The Research Of The Mice Model Of Primary ITP Qi Failing To Control Blood Disease Combined With Syndrome

Primary immune thrombocytopenia(ITP),which characterizes platelets decreasing,the body bleeding,the skin mucous membrane blood stasising and purple,epistaxis,and visceral hemorrhage,is an autoimmune disease.ITP animal model is the main method of studying the pathogenesis and treatment of ITP,and has played an important role in the study of ITP.The Disease-syndrome combined animal models is an important basis for the study of the mechanism of TCM treatment of ITP.At present,the disease-syndrome combination model for ITP is mainly composed of ITP excess heat syndrome model,ITP kidney-deficiency syndrome model,ITP QI failing to control blood model.However there is still little research on the animal model of ITP QI failing to control blood model.Therefore,this paper established a model of ITP QI failing to control blood model based on the previous research with Disease-syndrome combined and simulated clinical studies.The pathophysiological mechanism of the model was observed in order to provide a suitable animal model for the theoretical research of theory of Qi and Blood of traditional Chinese medicine and the evaluation of Chinese Herbal MedicineObjective1)This study used animal models contrast method and comparative analysis method to establish the model of ITP qi failing to control blood disease combined with syndrome.2)Explore the model of ITP qi failing to control blood disease combined with syndrome's pathogenesis of pathological and physiological molecular level,through the modeling's dynamic monitoring and comparison of physiological and pathological indxs.3)Through the monitoring of clinical index model and the contrast of clinical disease,makes comparative analysis reasonablely on the model of qi failing to control blood in accordance with the clinic.Contents and methods1.The establishment and evaluation of qi deficiency mice modelICR male mice were divided into normal control group,sleep deprivation group,exhausted swimming group,sleep deprivation + exhausted swimming group(abbreviate composite group),and each group have 12 mice.The sleep deprivation group used multi-platform water environment to deprive sleep for 9 hours a day;Exhausted swimming group adopted the method with 10%weight to swim once a day;The composite group Combined two methods for 4 weeks continuously.After the experiment,observed the behavior and weight of mice,then used mice holding power equipment to measure grasping force of mice,then used digital camera to collect the surface image of tongue,used small animal noninvasive monitoring equipment to detect the pulse signals(heart rate,breathing rate,respiratory amplitude,pulse amplitude),and detected the spleen T cell subgroupand B cells by flow cytometry.2.The establishment of mice primary immune thrombocytopenia(ITP)modelCBA/J female mice were divided into normal control group and model group,and each group had 5 mice.Model group's mice model was established by immunization after intraperitoneal injection lactation female wistar rats platelets.Model group mice were immune for the first time by intraperitoneal injection of 0.5 ml 0.9%sodium chloride solution that contained 1 X108 rat platelets,then injected 0.5 X108 platelets to streng immunity once a week.The control group only injected 0.9%sodium chloride solution.The two groups mice were all continuously injected for 4 weeks.Mice were extracted their blood from tail top to measure the platelet number before immunity injection every week.3.The establishment and evaluation of the model of mice primary immune thrombocytopenia(ITP)qi failing to control blood disease combined with syndromeThe CBA/J female mice were divided into normal control group,sleep deprivation group,immune group,sleep deprivation-immune group,and supplementing qi and photoing blood group,and each group had 12 mice.The sleep deprivation group used multi-platform water environment to deprivesl eep for 16 hours a day;Immune group's mice model was established by immunization after intraperitoneal injection lactation female wistar rats platelets;sleep deprivation-immune group combined two methods;traditional Chinese medicine group and sleep deprivation-immune group had the same interventions,and they were given by gavage once a day for 35 days with Yiqi Shexue Prescription to disproof the treatment of disease model.After the experiment,observed the behavior and weight of mice,then used mice holding power equipment to measure grasping force of mice,then used digital camera to collect the surface image of tongue,then used small animal noninvasive monitoring equipment to detect the pulse signals(heart rate,breathing rate,respiratory amplitude,pulse amplitude);weight the spleen index and thymus index;Detected the spleen T cell subgroupand B cells by flow cytometry;The indexes about spleen-Pathological and blood:platelet count;Serum ELISA detection.4.The dynamic research of ITP qi failing to control blood disease combined with syndrome modelThe CBA/J female mice were divided into normal group,normal group and each group had 48 mice.The model group used multi-platform water environment to deprive sleep for 16 hours a day and in the second week combined with intraperitoneal injection lactation female wistar rats platelets to establish the integrated disease and syndrome of ITP.Collected materials on normal group and model group in 2,3,4,5-weekend.Observed the behavior and weight of mice,then used mice holding power equipment to measure grasping force of mice,then used digital camera to collect the surface image of tongue,then used small animal noninvasive monitoring equipment to detect the pulse signals(heart rate,breathing rate,respiratory amplitude,pulse amplitude);weight the spleen index and thymus index;Detected the spleen B cells and T cell subgroup by flow cytometry;The indexes about spleen-Pathological and blood:platelet count;Serum ELISA detection.Result1.Establishment and evaluation of qi deficiency model in mice1)The main symptoms:Compared with the normal control group,the activity