Experimental Research Of Micro Transplantation On The Hematopoietic Recovery Of H-2Haploidentical Mice

Research objective:Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the important medical means to treat hematologic malignancies, certainmalignant tumors, immune and genetic diseases. However, traditional hematopoieticstem cell transplantation can be carried out until the pretreatment of radiation of largedose and immune inhibitor is completed. Toxic reaction, complications such asGVHD, rejection and infection related to pretreatment after transplantation seriouslyrestrict the curative effect of allo-HSCT. Compared with the traditionalmyeloablative scheme, even trough nonmyeloablative scheme significantly decreasesthe complications related to pretreatment, the pretreatment itself including theseverely bacteria, epiphyte and viruses infection complications caused by theapplication of immunosuppressant after radiation and chemotherapy andtransplantation as well as the low immune function still seriously threat the patients’health of life. It is found from the previous patients’ treatment that hematopoietic stemcell implants have the effect of support of hematopoiesis and the occurrence ofGVHD is closely associated with the receiver’s implanted ratio of donor’shematopoietic stem cells. Therefore, how to make hematopoietic stem cell implantedin the receiver for short time and low ratio to promote the recipients’ ownhematopoietic recovery and at the same to avoid the occurrence of GVHD hasbecome our focus. Originally proposes the new concept of “micro transplantation"and new means of radiation injury treatment in the world. Micro transplantation doesnot carry out deadly or fatal immune inhibition and pretreatment for the recipients anddoes not require complete implantation or mixed implantation of donor cells, onlyadopts infinitesimal immune inhibition pretreatment to form trace donor chimera. Thisis different from the ideas and concepts of traditional transplantation pretreatment andimmune toleranceThis topic is to discuss the safety of micro transplantation and its impact onhematopoietic function to provide animal experimental foundation and theoreticalbasis for clinical application of micro transplantation.Content of research:Under the condition of micro transplantation and H-2 haploidentical donor cells of different quantity (1×1078×107), peripheral blood inmice and donor implantation and GVHD are studied and peripheral blood, bonemarrow colony formation and impact of spleen of H-2haploidentical mice are mainlydiscussed.Research methods:1.The chemotherapy dose:Female CB6F1mice of6-8weeks were performed intraperitoneal injection ofAra-C of large dose,200mg/kg, once a day, for2days. On the third day,intraperitoneal injection was continued with50mg/kg as the maintenance dose, oncea day, for3days. The induction time is5days.2.Experiment on the effect of quantity of donor cells on the blood picture,implantation and GVHD of mice treated with micro transplantation.Female CB6F1were randomly divided into5groups according to the weight, i.e.,Ara-C group(chemotherapy-control group), Ara-C＋1×107group, Ara-C＋2×107group, Ara-C＋4×107group and Ara-C＋8×107group. Each group has10mice. Allthe mice in cell infusion group were performed tail vein infusion G-PBSC2h after lastchemotherapy, and the mice in control group were performed tail vein infusion ofnormal saline of the same amount. The difference of different quantity of donor cellsinfusion on the peripheral blood picture, implantation and GVHD of mice wascompared.3.Experiment on the comparison of effect between micro transplantation andG-CSF on peripheral blood pictureFemale CB6F1were randomly divided into3groups according to the weight, i.e.,Ara-C group(chemotherapy-control group), Ara-C＋4×107cells infusion group (microtransplantation group), Ara-C＋G-CSF group(G-CSF group),10mice in each group.The control group was performed tail vein infusion of normal saline. The mice in cellinjection group were performed tail vein infusion of G-PBSC2h after lastchemotherapy. The mice of G-CSF group were performed subcutaneous injection ofrhG-CSF10μg/kg, once a day, for6days. Peripheral blood of20μl of mice in all thegroups on1d,3d,5d,7d,9d,11d,13d after chemotherapy stopped was collected andperformed EDTA anticoagulant, and blood cell analyzer was used to detect thequantity of WBC, Hb, PLT of peripheral blood of mice.4.Comparison experiment of bone marrow pathology, bone marrow cell colonygenerated unit and spleen structure between micro transplantation group and chemotherapy control group3mice in each group were executed5d and9d after thechemotherapy stopped and femoral bone marrow was maintained to observe thepathological morphological changes, and in the sterile condition,2×10~5bone marrowcells were maintained to perform