| The First Part Reproductive toxicity of recombinant human thrombopoietinIntroduction:Primary immune thrombocytopenia(ITP)is the most common hemorrhagic disease in clinic.The incidence of thrombocytopenia is 5-10/100,000.Females of childbearing age are more than males of the same age.ITP is the main cause of thrombocytopenia in early pregnancy,accounting for 3%～5%of the total thrombocytopenia in pregnancy.Platelet count decreased progressively with the increase of gestational weeks,reaching a low point in the third trimester of pregnancy,and 15%～35%of patients needed treatment.Similar to non-pregnant ITP patients,the first-line treatment for pregnancy with ITP includes glucocorticoid and intravenous gamma globulin infusion.However,the second-line treatment is very limited for patients who are ineffective in the first-line treatment.The only second-line treatment recommended by domestic and foreign guidelines is hormone combined with gamma globulin.A recent clinical trial of rhTPO in pregnancy with ITP has preliminarily proved that rhTPO can significantly increase platelet count and has no significant toxic and side effects on pregnant women and fetuses/newborns.At present,there is no systematic and rigorous animal reproductive toxicity test for rhTPO in the treatment of pregnancy complicated with ITP.The safety of rhTPO still needs to be confirmed by the study of mouse models.Therefore,we conducted a study on the reproductive and developmental toxicity of rhTPO.Research objective:1.Reproductive Toxicity of Female Mice in Lactation--Fertility and Early Embryo Development Toxicity Test.2.Prenatal developmental toxicity:embryo-fetus developmental toxicity test3.Study on perinatal toxicityMethod:1.Fertility and early embryo development toxicity test.Female mice and male mice were caged,and female mice were randomly divided into four groups:control group and rhTPO low,medium and high dose group.From 14 days before mating to the 5th day of pregnancy(G5),rhTPO was injected subcutaneously every day(control group),150 U/kg(low dose group),1500 U/kg(medium dose group)and 15 000 U/kg(high dose group).The daily activities from 14 days to G18 days before mating were observed and the weight and food intake were recorded.No uterine weighing,corpus luteum number,implantation number,live fetus number,absorbed fetus number and sex number were recorded in pregnant mice at G18 autopsy.The pregnancy and abortion of female rats were observed.Fertility index,implant loss rate and post-implant loss rate were calculated.Anatomy abortion female rats,analysis of the causes of abortion.2.Embryo-fetal developmental toxicity test.Eighty successfully mated female mice were randomly divided into four groups.Different doses of rhTPO were subcutaneously administered from G6 to G15.The changes of normal behavior,body weight and food intake were recorded from GO to G18.G18 pregnant mice were autopsied.The weight,number of corpus luteum,number of implants,number of live births,number of stillbirths and number of early and late abortions were recorded,and the loss rate after implantation and implantation was calculated.Anatomical microscopy was used to examine the abnormal appearance,viscera and skeleton of the fetus.Emphasis was placed on whether there were deformities such as curvature of tail,cleft palate,small thymus,nasal cavity dilatation,enlargement of lateral ventricle,pyelomegaly,multiple ribs,incomplete ossification of temporal bone,suprapubic perforation,etc.3.Study on perinatal toxicity.Female mice and male mice were caged,and female mice were randomly divided into four groups.From 14 days before mating to 21 days during lactation,rhTPO was given at different doses.Observe the general situation of the mother rats and pay attention to the delivery.The number of live births,stillbirths and deformities were recorded,the dead pups were dissected and the causes of death were analyzed.The physiological status and behavioral development of the pups were observed.The time points of pups’development progress were recorded,including eye opening,tooth eruption,plane inversion reflex,negative geotaxis,cliff avoidance,visual localization and auditory startle reflex.Results:1.Study on reproductive toxicity of female offspring mice.At this stage,no daily abnormalities of FO female mice were observed in all experimental groups.Six female mice died of dystocia due to abnormal fetal position during delivery,including 1 in the control group and 1 in the middle dose group,2 in the low dose group and 2 in the high dose group.No correlation with medication was found through autopsy.Four mice failed to mate,one in the control group and one in the low dose group,and two in the high dose group.Chi-square test showed that there was no significant difference between the control group and each dose group(P>0.05).With the physiological changes of mice after conception,implantation and pregnancy,the weight gain of female mice in each group was equivalent(Table 1.A).In the G18 autopsy female mice,the weight,corpus luteum,number of implantation,number of fetal deaths,number of early abortions,number of abortions,number of abortions,number of fetuses and fetuses and weight of fetal rats were recorded and analyzed.The implantation loss rate,post implantation loss rate and sex ratio were calculated,and the mean deviation was calculated.The one-way ANOVA test was used to combine Tu.The results of Tukey’s multiple comparisons test showed that there was no significant difference between the dose groups and the control group(P>0.05)(Table 1.B).2.Study on reproductive toxicity of prenatal development.Female FO mice accorded with physiological changes during