| BACKGROUNDHematopoietic stem cell transplantation (HSCT) is efficient to clear the tumor or abnormal cells in recipients by conditioning regimen composed of high-dose chemotherapy and/or radiotherapy, then transplant with autologous or allogeneic hematopoietic stem cell (HSCs) or bone marrow (BM) cells to recipients to rebuild normal hematopoietic and immune systems. HSCT is widely used in the treatment of hematological disease, genetic disease, solid tumor and autoimmune disease and can be divided into autotransplantation and allotransplantation or bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT) and cord blood transplantation (CBT), etc. Rapid and effective hematopoietic reconstruction after HSCT is important to improve the survival rate and quality of life.Currently, possible strategies as follows can promote reconstruction after HSCT: (1)Increasing the number of transplanted cells:HSCs’ homing and implantation can thus be improved directly, which affects the reconstruction of hematopoietic and immune systems after HSCT. However, the proportion of HSCs in bone marrow is extremely low, only about 1% according to the literature. So the increase of transplanted cells will also enter a lot of allogenetic cells as well as raise the probability of graft versus host disease (GVHD), leading to complications and even threatening to life. (2)Transplantation of the purified HSCs:Transplanting of the purified HSCs can increase the proportion of HSCs and improve the homing and implantation rate in BM. However, the separation and purification of HSCs is complex and expensive. And related research also showed that not only HSCs played an important role in the recovery of hematopoietic and immune systems after HSCT, but also some stromal cells. Excessive purification may be harmful to the transplantation. (3) Amplification of the HSCs in vitro:Amplifying of HSCs in vitro can increase the number of "HSCs" and even improve the homing and implantation rate. But studies showed that the surface antigen and some features of HSCs might change due to cell differentiation and it is not good for transplantation.(4)Intra-bone marrow injection transplantation:In 2001, Kushida reported that intra-bone marrow (IBM) injection could improve the homing rate, since then lots of researchers carried out relevant research repeatedly to confirm that intra-bone marrow injection can markedly improve the homing and survival rate comparing to intra-venous transplantation, IBM injection can reduce the HSCs required for transplantation and the incidence of GVHD. (5)Increasing the percentage of HSCs homing to bone marrow:This strategy is promising and the source of targeted transplantation. After venous transplantation, HSCs travel all over the body and many are arrested in the lungs, liver, spleen and other organs. Few HSCs home to bone marrow, implant and differentiate into precursor cells, then reconstruct the hematopoietic and immune systems. How to help more HSCs to home back and engraft in the bone marrow? Our proposal named magnetism-induced cell target transplantation (Magic-TT) meets this requirement. The concept of Magic-TT is that:Donor cells are marked by magnetic beads and transplanted to recipients. Then a temporary magnetic field is applied around the bone marrow of recipients. Under the action of magnetic field, donor cells homed to the bone marrow rapidly and implanted in the niche. The advantages of Magic-TT may be as follows:(1) It can improve the homing rate, shorten the time of reconstructing hematopoietic and immune system and might raise survival rate. (2) Combining with intra-bone marrow transplantation, Magic-TT can significantly improve the homing rate after BMT, which will reduce the amount of transplanted cells and the incidence of GVHD.OBJECTIVE1. To establish the eGFP donor cell auto-transplantation murine model and observe the spatial distribution of HSCs in bone marrow after BMT.2. To establish the mRFP to eGFP allo-transplantation murine model and observe the spatial distribution of HSCs in bone marrow after BMT.3. To explore whether Magic-TT can improve the transplantation efficiency and shorten the hematopoietic reconstruction duration in eGFP donor cell auto-transplantation murine model.CONTENTSPart 1 To establish the eGFP donor cell auto-transplantation murine model and observe the spatial distribution of HSCs in the bone marrow after BMT[Methods] (1)20 C57BL/6 female recipient mice were randomly divided into experimental group and control group, each 10, which had bowel preparation for 3 days before transplantation. (2)5 C57BL/6 male eGFP transgenic donor mice were killed and bilateral femoral were removed to prepare bone marrow cells suspensions. (3) After 7Gy myeloablative irradiation, each mouse in experimental group was infused 0.1ml suspensions PBS with 5×106 bone marrow cells, each in control group was infused 0.1ml PBS through tail vein. (4)Then the general characters, survival and engraftment levels, etc of recipients were evaluated after transplantation. In the recovery process, semi-solid decalcification (SSD) was used to treat the femora from experimental mice to observe the distribution of cells, morphology and so on under confocal microscope directly or after frozen section was made.[Results] (1)The results showed that experimental group gained WBC recovery on (19.87±1.81) d and (92.67±6.65)% peripheral cells were RFP+(n=10). (2)The control group mice began to die 5 days after transplantation and all dead on the 11th day. The median survival time is 8 d and all of them died of myelosuppression. The WBC was less than 0.5×109/L when they died and the mortality was 100%.(3)uring SSD processing, the hard components were replaced gradually and donor cells’ location, morphology and fluorescence were retained in situ by confocal microscope.