and blocker,can produce cytotoxicity,which will also decrease the results of the detection.Obviously,it is important to choose the appropriate concentration of both stimulants and blocker.What's more,because of the reduction of CD4 when using PMA and Ion,and the extra detection of Nk cell and double negative T cell when using the CD4 to set the gate,we suggest using CD3 and CD8 to set the gate during the detection of the Thl/Th2.Anaphylaxis(allergic reactions)is a multi-systemic syndrome that poses a serious threat to human health.The inducement of this disease is a variety of inflammatory mediators that mast cells and basophils release in the systemic circulation.According to whether the reaction has the involvement of the immune system,such as the presence or absence of immunoglobulin,it is divided into immune system-mediated allergic reactions,and non-immune system-mediated allergic reactions(anaphylactoid reactions).5-Hydroxymethylfurfural(5-HMF,C3H6O3)is a small five-carbon molecule consisting of a furan ring that can be generated by a Maillard reaction of sugars at high temperatures21.As a by-product formed during high-temperature sterilization,5-HMF can be found in various foods and pharmaceutical preparations containing saccharides.Besides,with high temperature,the dimer of 5-HMF,namely,5,5'-oxydimethylenebis(2-furfural)(OMBF,C12H10O5),can be easily generated by polyreaction of 5-HMF.Our previous research confirmed using a reporter antigen popliteal lymph node assay(RA-PLNA)that 5-HMF and OMBF causes anaphylaxis in mice.Therefore,based on our former assumption that some small molecular compounds may cause both type I hypersensitivity and anaphylactoid reactions and previous research on 5-HMF and OMBF,the present study further investigates the mechanism underlying anaphylaxis due to 5-HMF and OMBF,including both type I hypersensitivity and anaphylactoid reactions.This research will also research the protein binding situation of both 5-HMF and OMBF in vivo.Firstly,we converted the equivalent dose of rats according to the limit of 5-HMF in glucose injection in the Chinese Pharmacopoeia,and based on this,designed the concentration of each test.This part of the study includes four parts of the trial content.1)SPF grade male Brown Norwegian rats(BN),170 ± 20g,were used to build the animal model of both type I hypersensitivity and anaphylactoid reactions by positive drugs.The detection conditions of each indicator were also ensured.2)Under the above experimental conditions,5-HMF and OMBF were directly used to induce anaphylactoid reaction in BN rats.By observing changes in behaviors of animals,detecting changes in related cytokines,and combining the results of pathological sections,we tended to study the ability of 5-HMF and OMBF to induce the anaphylactoid reactions;3)Under the above experimental conditions,5-HMF and OMBF were used to induce type I hypersensitivity reaction in BN rats,by observing changes in animal behavior,detecting changes in related cytokines,observing results of related pathological sections,and detecting Th1/Th2 cell population ratio change by flow cytometry,the ability of 5-HMF and OMBF to induce type I hypersensitivity reaction were studied;4)The ability of plasma protein binding of both 5-HMF and OMBF were tested.The molecular docking test was also be conducted to test the binding ability of 5-HMF and OMBF to human serum albumin(HSA)and bovine serum albumin(BSA).By establishing the animal disease model of type I hypersensitivity and anaphylactoid reaction and giving 5-HMF and OMBF to the test animals under such conditions,we preliminarily determined that 5-HMF and OMBF can cause both type I hypersensitivity and anaphylactoid reaction animal model of BN rats.The mechanisms by which it triggers allergic reactions may include direct stimulation and stimulation of mast cells or basophils via the complement activation pathway.The type I hypersensitivity induced by 5-HMF and OMBF may be mediated by both IgE and IgG.In anaphylactoid reaction,both low dose groups show stronger effect.In the type I hypersensitivity reaction,the reaction intensity of the high-dose groups is stronger than that of the low-dose groups.Therefore,we suggest that the two substances may directly cause the degranulation of mast cells without the assistance of the immunoglobulin at relatively low concentrations.At the high dose,5-HMF and OMBF act on the immune system,causing the release of immunoglobulin,triggering type I hypersensitivity reaction.In addition,according to the results of plasma protein binding experiments and molecular docking experiments,we believe that the toxicity of OMBF is higher than that of 5-HMF.Former researches about anaphylaxis always separate the type I hypersensitivity reactions with anaphylactoid reaction,and few studies try to find the links or overlaps between two reactions.Based on our study,we proved that two small molecule compounds can trigger both reactions,by in vivo and in vitro models.We suggest that it may have some links between these two reactions.Allergic-related adverse reaction is one of the serious reasons that is threatening our lives.We hope to find the ingredients which are commonly exist in products,such as 5-HMF and OMBF,and to determine their ability to cause anaphylaxis,so as to improve the safety of the products and warn people to use products containing such substances more carefully.In this experiment,recombinant human coagulation factor VIII was administered intravenously to Macaca fascicularis in a single and 30-day period to observe the potential toxicity and extent of the toxicity,and also the development and recovery of toxicity,together with providing reference information for the dose design of clinical and clinical adverse drug reactions monitoring.The acute toxicity test was performed by lethal dose method.After single intravenous administration,the acute toxicity of Macaca fascicularis was observed.In the 30 days repeated-dose toxicity experiment,1 time/day administration was carried out for 30 days,and the recovery period was four weeks.Changes in various indexes of the Macaca fascicularis were observed.In the acute toxicity test,only the 61 250 IU#kg-1 group animals were observed lactate dehydrogenase(LDH)increased compared with self-control before drug giving.In the 30-day administration experiment,clinical symptoms associated with factor VIII antibodies and inhibitors,such as prolongation of APTT,hemorrhage,A/G reduction were found,and there are no toxic pathological changes clearly associated with administration.Also,no delayed or accumulation toxicity were observed.The maximum tolerated dose of recombinant human factor ? for single intravenous injection to Macaca fascicularis is greater than 61 250 IU·kg-1;No observed adverse effect level(NOAEL)of recombinant human factor VIII is 250 IU·kg-1 for the 30 days of repeated administration. |
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