The Effect And Mechanism Of Mouse Bone Marrow-Derived Mesenchymal Stem Cells On The Proliferation Of Leukemia Cells

Objective Allogeneic hematopoietic stem cell transplantation is the preferred treatment for many hematologic malignancies,which mostly depend on the intensive chemotherapy and graft versus leukemia effects.However,many leukemias will relapse in some patients due to the existence of leukemia stem cells.With the increasing of the radiotherapy and chemotherapy intensities,the patients'survival rates can't be boosted.Accordingly,the graft versus leukemia effects play an important role in hematopoietic stem cell transplantations.Bone marrow derived mesenchymal stem cells are known for their characteristics of being postnatal stem cells,having the capacity of self-renewal and differentiation into the mesenchymal lineage.Many experiments prove that they have immunosuppressive functions in vivo and in vitro,and they can alleviate GVHD.But the effects of mesenchymal stem cells on leukemia cells have not been known,which will play an important role in the clinical application of the transplantations of both MSC and hematopoietic stem cells.Based on such a status quo,our experiment will solve three problems:1.we will detect the effects of mMSC on the proliferation and biology of leukemia cells through the experiment ex vivo;2. Intravenously infusion of a small amount of A20 cells,to establish a minimal residual leukemia in allogeneic bone marrow transplantation model, we study infusion of allogeneic bone marrow-derived MSC on the A20 cells proliferation in allo-BMT model;3.To establishment of a BABL/c mouse subcutaneous A20 tumor model, we study intravenously infusion of or local injection of allogeneic bone marrow-derived MSC on the effect of proliferation of A20 subcutaneous tumor.Then we can provide some experimental basis for clinical hematopoietic stem cells and mesenchymal stem cells transplantation of treating malignant hematological diseases.Part one mMSC can inhibit the proliferation of A20 cells in allogeneic bone marrow modelMethods 1.Allogeneic BMT model was established as follows:lethally irradiated BABL/c recipients received allogeneic BM cells of C57BL/6 origin.The mice were divided into 5 groups according to the infusion cell types:①PBS group(n=10):PBS 0.6ml;②BM group(n=10):1×107 BM cells per mice;③MSC-BM group(n=10):1×107 BM cells+5×105 MSCs;④A20 group(n=17):1×107 BM cells+1×104 A20 cells;⑤MSC-A20 group(n=17):1×107 BM cells+1×104 A20 cells+5×105 MSCs.2.Compare the weight curve,survival rate,and tumor incidence,and analyze the T cells subset in peripheral blood at day 7 and day 14 after allogeneic BMT.3. Liver,spleen,small intestine and lung biopsy were stained with Hematoxylin-eosin,and compare the infiltration of A20 cells and the organ injuries.4.PKH26 labeled MSCs were transplanted into allo-BMT model to observe the distribution of mMSCs.Results 1.The mean survival time in PBS group was 15 days,while 90% mice in BM group and 100% mice in BM-MSC group were survived over 29 days.2.The tumor incidence of MSC-A20 group at day 28 after allo-BMT was 58.28%,which was lower than that of A20 group(94.12%)(P<0.05);3.Allogeneic MSCs infusion could increase the CD3+CD8+T cell subset in peripheral blood to (37.40±9.03)%,which was significantly higher than A20 group(4.90±1.74)% at day 7 after BMT(P<0.01);The percent of CD3+CD4+T cell was (50.85±11.85)%,and CD4+CD25+T cell was (1.88±0.43)% in MSC-A20 group,which were both lower than in A20 group(P<0.05).But MSCs infusion had no influence on the percentage of T cell subset at day 14 after allogeneic BMTAmMSCs were homing to the spleen,liver,lung and kidney,perhaps related with repairment the injury organs.Part two The effects of mMSC on the proliferation and biology of leukemia cells in vitroMethods 1.Reanimate the freezing and preserving mesenchymal stem cells of passage ten to fifteen,and the cell's phenotypes