Comparison Of Influence Of MSC On HL-60 Under Circumstances Of Leukemia And Non-Leukemia

MSC is another marrow adult stem cell after the discovery of HBSC, which plays a big role in the haematogenesis and immunological regulation. Meanwhile it exerts some influence on the normal HSC and leukemia cells.Objectives1. Study on influence of non-leukemia MSC on proliferation and differentiation of HL-60 and its working mechanism.2. Comparison of some biology characteristics in MSC in AML, MSC after relief and MSC in non-leukemia.3. Comparison of influence of the above-mentioned MSCs on HL-60 cells.Methods (I)1. Isolation of mononuclear cell with the method of density gradient centrifugation, and isolate MSC by adherent culture. Co-culture of HL-60 and MSC after planting HL-60 on the film of MSC.2. Illustration of changes of quantity of proliferation of HL-60 cells cultured solely and those co-cultured with MSC by time by direct counting method.3. FCM to detect cell life cycle and apoptosis of HL-60 cells cultured soley and those co-cultured with MSC.4. FCM to detect expression of CD11b and CD14 of HL-60 cultured solely and co-cultured with MSC.5. FCM to detect expression of Survivin and Bcl-2 of HL-60 cultured solely and co-cultured with MSC.6. Taking HL-60 cultured solely as control group, calculation of differentiation rate after observing HL-60 co-cultured with MSC after Wright-Giemsa staining.7. Ink-swallowing to detect functions of HL-60 cells co-cultured with MSC.Methods (II)1. Observation of cell morphous in MSC in AML, MSC after relief and MSC in non-leukemia after situ Wright-Giemsa staining.2. Calculation of number of CFU-F after situ Wright-Giemsa staining. 3. Calculation time between planting mononuclear cell to cell-fusion of MSC.4. Illustration of changes of quantity of proliferation of MSC in the above-mentioned different groups by time by direct counting method.5. FCM to detect cell life cycle and immunophenotype of MSC in the above-mentioned different groups and then calculation of DI.6. Calculation of number of HL-60 cells co-culturd with the above-mentioned MSCs of different groups by direct counting method.7. FCM to detect expression and distribution of cell life cycle of CD11b and Survivin of HL-60 co-cultured with the above-mentioned MSCs of different groups, and then comparison of their cell life cycle.8. Calculation of differentiation rate after observing HL-60 co-cultured with the above-mentioned MSCs of different groups after Wright-Giemsa staining and then comparison of cell morphous of all these HL-60 cells.Statistical treatmentRecord consecutive variance of different groups as mean±standard error after statistical analysis helped with STATA software. Adopt t-test to compare the HL-60 co-cultured with non-leukemia MSC and those HL-60 cells cultured solely. Adopt variance analysis to compare HL-60 cells co-cultured with MSC of different groups. Adopt linear regression to assess the dependability of the entire index. It is taken as different in sense of statisitics when P<0.05.Result (I)1. Proliferation of HL-60 cell restrained significantly after co-cultured with MSC, and the restrain is outstanding after 5-7 days. ( 3rd day P=0.07, 5th day P=0.01 and 7th day P<0.01)2. Significant increase of percentage of HL-60 cells (for control group 47.0±9.0%, experiment group 70.0±16.0%, P=0.003 )in phase of G0/G1 after co-cultivation by checking cell life cycle, and appearance of fading-wave crest left to phase of G0/G1.3. Decrease of expression of Survivin and Bcl-2 of HL-60 cells after co-cultured with MSC (Bcl-2: control group 63.0±9.1%, experiment group 50.0±14.1%, P=0.045; Survivin: control group 94.0±9.3%, experiment group 7.0±11.8%, P=0.006).4. Significant increase of expression of CD11b and CD14 of HL-60 cells after co-cultivation with MSC (negative for control group and experiment group 25.0±3.2%).5. Negative expression of Survivin in a group of CD11b positive cells after being marked with monoclonal antibody of Survivin and CD11b with the percentage of 16.0±4.9%. 6. 18.0±9.0% of cells differentiating to myelocyte or metagranulocyte analysized morphologically.7. The cells of HL-60 can not phagocytose ink after co-cultured with MSC.Result (II)1. Number of CFU-F after 14 days: for leukemia group, it is 20.0±2.1/107MNC; for non-leukemia group, it is 25.0±3.0/107MNC; and for CR leukemia group, it is 23.0±4.1/107MNC. For leukemia group, it is the least. And the number is quite different for the three groups, P=0.01. For non-leukemia and CR leukemia groups, there is not much difference and P>0.05.2. Confluence time for MSC in different groups: for leukemia group, it is 26.0±3.0 days; for non-leukemia group, it is 21.0±1.0 days, and for CR leukemia group, it is 22.0±2.0 days. The time for leukemia group is the longest, and there is significant difference for these three groups (P<0.01).3. No difference for proliferation of MSC in different groups after screening and going down to posterity. On 3rd, 5th and 7th days, compare cell counting for these three groups, P>0.05 and no difference.4. No difference for cell morphous of different groups under ligh microscope.5. High expression of CD 105 and CD 106 of MSC in different groups, and negative expression for CD45; compare the expression of CD 105 and CD 106 in different groups, P = 0.37 and 0.50, no difference.Percentage of cells in phase of G0/G1 in different groups: for leukemia group, it is 89.9±4.0%; for non-leukemia group, it is 90.2±3.0% and for CR leukemia group, it is 91.0±3.0%, P=0.79, no difference.DI for MSC in different groups is between 0.9-1.1.6. Cell couting of HL-60 after co-cultured with MSC in different groups: no difference after comparing on 5th and 7th day, P=0.77 and 0.96.7. No difference for cell life cycle distribution, Survivin expression and CD11b expression of HL-60 after co-cultured with MSC in different groups, P=0.89, 0.08 and 0.19.8. Cell morphous of HL-60 cells turn to maturity after co-cultured with MSC in different groups and the differentiating rate is 18.0±3, 117.0±1.3 and 19.0±2.0, P=0.23, no difference. Conclusion (I)1. Bone marrow MSC restraining HL-60 cell proliferation: (a.) by preventing cells taking part in periodical circulation or delaying speed of periodical circulation (b.) urging cells exiting proliferation cycle by inducing differentiation of tumor cells, which kind of restraining is realized by cell factors and direct contact.2. Bone marrow MSC can cause apoptosis of HL-60 cells, which is accompanied by differentiation, and highly related to differentiation. Cell apoptosis is accompanied by decrease of Bcl-2 and Survivin. It is estimated that there is maybe some relationship between the apoptosis of these two proteins.3. Enhancing of bone marrow MSC on HL-60 cells may be related to blockage of cell life cycle, but it could not finish terminal differentiation.4. Restraining of bone marrow MSC on HL-60 proliferation and enhancing differentiation may play a role in exploring new treatments for leukemia. But the blockage of cell cycle prompts us that it may be sheltered under chemotherapy.Conclusion (II)1. There are no essential changes for MSC in leukemia circumstances.2. MSC in leukemia circumstances can also restrain proliferation of leukemia cells and enhance differentiation.3. MSC doesn't play a big role in the onset and development of leukemia.4. Self-transfusion of MSC is safe in leukemia circumstances.