| The incidence of hematological malignancies is the highest in children with malignanttumolors. Hematopoietic stem cell transplantation is a standard treatment for manyhematologic malignancies, and this therapy is the only effective method to cure hematologicalmalignancies. Currently, children with hematological diseases are mainly treated bychemotherapy and hematopoietic stem cell transplantation. For some hematological diseases,such as acute lymphoblastic leukemia,85%patients could survive by chemotherapy, while forthe acute myeloid leukemia, high risk acute lymphoblastic leukemia, refractory and relapsedleukemia, chemotherapy could not achieve long-term survival, thus allogeneic hematopoieticstem cell transplantation should be applied. The biggest advantage of allogeneichematopoietic stem cell transplantation is to eliminate most tumolor cells by high doses ofchemotherapy pretreatment. After transplantation the remained few tumolor cells will becleared by the donor graft versus leukemia (GVL) effect, thus the risk of recurrence could bequite low. However, allogeneic donor cells might cause graft versus host disease (GVHD),which would reduce the quality of life of the recipients or even be life-threatening. For thehematopoietic stem cell transplantation, a large portion of the donors are the parents, namelyhaplotype hematopoietic stem cell transplantation. In this circumolstance, the failure rate andthe incidence of GVHD could both be higher than allogeneic HLA matched stem celltransplantation.It’s reported that the immune tolerance effect is good when using umolbilical cord bloodcells infusion as the third party cells in haploidentical hematopoietic stem cell transplantation.The incidence and severity of GVHD could be effectively reduced after allogeneichematopoietic stem cell transplantation, but its mechanism is still unknown. This study aimsto establish murine hematopoietic stem cell transplantation model, and then to explore theimmune tolerance mechanism and effect in haploidentical hematopoietic stem celltransplantation with umolbilical cord blood cells infusion as the third party cells.In this study, humolan umolbilical cord blood cells were labeled with CFSE and infusedas third party cells in haploidentical hematopoietic stem cell transplantation. CFSE wouldfacilitate tracing in vivo so that it would be easy to analyze the distribution and differentiationof umolbilical cord blood cells after transplantation. CFSE labeling effect was evaluatedbefore transfusion. The final concentration of CFSE cell staining was set to5umol,10umol,15umol,20umol,25umol,30umol respectively. During cell culturing, every10d the cells were observed under fluorescence microscope and cell viability was detected. The resultsindicated that CFSE labeling is effective and easy to operate. In addition, non-cellularcomponents would not be stained, thus effectively reduce the false positive rate. CFSEstaining is final concentration dependent, so it’s easy to adjust the fluorescence intensityaccording to need. CFSE fluorescence was still visible at+10d,+20d, so it would be possibleto discriminate between CFSE and other interference. The cell viability decreased graduallywith the increasing final concentration of CFSE. It suggested that final concentration of CFSEhigher than30umol could induce more serious cell toxicity. The low concentration of CFSEcan be tolerated by umolbilical cord blood cells. In conclusion, the best CFSE finalconcentration for labeling is20umol.In this study, female BALB/C mice as aGVHD model were used as recipients. MaleCB6F1mice (F1generation of female BALB/C mice and male C57mice) were used asdonors. All mice were SPF grade and fed in the SPF animal lab. Bone marrow and spleencells of donor mice were harvested and1:1mixed, ready to be used as donor stem cells.Recipient mice were treated with8Gy Co60irradiation. After irradiation, recipients inexperimental group were infused with CFSE labeled umolbilical cord blood cells and donorstem cells. Recipients in the control group were only infused with donor stem cells. After theinfusion, manifestations and reactions of the mice in these two groups were observed andrecorded. The proportion of T-reg cells was detected by complete blood count test and FACSanalysis weekly. Mouse skins, livers and intestines were harvested weekly for histopathologyexamination.According to the clinical aGVHD score at different time points after transplantation (+7d,+14d,+21d,+28d,+50d), the results indicated that the incidence and severity of aGVHD inexperimental group was significantly lower than that in control group (P <0.05). The resultsof histopathology examination of aGVHD target organs (skins, livers and intestines) alsoindicated that the incidence and severity of aGVHD in experimental group was significantlylower than that in control group. The result of tracking CFSE labeled umolbilical cord bloodcells indicated that CFSE labeled cells disappeared quickly in the blood, while these cellscould be found in frozen sections of aGVHD target organs (intestines, livers). The resultsuggested that some or all of umolbilical cord blood cells got into aGVHD target organs, andthese cells might play a certain role in the local tissue so that reduce the incidence andseverity of aGVHD in experimental group.The results of implanting cells analysis indicated that WBC implanted at+7d in both groups. At+14d, WBC of mice in experimental group was significantly higher than that incontrol group (P <0.05). This result suggested that WBC of mice in both groups had nosignificant difference before+7d (P>0.05). But at+7d~+14d mice cells recovered fasterthan that in control group, suggesting that in the early stage after transplantation, peripheralblood WBC of mice in experimental group recovered faster than that in control group. PLT ofmice in experimental group at+7d,+14d recovered faster than that in control group (P <0.05),while at the late stage of transplantation (+21d,+28d,+50d) PLT recovery levels in bothgroups had no significant difference (P>0.05). At+7d,+28d, T-reg cells of mice inexperimental group were significantly more than that in control group (P <0.05), while atother time points (+14d,+21d,+50d) T-reg cell numolber in both groups had no significantdifference (P>0.05).Comparison of the survival time after transplantation of mice in these two groupsshowed no significant difference (P>0.05). This result indicated that umolbilical cord bloodcells infusion as third party cells could not improve long-term survival rate and survival timeof mice in haploidentical hematopoietic stem cell transplantation.Hence, umolbilical cord blood infusion as the third party cells could effectively promotethe early hematopoietic recovery. The faster early recovery of WBC, PLT could reduce therisk of infection, bleeding and other complications. Umolbilical cord blood infusion as thethird party cells could also reduce the mouse aGVHD, supported by both the clinicalmanifestations and pathological results. CFSE labeled cells in aGVHD target organssuggested umolbilical cord blood cells could get into aGVHD target organs, and play aprotective role through some mechanism to reduce local aGVHD reaction. The difference ofT-reg cell numolbers between the two groups suggested that umolbilical cord blood cellsmight induce the production of more effective T-reg cells through a certain mechanism, thusincrease immune tolerance after transplantation, so the systemic aGVHD reaction andaGVHD reaction of local target organs both were reduced. Umolbilical cord blood infusion asthe third party cells in hematopoietic stem cell transplantation cannot improve the survivalrate and prolong the survival time of mice. Considering the survival rate and survival timewere affected by many factors, umolbilical cord blood infusion currently has not yet beenreported to have impact on these two indicators. |
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