were demonstrated.MSCs were inactivated in MSC medium supplemented with mitomycin(25μg/ml) for 20 minutes at 37℃in advance, then were washed with PBS three times.They were inoculated in 96-well flat-bottomed microplates in different quantities(i.e.2×103,5×103,2×104,5×104/well).2×104 leukemia cells (A20,FBL3,P388)were cultured with or without a increasing number of MMC-treated MSCs in 96-well plates for 48h(n=6).Proliferation was assessed by CCK-8 assay during the final of 4h of culture.2.Time-dependent analysis:2×104 leukemia cells were cultured in 96-well plates for different time(24h,48h,72h) in the presence of 2x104 MMC-treated MSCs,and proliferations of leukemia cells were analyzed by CCK-8 assay.3.The proliferation rate of A20 cells was evaluated in different groups for 48h. Control group:A20 cells only;Group1:A20+MMC-MSCs(1:1);Group2:A20+Akt inhibitor(5μM);Group3:A20 +MSCs(1:1)+Akt inhibitor(5μM);Group4:A20+transwell-seperated MSCs.The results are shown as percentage of cell proliferation in comparison with control leukemia cells proliferation.4. 1×105 leukemia cells were cultured for 72h alone or in presence of MMC-treated MSCs at ratio 1:1 (MSCs:Leukemia cells).Leukemia cells were harvested and analyzed for the percent of early apoptosis and cell cycle distribution.5. 1×105A20 cells were cultured as a ratio of 1:1 physically separated from MMC-MSCs using a transwell-system for 72h,then A20 cells were harvested and analyzed for the percent of early apoptosis and cell cycle.6.After cultured with or without MMC-MSCs for 72h,A20 cells of the two groups were harvested and extracted for total RNA,then was analyzed for p21 and caspase 3 gene expression with Real time PCR.7. 1×105 A20 cells were cultured with or without MMC-treated MSCs in 6-well plates at ratio 1:1 for 24h,48h,or 72h,respectively (n=5).Then the supernatants were harvested, and cytokines'levels (IL-10,TGF-β,TNF-a and IFN-y) in cultural supernatants of different time were measured by ELISA arrays.8.Expression of A20 cells from cultures with or without MMC-treated MSCs for 48h using IL-10 staining on CD19-gated cells in flow cytometry.Results 1.The cells population consisted of spindle-and star-shaped cells.Flow-cytometric analysis showed that they were high positive for CD29,CD44,Scal-1,MHC-I, moderate positive for CD31,CD90.2 and negative for CD117,CD45,Flk-1,MHC-II.MSCs were of no tumorigenicity in BABL/c nude mice.2. MSCs exhibited a number-dependent growth inhibitory effects on different hematopoietc original leukemia cells.When the ratio of mMSC and leukemia cells was 1:0.4,the inhibition effect was most obvious.Compared with the control groups,the relative proliferation rate of A20,FBL3,and P388 cells in cocultural groups were respectively (78.32±9.16)%,(52.43±17.10)%, (58.47±5.68)% (P<0.01).3.The inhibitory effect of mMSC on different leukemia cells was time-dependent when seeded at a ratio 1:1(P<0.05);4.MSCs can not inhibit the activation of the Akt protein kinase;5.Apoptosis of leukemia cells was dramatically increased after coclutured with MMC-MSCs for 72h,the early apoptosis rates of A20,FBL3 and P388 cells were (4.80±0.78)%,(12.88±2.78)%,and (18.28±1.72)% respectively,while the rates of controls were (1.06±0.21)%,(4.64±0.61)% and (7.70±0.61)% respectively.6.When cocultured with MMC-MSCs for 72h,the DNA synthesis of leukemia cells were dramatically decreased.The perecent of A20,FBL3 and P388 cells in S phase were (45.77±1.56)%,(38.40±7.25)% and (51.03±2.10)% respectively,while the rates of control were (62.93±1.20)%,(63.38±14.47)% and (56.27±0.55)%(P<0.05);7.The inhibitoy effect on the proliferation of leukemia cells, the arrest effect on the leukemia cell cycle and the promoting early apoptosis effect require MSCs to make direct cell-cell contact;8.The results of Real-time PCR demonstrated that after coclutured for 72h,MSCs could upregulated mRNA expression of p21 and caspase 3 in leukemia