| Background and purpose Background Aplastic anemia(AA)is a bone marrow hematopoietic disease,by a group of physiologic factors,biological factors and unexplained factors,which has high mortality,poor prognosis characteristics,and hematopoietic stem cell transplantation offers the most effective therapy for patient with aplastic anemia.However,due to family planning policy,fewer sibling donor,the patient's parents and brothers and sisters can be used as a donor source for haplotype hematopoietic stem cell transplantation,which making haplotype hematopoietic stem cell transplantation into the mainstream force.Yet the occurrence of graft versus host disease(GVHD)and graft rejection is a major obstacle,which directly affects the long-term survival rate after transplantation.Cyclophosphamide(CTX)has potent immunosuppressive anti-tumor effects,high-dose CTX-induced immune tolerance after transplantation is of concern in haplotype transplantation,but the timing and dose are important.CTX-induced tolerant new method plays an important role in haplotype transplantation,but the mechanism and characteristics of CTX-induced immune tolerance are not yet fully understood,especially worrying about hematopoietic reconstitution.In this study,the effect of CTX on hematopoietic stem cells was observed by mouse solid experiment,which provided the theoretical basis for transplantation.Purpose 1.To evaluate the clinical efficacy of high-dose CTX-induced immune tolerance in the treatment of severe aplastic anemia(SAA)after haplotype transplantation;2.To observe the effects of different doses of cyclophosphamide on hematopoietic function in mice by animal experiment.Research methods 1.Single Center 16 patients with SAA who were treated with high-dose CTX-induced immune tolerance haploidentical hematopoietic stem cell transplantation after hematopoietic transplantation in the Army General Hospital of the Chinese People's Liberation Army from Jan 2014 to August 2015,including 7 males and 9 females,average age of 14.5 years(4~30 years old).All were relatives of haplotype donors,including father donor 9 cases,mother donor 5 cases,sibling donor 2 cases,and median age of donor,average age of 31 years(20~45 years old).To observe implant situation,hematopoietic reconstitution(including neutrophils and platelet planting time),transplant-related complications(graft-versus-host disease,infection and hemorrhage),survival and follow-up time and so on in all patients after transplantation.2.Female pure BALB /C mice were selected to make a model of aplastic anemia according to the conventional method.All mice after successful modeling were randomly divided for experimental purposes.The rats in the experimental group(HD-CTX group,MD-CTX group,LD-CTX group)were treated with CTX treatment dose(380mg / kg,200 mg / kg,100 mg / kg)and observe general state?blood routine change,femoral bone marrow biopsy,and the number of granulocyte and macrophagocyte colony-forming unit(CFU-GM),the number of erythrocyte colony-forming unit(CFU-E)by semi-solid methylcellulose culture method and the changes of CD34+cells in peripheral blood and bone marrow by flow cytometry in the experimental group,the AA model group and the normal control group. 1.All 16 patients except for 1 case of primary rejection,more than 15 cases were completely implanted,of which 2 patients were not implanted for the first time,the second transplant to achieve complete donor implantation.The median time of neutrophil migration was 15.5(14~26)days,and the median time of platelet implantation was 22.0(17~32)days.Follow-up to August 2016,the median follow-up time of 17.4(9~28)months,surviving patients were followed up for more than 6 months,the longest follow-up time of 28 months.A total of 8 cases of acute GVHD after transplantation,including IV acute GVHD 2 cases,III degree of acute GVHD 3 cases,I ~ II degree of acute GVHD 3 cases,according to the site is divided into 3 cases of intestinal,liver 3 cases,skin 2 cases;Chronic GVHD occurs both in the skin.7 cases of pulmonary infection;cytomegalovirus infection in 3 cases,of which 2 cases of development of giant cell pneumonia,which improved after treatment with ganciclovir,sodium phosphate,no hepatic venous syndrome and lymph nodeosis after transplantation;Hemorrhagic cystitis in 4 cases.One patient died due to no implantation,one died of severe pulmonary infection,one died due to acute GVHD,and the remaining 13 survived.2.All the rats were successfully constructed according to the pathogenesis of AA aplastic anemia.2)The changes of white blood cells and platelets in AA model group were similar,and decreased gradually on the 5th day,was(0.46±0.32)×109/L?(33.4±14.67)×109/L,respectively(P<0.05).The number of white blood cells in LD-CTX and MD-CTX groups began to rise on day 9.The number of white blood cells in HD-CTX group was approximately the same as that in model group.Bone marrow and peripheral blood CD34 + cells in AA model group had a significant decrease,decreased to a lower level on day 5,and began to rise on day 7,but still lower than normal,was(1.76±0.28)×109/L,(2.86±0.45)×109/L,respectively(P<0.05).Bone marrow and peripheral blood CD34 + cells of LD-CTX and MD-CTX group to a minimum on Results the day 3,and began to rise on day 5,was(0.66±0.30)×109/L,(0.72±0.40)×109/L,respectively(P<0.01).The bone marrow and peripheral blood CD34+cells in HD-CTX group were the lowest at day 3,was(0.49±0.38)×109/L,and then remained low.The number of CFU-GM and CFU-E colonies in model group AA decreased significantly at 48 h and 96 h,was(10.00±5.00),(5.00±3.00),(12.67±3.79),(5.33±3.21)(P<0.05).LD-CTX,MD-CTX and HD-CTX group were not significantly different from the model group.Conclusions 1.High dose of CTX-induced immune tolerance haplotype allogeneic hematopoietic stem cell transplantation in the treatment of severe aplastic anemia,hematopoietic reconstruction and stability,have a good clinical efficacy.2.Haplotype hematopoietic stem cell transplantation after transplantation with high dose of CTX induced immune tolerance can reduce the transplant-related complications,is a new method to prevent GVHD.3.BALB / C mice were irradiated with Co60? radiation sub-lethal dose(5.5Gy),and the lymphocyte suspension from DBA / 2 mice was injected into the tail vein at 4 o'clock.This process roughly simulated the pathogenesis of AA.The AA mouse model was used to observe the changes of peripheral blood in AA mice.The pathological changes of bone marrow nucleated cells and bone marrow were consistent with the pathological changes of AA,indicating that mouse AA was successful.4.Animal experiments confirmed that CTX had no significant damage to bone marrow hematopoietic function,and provided theoretical basis for the application of HD-CTX to induce immune tolerance in clinical transplantation. |
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