[Conclusions] We successfully established eGFP auto-transplantation murine model, together with SSD, we could visually observe GFP+cells and their morphology in the bone marrow of recipients. This model helps to explore the migration and distribution of donor cells after BMT. It provides a new way for thinking about the migration and distribution of donor cells, which will be helpful to study the HSCs niche.Part 2 To establish the mRFP to eGFP allotransplantation murine model and observe the spatial distribution of HSCs in the bone marrow after BMT[Methods] (1)20 C57BL/6 female eGFP transgenic 6recipient mice were randomly divided into experimental group and control group, each 10, which had bowel preparation for 3 days before transplantation. (2)5 C57BL/6 male mRFP transgenic donor mice were killed and bilateral femoral were removed to prepare bone marrow cells suspensions. (3) After 7.5Gy myeloablative irradiation, each mouse in experimental group was infused 0.2ml suspensions PBS with 5×106 bone marrow cells, each in control group was infused 0.2ml PBS through tail vein. (4)Then the general characters, survival and engraftment levels and occurrence of GVHD, etc of recipients were evaluated after transplantation. In the recovery process, SSD was used to treat the femora to observe the distribution of double fluorescent cells, morphology and interaction under confocal microscope directly or after frozen section was made.[Results](1)The results showed that experimental group gained WBC recovery on (20±3.07) d and (93.94±1.59) % peripheral cells were RFP+(n=10),4 of 10 developed GVHD within 1 month. (2)The control group mice began to die 5 days after transplantation and all dead on the 11th day. The median survival time is 8.5 d and all of them died of myelosuppression. The WBC was less than 0.5×10/L when they died and the mortality was 100%. (3)During SSD processing, mRFP+ cells could be seen mainly in the bone trabecula and surrounded by eGFP cells, their interactions could be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction.[Conclusions] We successfully established mRFP to eGFP allo-transplantation murine model, together with SSD, we could visually observe the distribution of donor cells and cellular interaction, which will provide the basis for clinical studies of the distribution of donor cells, homing and related research after transplantation.Part 3 To explore whether Magic-TT can improve the transplantation efficiency and shorten the hematopoietic reconstruction duration in eGFP donor cell auto-transplantation murine model[Methods] (1)40 C57BL/6 female eGFP recipient mice were randomly divided into 4 groups, the group A:Intravenous transplantation and beads; the group B: Intravenous transplantation, beads and small magnetic field; the group C:Intravenous transplantation, beads and middle magnetic field; the group D:Intravenous transplantation, beads and large magnetic field, each 10, and had bowel preparation before transplantation. (2)5 C57BL/6 male eGFP transgenic donor mice were killed and bilateral femoral were removed to prepare bone marrow cells suspensions. (3) Anti-Sca-1-FITC and Anti-FITC beads were added into the bone marrow suspensions to mark bone marrow cells as many as possible. (4) After 7Gy myeloablative irradiation, each group was infused 0.1ml suspensions liquid with 5×106 bone marrow cells through tail vein.(5)Four groups of mice were added different intensity of magnetic field after BMT, without magnetic field, small magnetic field, middle magnetic field and large magnetic field respectively, the magnetic fields were removed after 24h.(6)Then the general characters, engraftment levels, hematopoietic recovery and survival of recipients were evaluated after transplantation.[Results] All of mice lost weight and reduced activities after radiotherapy and recovered 1 week after. Parts of mice fall of their magnets and parts of mice had lower extremity swelling and even fractures due to the tight magnets.(1) Survival of four groups was as follows:Two mice died on 17th d after BMT and the rest lived more than 30d, survival rate of 80% in group A; all mice were alive in group B, survival rate of 100%; one mice died in 15th d after BMT and the rest lived more than 30d, survival rate of 90% in group C; one mice died in 10th d after BMT and the rest lived more than 30d, survival rate of 90% in group D. The survival rate of four groups had no statistical difference. (2)When four groups of mice had gotten hematopoietic recovery, the proportions of RFP+ cells in peripheral were(91.35±9.81)%、 (95.13±1.50)%、(94.23±0.86)% and (91.53±3.30)% respectively. (3)Take the routine blood parameters of four groups for comparison:WBC had no statistical difference (P=0.081>0.05);HGB had no statistical difference (P=0.328>0.05) and PLT was significantly different in four groups(P=0.007<0.05).[Conclusions] From this part of research we added different intensity of magnetic field in four groups of mice after BMT to explore the influence of magnetic field on the hematopoietic recovery. Our results showed that Magic-TT can help to shorten the recovery time of PLT after transplantation, which have great importance on improving hematopoietic recovery after transplantation, however, without significant effect on the recovery of HGB and WBC.SUMMARY AND PROSPECTThrough three parts of experiment, we successfully established the murine models of eGFP auto-transplantation and double-fluorescent allo-transplantation. We could clearly observe the distribution of double fluorescent cells, morphology and interaction with the help of SSD after transplantation. Our research is helpful to explore the distribution, migration, retention and other related researches, which is important to track HSCs homing and explore the interaction of donor and recipient cells after transplantation.The 3th part of experiments explored Magic-TT’s influence on the hematopoietic construction in eGFP auto-transplantation murine model. It showed that Magic-TT could shorten the recovery time of PLT but had no effect on the recovery of HGB and WBC. During the myelosuppression, we have better solutions for lower WBC and HGB count, but not to PLT decrease. Lower PLT always related with more motalities and morbidities, as well as poor clinical result, died of bleeding. Magic-TT could shorten the recovery time of PLT, reducing bleeding, improving survival rate, the further research will provide help for improving the recovery of PLT after transplantation.The following work will combine Magic-TT with intra-bone marrow transplantation. We predict that it can significantly improve the homing rate of HSCs after transplantation, reducing the number of transplantation cells, shortening the recovery time and might reducing the occurrence of GVHD. If so, it can largely improve the overall survival rate and the quality of life. |
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