cells significantly(P<0.05);9.ELISA assay showed that MSCs could decrease the level of IL-10 in supernantant and was time-dependent.The IL-10 concentrations of coculture supernatants of 24h,48h,and 72h were (236.99±15.91)pg/ml, (318.76±15.91) pg/ml, and(680.41±78.07)pg/ml,while the controls'were (301.47±30.31)pg/ml,(554.62±19.79) pg/ml,and (1892.97±176.22)pg/ml respectively; 10.After cocultured with mMSC for 48h,the intracellular IL-10 of A20 cells was significantly increased to (4.64±0.89)%,while the control's was (1.74±0.59)%(P<0.01).Part three The effect of mMSCs on the proliferation of A20 subcutanous tumor Methods 1.MSCs were either coimplanted as a mix(5x105MSC cells) with A20 cells(1×106) subcutaneously or injected alone via the tail vein.Groups were divided as follows:①(MSC+A20)/SC:MSCs and A20 cells were coimplanted subcutaneously.②MSC(IV)+A20(SC):MSCs were injected intravenously and A20 cells were implanted subcutaneously.③A20 alone:A20 cells were implanted subcutaneously without MSCs.Recipient mices were BABL/c mice or BALB/c nude mice.2.The tumor incidence,tumor mean volumes,tumor histology,immunohistochemitry of PCNA and CD31 of tumors and T cell subset of day 7 in peripheral blood were detected after implantation.Results 1.Coimplantion with MSCs and A20 cells could promote the proliferation of A20 cells in BALB/c mice.The mean volume of (MSC+A20)/SC in BALB/c mice at day 24 after implantation was (1511.43±467.85)mm3,while that in A20 alone group was (615.36±201.22)mm3(P<0.05);Tumor incidence reached 100% at day 13 after MSCs and A20 cells were coimplanted subcutaneously,while that in A20 alone group was only 83%;The T cell subset at day 7 in peripheral blood were of no significant difference(P>0.05);Immunohistochemitry analysis showed that PCNA and CD31 expression in (MSC+A20)/SC was higher than A20 alone group;2.Coimplantion with MSCs and A20 cells could promote the proliferation of A20 cells in BALB/c nude mice.The mean volume of (MSC+A20)/SC in BALB/c mice at day 20 after implantation was (1993.34±1009.89)mm3, while that in A20 alone group was (604.88±235.92)mm3 (P<0.05);Tumor incidence reached 100% at day 16 after MSCs and A20 cells were coimplanted subcutaneously,while that in A20 alone group was only 40%; Immunohistochemitry analysis showed that PCNA and CD31 expression in (MSC+A20)/SC was higher than A20 alone group;3.There were no significant difference of mean tumor volumes and tumor incidence when MSCs were injected intravenously while A20 cells were implanted subcutaneously both in BABL/c mice and in BALB/c nude mice;Allogeneic MSCs had no effect on the T cell subset at day 7 after implantation when they were injected intravenously.Conclusions 1.When MSCs were injected via vein,they could decrease the tumor incidence of A20 tumor in allogeneic BMT model through increasing the percent of CD3+CD8+T cells and decreasing the percent of CD3+CD4+T and CD4+CD25+T cells;2.MSCs could be homing to the spleen,liver,lung and kidney,perhaps related with repairing the injury organs.3. Mouse bone marrow-derived MSCs could inhibit the proliferation of different leukemia cells and was concentration-dependent and time-dependent;4. Mouse bone marrow-derived MSCs can promote the early apoptosis of different leukemia cells and arrest the cell cycle of leukemia cells;5.The inhibitoy effect on the proliferation of leukemia cells, the arrest effect on the leukemia cell cycle and the promoting early apoptosis effect require MSCs to make direct cell-cell contact;6. MSCs could inhibit the level of IL-10 in A20 culture supernantant and was time-dependent; 7.MSCs could increase the tumor incidence and the mean tumor volumes when they were coimplanted with A20 cells subcutaneously both in BALB/c mice and in BALB/c nude mice,which were not irrelevant with T cell immunity;There was no effect on the proliferation of A20 subcutous tumors when MSCs were injected intravenously.