Primarily Study On The Engraftment Kinetics And Mechanism In Double Unrelated Cord Blood Stem Cell Transplantation

Background and objective Umbilical cord blood (UCB) is a good source of hematopoietic stem cells (HSC), but the numbers of HSC are relatively small thus limiting its application in adults. The successful treatment for leukemia with double units umbilical cord blood transplantation (DUCBT) aroused the research on the mechanism of interaction between the two units UCB. Practically, graft failure, disease relapse are major obstacles that affect prognosis and survival of patients undergoing UCB .Serial and quantitative analysis of chimerism has been propsed as an important method in early monitoring these negative events in order to set up the relevant preventive therapeutics. On the other hand, previous research revealed dominancy of one of two units from the day of engraftment and the interaction between the two units may exist. Our research is to explore the influence of co-culture ex vivo of CD34+ cells and T-lymphocyte from different unit UCB on the differentiation ability.Methods 1,Amplified fluorescence labeled multiplex-STR with 16 locus 1 sexual gene locus was used to detected chimerism at different time after UCBT for 12 hematologic malignancies patients who received UCBT in Anhui provincial hospital between 2006-2008. To build the STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique for personal identification. 2,Mesenchymal stem cells (MSCs) were obtained from human bone marrow and their morphology and phenotype were identified. CB CD34+ cells and T-lymphocyte from different unit were co-culture on 12Gy X-Gy irradiated MSC layer. Their differentiation ability (CD33, CD41, CD71) were assessed respectively by flow cytometry.Results 1. Engraftment kinetics analyzed by the serial chimerism Patients who received UCBT and detected donor DNA at 7 or 14 days ,successfully had full donor chimera at last, independent on donor DNA percentage; while, patients who received Unrelated duplicate Cord blood transplantation and only detected donor DNA 100% at 14 days ,successfully had full donor chimera at last. 2. Establishment and identification of MSC layer After cultured for 12days, MSC integrated of 70% to 80% with a whirlpool-like growth. Cells were transferred to the third passage and then phenotype was identified. CD90, CD105, CD166, CD29, CD44, CD13 and CD49e are highly expressed on MSC with no expression of CD14, CD34, CD31, CD45 and HLA-DR. 3. The differentiation ability change of CD34+ cells During the co-culture, the expression of CD33,CD41,CD71 on CD34+ cells was gradually upregulated . However, there was still no significant difference from the control groups.Conclusion 1. The STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique can dudge graft accurately at early time after UCBT. The method can reveal UCBT engraftment kinetics. Detect the Chimerism at 14 days after UCBT can provide the information of graft implantation, Also,the method can predict graft implantation permanently and disease prognosis by Chimerism track after UCBT, to prove reliable experimental basis for clinical treatment. 2. Co-culture with CD34+ cells and T-lymphocyte from different unit may have no effects on the differentiation ability of each other. Therefore, we suppose the interaction between cord bloods may not be induced by the interaction between the CD34+ cells and T-lymphocyte from different unit.