of the mice in the three groups were decreased,showing the fatigue characteristics,the weight and grasping force significantly reduced(P<0.05-0.01).2)Tongue picture:The lingual surface of mice were seen white in different degree,only compound group has a significant difference(P<0.05).3)Pulse condition:The heart rate,respiratory rate,pulse amplitude and respiration rate of sleep deprivation group and compound group were significantly decreased(P<0.05-0.01).4)Immunology:The CD3+,CD4+,CD8+T cell subsets in the three groups were significantly increased(P<0.01),the CD19+B cells in the sleep deprivation group were significantly reduced(P<0.01).2.Establishment of primary immune thrombocytopenia model in mice1)Compared with the normal control group,the number of platelets in the model group reached the lowest point in the second week,and the results were significantly different(P<0.01).3.Establishment and evaluation of the combination model of primary immune thrombocytopenia syndrome and Qi failing to control blood syndrome in mice1)The main symptoms:Compared with the normal control group,the activity of the mice in each group were decreased,showing the fatigue characteristics,the weight and grasping force significantly reduced(P<0.05-0.01).2)Tongue picture:The lingual surface of mice were seen white in different degree.3)Pulse condition:The oxygen saturation of each group was significantly decreased(P<0.01-0.05),the heart rate of the sleep deprivation group,the immune group and the sleep deprivation group-immune decreased,while the heart rate of the drug group increased,and the respiratory rate of the sleep deprivation group increased(P<0.05),there was no significant change in the other groups(P>0.05).The pulse amplitude was significantly reduced in the sleep deprivation group(P<0.05),the other groups had no significant change(P>0.05).Hematology:The megakaryocyte of model groups mature hindrance,and apoptotic bodies were detected.4)The platelet of the drug group was significantly higher in the first 3 weeks(P<0.05),and the lowest in the sleep deprivation-immunized group(P<0.01)in 4 and 5 weeks.4)The spleen index of each group wad decreased(P<0.05-0.01).The thymus index of sleep deprivation group and sleep stripping polysaccharide group was decreased,and the difference was significant(P<0.01-0.05).5)Immunology:The CD3+,CD4+,CD8+T cell subsets in each groups were significantly increased(P<0.01),the CD19+B cells in the sleep deprivation group were significantly reduced(P<0.01).6)Platele associated antibodies in mice have no significant difference.7)The IL-6 secreted by each serum cytokines group were higher than those in the normal group(P<0.01),the difference between the sleep deprivation-immune group was the largest,and the ratio of IFN-to/IL-4 in each group was larger than that in the normal group.4.Dynamic research of the combination model of primary immune thrombocytopenia syndrome and Qi failing to control blood syndrome in mice1)The main symptoms:Compared with the normal control group,the activity of the mice in each group were decreased,showing the fatigue characteristics,the weight and grasping force significantly reduced(P<0.05-0.01).2)Tongue picture:The lingual surface of mice were seen white in different degree3)Pulse condition:The heart rate of the model group was higher than that of the normal group(P<0.01)in third and fourth weeks.After the third weeks,the pulse amplitude was significantly lower than that in the normal group(P<0.01).4)Hemotology:The megakaryocyte of model groups mature hindrance.And the difference in the 5th week is greatest.5)In the first 1-2 weeks,platelet count of model group was higher than the normal group and differented significantly(P<0.01-0.05),in 3-5 weeks,the number of platelets in the model group was lower than that in the normal group,and the difference was significant(P<0.01).In fourth weeks,the platelet was reduced to the lowest.6)Immunology:The spleen index and thymus index of the model group were lower than those of the normal group,and the difference was significant in 2,3 weeks(P<0.05).7)The CD3+,CD4+ and CD8+T cell subsets of the model group increased significantly(P<0.01),and CD4+/CD8+ was significantly higher than that in the normal group in fourth and fifth weeks(P<0.01).The CD19+B cells in the model group were significantly reduced(P<0.01).8)The platelet-associated antibodiey PAIgA,PAIgG of model mice were higher than those of normal group.The difference increased with the growth of modeling time.The PAIgG difference was significant in the 5th week.9)The IL-6 secreted by each serum cytokines group were higher than those in the normal group(P<0.01),the difference was significant,and the ratio of IFN-to/IL-4 was larger than that in the normal group,the difference was significant in 3,4 weeks.Conclusions1.Sleep deprivation,exhaustive swimming,sleep deprivation and exhaustive swimming can be successfully used to produce the model of qi deficiency in mice,and sleep deprivation is the best way of the three.2.Based on the principle of immunological antigen cross simulation,The platelets that were extracted from Wistar female rat were injected into the CBA female mice by intraperitoneal injection to simulate the chronic ITP disease,and it was best for the experiment to two-three weeks.3.Sleep deprivation and platelets immune for 5 weeks could prepare a kind of the model of ITP qi failing to control blood disease combined with syndrome.This provided a nearer match clinical animal model for the subsequent study of ITP mechanism research and pharmacodynamic evaluation of TCM research.4.Indexes of the model of ITP qi failing to control blood disease combined with syndrome began having pathological changes since the second week,than the fourth week was the most serious,and the symptoms and related indexes recovered on the fifth week.To sum up,this paper initially built a nearer match clinical animal model of ITP qi failing to control blood disease combined with syndrome through the methods such as the selection and optimization of modelling method,animal strains,modelling time and other factors.