pregnancy.During delivery,two mice died of dystocia due to improper fetal position,one in the control group and one in the low dose group,and there was no significant difference in food intake and weight gain among the groups(P>0.05)(Table 2.A).The pregnant mice were killed in G18.After autopsy,all the mice were successfully mated and conceived,and the mating index was 100%.After calculating the mean±standard deviation,statistical analysis showed that there was no significant difference among the four groups(P>0.05),except for the weight after uterus,the number of corpus luteum,the number of implantation,the number of live births,the number of stillbirths,the number of early abortions,the number of late abortions and the number of male and female.There was no statistical difference in the rate of implantation loss,post implantation loss rate and sex ratio between the dose groups(P>0.05)(Table 2.B).Fetal rats were examined by Kruskal-Wallis test combined with Dunn’s multiple comparisons test.There was no significant difference in the number of live births and the weight of fetal rats between the control group and each dose group(P>0.05).The observation of abnormal variation of appearance,viscera and skeleton showed that there was a small thymus abnormality in the middle dose group,and the incidence was within the normal range of fetal growth and development abnormality(P>0.05).Four fetal rats had multiple ribs,including two in the control group,one in the middle and high dose group,and one in the low dose group.Incomplete ossification of the temporal bone occurred in the low dose group.The incidence of these deformities was not related to the medication in the normal range of embryonic development.There was no significant difference between the control group and each dose group(P>0.05).There were no abnormal variations in other systems(Table 2.C).3.Study on perinatal reproductive toxicity.In this stage of study,there are still pregnant mice died of dystocia due to improper fetal position.One in the control group and one in the middle dose group.Chi-square test shows that the analysis is not related to medication,and it is a normal adverse phenomenon of childbirth.There was no significant difference in the body weight between the four groups(P>0.05)(Table 3.A).The number of stillbirths of offspring mice in each group was the same,except that there was no stillbirths in the middle dose group,there was no significant difference between the control group and the low and high dose group(P>0.05).In the lactation stage,all groups of pups died,and the survival index was calculated.The proportion of each dose group was the same as that of the control group,with no statistical difference(P>0.05).The survival of young pups was normal during the lactation period.Careful observation of the time points for the occurrence of the key developmental phenomena showed that there was no statistical difference between the groups(P>0.05)(Table 3.B),such as opening eyes,erupting teeth,leveling reflex,negative orientation,cliff avoidance,visual localization and auditory startle reflex.Conclusions:To sum up,there was no significant difference between the control group and the three different dosage groups in the general appearance of mice at mating,conception,embryo implantation,embryo development,childbirth,breast-feeding,organ,embryo,fetal development and offspring growth after autopsy.In this study,the non-adverse reaction dose(NOAEL)of rhTPO on female fertility,embryonic development,delivery and lactation in mice was 15 000 U/kg/day.Significance:RhTPO has no adverse effect on reproductive function of female mice,which provides experimental basis for safe clinical treatment of pregnancy complicated with ITP.The Second Part Effects of rhTPO on Tregs in Pregnancy Mice with ITP Model and Related MechanismsIntroduction:Thrombopoietin(TPO),as an endogenous protein kinase,can activate JAK/STAT,Ras/MAPK signaling pathways on megakaryocytes by binding to c-Mpl receptor on megakaryocyte membrane,and induce tyrosine phosphorylation,thereby stimulating the proliferation,differentiation and maturation of megakaryocyte progenitor cells.In each stage of megakaryocyte formation,platelet production is regulated,the number of platelets is increased,and thrombocytopenia caused by various reasons is alleviated.Recombinant human thrombopoietin(rhTPO)has been widely used in the treatment of ITP and chemotherapy-related thrombocytopenia.Previous studies have found that rhTPO can be used to treat ITP pregnant mice without obvious toxic and side effects during pregnancy and perinatal period.The proportion of regulatory T lymphocyte(Treg)and the level of transforming growth factor-beta(TGF-p)were measured before and after rhTPO treatment.It is proved that rhTPO can effectively improve the level of TGF-β and the proportion of Tregs.However,the relationship between rhTPO and Tregs and TGF-p is not clear.This study focused on the relationship between rhTPO and Tregs and TGF-β,in order to further explore the immunoregulatory mechanism of rhTPO in the treatment of ITP.Research objective:1.To establish a stable model of pregnancy with ITP in mice,flow cytometry was used to detect the expression of TPO ligand c-Mpl on CD4 T cells in spleen single cell suspension,and to study whether TPO binding sites existed on T cells and whether rhTPO could directly act on T lymphocytes.2.To further verify the relationship between Treg and TGF-β in TPO immunoregulation.Flow cytometry was used to detect the proportion of Treg in CD4 T cells of spleen single cell suspension of pregnant ITP mice.After oral administration of TGF-β receptor inhibitor(LY2109761),the proportion of Treg