Establishment Of Micro - Transplantation Model Of Leukemia Mice And Its Related Mechanism

Objective To reproduce and identify a H-2completely mismatched microtrans-plantation model of leukemia mouse,and to explore the correlated preliminarymechanism.Contents1.To reproduce a model of leukemia mouse.To establish a acutemyelomonocytic mouse model according to the study past, aim to prove the modelis stable, and establish the diagnosis standard of leukemia mouse.2.To optimize thechemotherapy regimens. Using different chemotherapy(mitoxantrone+cytarabine),the early mortality, bone marrow suppression would be compared betweengroups,aim to choice suitable chemotherapy.3.To optimize the donor cell quantityand identify microtransplantation model. Transfusing different donor cells,the earlymortality, white blood cells recovery, leukemia suppression of the same time wouldbe compared between groups,aim to choice the optimized donor cells. Detection ofdonor chimerism by RT-PCR.From the clinical manifestation and pathologicalconditions to determine GVHD in microtransplantation model.4.To study thepreliminary mechanism of anti leukemia effect and avoid GVHD ofmicrothransplantation. Observation anti leukemia effect of microtransplantation, andthrogh testing the donor chimerism, cytokines to study the mechanism ofmicrotransplantation.Methods The recipients were female Balb/C mice while the donor were maleC57BL/6J mice.Firstly,to reproduce leukmia model,Balb/C mice were randomlydivided into two groups:leukemia model group, normal control group, leukemiamodel mice were inoculated intravenously with1×106of WEHI-3cells,a cell line ofmyelomonocytic leukemia while control mice were inoculated intravenously withequal normal saline. Three weeks after inoculation, the perpheral blood collected formorphological observation,and bone marrow cells were collected for morphologicalobservation, immunochemical and immunophenotyping test.The liver, spleen,kidney, lung,intestine, brain were collected for pathological observation.Moreover,the survival time would be recorded.Secondly,the chemogtherapy regimens wouldbe refined. The recipients were randomly divided into three groups:①mitoxantrone2mg/kg×1d+cytarabine200mg/kg×3d、②mitoxantrone4mg/kg×1d+cytarabine200mg/kg×3d、③mitoxantrone8mg/kg×1d+cytarabine200mg/kg×3d. The early mortality, bone marrow suppression would be compared between groups to choicesuitable chemotherapy. Thirdly,to choice the suitable donor cells. Donors werereceived100μg/kg G-CSF mobilize through hepodermic syringe,every12hours,last5days.The recipients were randomly divided into four groups:3×107microtransplantation group、6×107microthransplantation group、12×107microtransplantation group and chemotherapy control group. The recipients wereinoculated intravenously with1×106of WEHI-3cells, then received Chemotherapy(mitoxantrone+cytarabine),Continue4days after two days of leukemic cellsinoculation;the microtranspltation goups mice were received donor peripheral bloodstem cells (PBMC) after MA conditioning without GVHD prophylaxis whilechemotherapy control group received equal normal saline. To choice the suitabledonor cells,the death rate of early, recovery of peripheral blood and leukemiasuppression will be compared between chemotherapy and microtransplatationgroups.When the microtransplantation model were reproduced,the donor chimerismand GVHD would be tested to identify the micortransplantation model.Lastly,theearly mortality,hemogram recovery,leukemia suppression and survival time wouldbe compared between microtransplantation and chemotherapy groups,in additon,thechimerism and cytokines would be tested,to study the microtransplantationmechanism primarily.Results The leukemia model result:Mice inoculated with WEHI-3cells only died ofleukemia17-35days after inoculation,which similar to previous results.Two weeksafter inoculation,there were more than20%primitive and juvenile cells. Threeweeks after inoculation,the WBC counts of peripheral blood increased to85.09×109/L in the leukemia mice,which was significantly different from that of normalcontrol (P<0.05).A large number of primary and immature cells were found in themarrow, and peroxidase(POX), specific esterase(CE) and non-specific esterase(NSE)test were positive,while sodium fluoride(NaF)was nagative.There were a largenumber leukemia cells in mutiple organs.About the chemotherapy optimization, theearly mortality were①0%、②25%、③100%，respectively,there were no differentbetween①and②（P>0.05）,but the bone suppression of②was significantly differentfrom that of①（P<0.01）.So the chemotherapy is②mitoxantrone4mg/kg×1d+cytarabine200mg/kg×3d.About donor cells, chemothearpy group had a25%earlymortality,meanwhile the(3、6、12)×107groups were16.67%、8.33%、8.33%,there were no different form that of chemothearpy gourp（P>0.05）.(3、6)×107groups hasa stronger hematopoietic recovery skill than chemotherapy and12×107groups（P<0.05）.There were more leukemic cells in chemothearpy mice thanmicrotransplantation mice（P<0.01）,but（12、6）×107groups has lower leukemiasuppression than3×107group（P<0.05）,menawhile there was no different between6×107and12×107groups（P>0.05）. No signs of GVHD were observed inmicrotansplatation mice.The donor microchiemrism can be discovered in eraly2weeks after donor cells transfused. The phenomenon and mechanism ofmicrotransplantation effect that antileukemia and avoid GVHD at the same time hadbeen studied.The result showed microtransplantation mice had a decreased earlymortality rate, a strong recovery skill of white blood cell compared withchemotherapy group, and the time of onset were delayed about2weeks thanchemotherapy mice. The relevant examination showed that microtransplantationmechanism may be related with the microchimerism and cytokines changes.Donormicrochimerism could be detected after2weeks of transfusion; cytokines, especiallyIL-6, TNF, IL-2changes very severe within two weeks after transplantation,especially within one week after transfusion.Conclusion A leukemia murine model had been successfully reproduced.MA(M4mg/kg×1d+A200mg/kg×3d)had been selected as chemotherapyregimens,and6×107donor cells had been selected as the optimized transfusiondonor cells number. Finally a H-2completely mismatched microtransplantationmodel of leukemia mouse has been successfully reproduced and identified.what,smore, it was confirmed that microtransplantation played an anti-leukemia effect atthe same time without the occurrence of GVHD.The mechanism ofmicrotransplantation would be related with the ideal of microtransplantation、microchimerism and cytokines changes.This model and related mechanism researchcould provide foundation for animal experiment and clinical microtransplantationstudy.