bone marrow cell colony culture and observe mice’schanges of pathological tissue morphology and colony formed unit(CFU)after microtransplantation. In the sterile condition, mice’s spleen was separated and performedpathological and histological examination to observe the effect of microtransplantation on mice’s spleen structure.Results of research:1.Mice’s changes of blood picture and bone marrow pathology afterchemotherapy: after chemotherapy, no vomiting and loose stool occurred in the mice;one day after chemotherapy stopped, WBC was obviously decreased to2.56±0.38,significantly lower than that before chemotherapy(0.67±1.64, P<0.01), and3daysafter chemotherapy stopped, WBC was decreased to the lowest level(2.32±0.61,P<0.01)and then slowly recovered;13days after chemotherapy stopped, WBCcompletely recovered to the normal level.5days after chemotherapy stopped, Hbdecreased to the lowest level (123.32±4.32, P<0.01), and then slowly increased,11-13days after chemotherapy stopped, recovered to the normal level. After chemotherapy,PLT sharply decreased, and5days after chemotherapy stopped, decreased to thelowest level (455.44±108.42, P<0.01),13days after chemotherapy stopped, basicallyrecovered to the normal level.5days after chemotherapy stopped, bone marrowcavity was empty and the hematopoietic cells decreased significantly and fat cellswere replaced. In the process of experiment, no death occurred in the mice.2.The effect of injection quantity of donor cells on the mice’s peripheral bloodafter micro transplantation: compared with control group, in1×10~7group,2×10~7group and, there was no obvious effect to promote the recovery of WBC and in4×10~7group and8×10~7group, recovery of WBC was rapidly achieved, but in8×10~7group,WBC decreased after recovery. When the quantity of donor cells increased from1×10~7to4×10~7, the effect of micro transplantation to promote the recovery of Hbwas strengthened, but when increased to8×10~7, micro transplantation to promote therecovery of Hb did not continue to accelerate. The effect of micro transplantation forthe recovery of PLT was strengthened with the increase of injection quantity of donorcells. There was not significant difference of promotion of recovery of PLT between4×10~7group and8×10~7group. 3.Effect of injection quantity of donor cell on the mice’s implantation and GVHDafter micro transplantation: after treatment with micro transplantation, the mice in1×10~7group,2×10~7group and4×10~7group obtained micro mosaic of donor cells afterinfecting H-2haploidentical donor cells and the median time of donor cells existing inthe body was6w,8w and10w, respectively, and no GVHD occurred.80%of themice in8×10~7group were completely changed into CC within612w after cellinjection and no obvious GVHD occurred.4.In the comparison experiment of micro transplantation and G-CSF, both microtransplantation and G-CSF could promote the recovery of white blood cell afterchemotherapy. In the process of recovery, the speed to promote the recovery of whiteblood cell between two groups was not significant(P>0.05). Hb of microtransplantation group was significantly higher than that of G-CSF group andchemotherapy control group(P<0.01). On5d,7d,11d after chemotherapy stopped,the Hb of micro transplantation group was significantly higher than that of G-CSF(P<0.01). In the process of recovery of PLT,5d and7d after chemotherapy stopped,the PLT of micro transplantation group was higher than that of G-CSF group（P<0.05）, and9d and11d after chemotherapy stopped, the difference of PLTbetween micro transplantation group and G-CSF was significant(P<0.01).5.Mice’ s pathological histological changes showed that the mice’s hematopoieticfunction in micro transplantation was better than mice in the control group. Bonemarrow colony culture results also showed micro transplantation could promote themice to generate more CFU–Mix. Mice’s pathology result of spleen showed microtransplantation could reduce the damage of chemotherapy on mice’s spleen.Research conclusions:1.This study successfully establishes the H-2haploidentical mice microtransplantation model to provide a ideal platform to discuss and study the mechanismof micro transplantation to promote the hematopoietic recovery and immunereconstruction.2.Micro transplantation is effective on the recovery of mice’s WBC, Hb, andPLT after chemotherapy. And the promotion of micro transplantation on the recoveryof WBC matches the curative effect of universally accepted G-CSF. The effect ofmicro transplantation to promote the recovery of Hb, PLT is better than G-CSF;3.Micro transplantation can promote mice’s bone marrow to generate more CFU–Mix and may protect the structure of the spleen. 4.This experiment provides animal experimental basis and theoretical basis forthe clinical application of micro transplantation.