in spleen CD4 T cells was detected after subcutaneous injection of rhTPO,and the relationship between Treg and TGF-p was explored.3.Establish the ITP mice model of conditionally knock-out TGF-β gene on platelet surface,detect the effect of rhTPO on peripheral platelet count,Tregs ratio and TGF-β level,and explore the mechanism of rhTPO on Tregs.Method:1.Establish a stable model of passive pregnancy with ITP in mice.According to the relevant research experience and methods of the previous experiment,the ratio of male to female was 2:1 to confirm pregnancy,and anti-CD41 antibody(MWReg30)5.g/200μL/mouse was injected into tail vein to confirm the successful establishment of ITP model mice during pregnancy.Different doses of rhTPO were used to treat model mice,and peripheral platelet count was measured to verify the therapeutic effect of rhTPO.2.Propagation of platelet-specific TGF-β knockout mice and establishment of passive conditioned TGF-β knockout ITP mice model.The peripheral platelet counts of mice before and after the establishment of the model were measured to determine the establishment of the model.3.Pregnancy combined with ITP model mice were executed after peripheral blood was collected at the end of pregnancy.Spleen was dissected and single cell suspension of spleen was extracted by screen grinding method.Flow cytometry was used to detect the presence of TPO ligand c-Mpl on splenic CD4 T cells.4.CD4 T cells were isolated from spleen of pregnant mice with ITP by CD4+immunomagnetic beads method.The spleen CD4+T cells were cultured with rhTPO of 0 and 2 ug/L for 3 days.The proportion of Tregs in spleen CD4 T cells was detected by flow cytometry.5.Twenty pregnant mice with ITP were randomly divided into two groups.The experimental group was given 100mg/kg TGF-β receptor inhibitor(LY2109761)orally and the control group was given 0.5%sodium carboxymethyl cellulose solution orally for 14 days.From the first day,the mice in both groups were subcutaneously injected with 1500 U/kg rhTPO for 14 days.All the mice were killed.Platelet count was measured.6.Flow cytometry was used to detect the proportion of Tregs in CD4 T cells of spleen in rhTPO treatment model of ITP mice.7.The passive conditioned TGF-β gene knockout ITP mice were subcutaneously injected with rhTPO 15000U/Kg/day.After 14 days,the mice were killed and the tail blood and spleen single cell suspension were extracted.Peripheral platelet count was measured.The level of TGF-β1 in serum was detected by Elisa method.Tregs ratio in peripheral blood and spleen cells was detected by flow cytometry.Results:1.Pregnancy-associated ITP model mice were successfully established.2.After different doses of rhTPO were used to treat ITP pregnant mice,the peripheral platelet count of mice treated with rhTPO was significantly higher than that of control group.3.The model of passive conditioned TGF-β gene knockout ITP mice was successfully established.The peripheral platelet count of passive conditioned TGF-βgene knockout ITP model group and Flox ITP control group decreased significantly 24 hours after injection of antibody.At the 14th day,the platelet count was lower than that before the model establishment.There was no significant difference between ITP model group and Flox ITP control group.4.The plasma level of TGF-β in rhTPO group was significantly lower than that in rhTPO group without gene knockout(P=0.040).5.Flow cytometry analysis showed that CD4 T cells and c-Mpl did not co-express in the whole lymphocyte of ITP pregnant mice spleen single cell suspension,suggesting that c-Mpl on the surface of CD4 T cells was negative.6.There was no significant difference in the percentage of CD4+/CD25hi/Foxp3+Tregs cells in spleen lymphocytes of ITP pregnant rats in vitro between rhTPO culture group and control group(P=0.619).7.Increasing platelet count by rhTPO does not require the involvement of TGF-β7.1 There was no significant difference in platelet count between TGF-β receptor inhibitor and the rhTPO alone control group(P=0.347).7.2 The platelet count in rhTPO treatment group and rhTPO treatment group of ITP model mice with and without TGF-β gene knockout increased,and there was no statistical difference(P=0.685).8.Increased Tregs ratio is associated with elevated levels of TGF-β8.1 During the treatment of rhTPO in ITP pregnant mice,the Tregs ratio of CD4+T cells in spleen of mice treated with oral TGF-β receptor inhibitor was significantly lower than that of ITP pregnant mice treated with rhTPO alone(P=0.044).8.2 The proportion of Tregs in CD4 T cells of spleen in rhTPO group was lower than that in rhTPO group(P=0.040).Conclusions:The passive model of pregnant ITP mice can simulate the pathogenesis of ITP mediated by human anti-platelet antibody to a certain extent.As a second-line drug for the treatment of primary immune thrombocytopenia,rhTPO can not directly act on CD4 T cells,but through c-Mpl on the surface of megakaryocytes,stimulate the activation of megakaryocytes to produce platelets,increase the release of TGF-β1 by platelets,and then increase the proportion of Tregs in CD4 T cells,and regulate the immune function.Significance:Recombinant human thrombopoietin has a definite effect on improving platelet count in pregnant mice with ITP.It promotes the activation and proliferation of megakaryocytes,promotes platelet production,increases the platelet release of TGF-β1,further increases the proportion of Treg in T cells,induces immune tolerance and promotes long-term retardation of ITP patients by binding with megakaryocyte surface receptor c-Mpl.The solution provides an effective theoretical basis. |
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