The Study Of The Effect Of Different Culture Modes On The Proliferation Of Hematopoietic Stem Cells By Mesenchymal Stem Cells And Its Mechanism

Objective:By studying the effect of mesenchymal stem cells(MSC)on the proliferation of hematopoietic stem cells(HSC)in vitro in different co-culture modes.Thus,the bone marrow microenvironment of HSC proliferation in vivo is simulated as true as possible.the mechanism was explored to find a better way to promote the proliferation of HSC in vitro.Methods:1.Separation and cultivation of MSC:The marrow cells of C57BL/6 mice were cultured in 10%FBS,1%-streptomycin,DMEM-F12 medium,half amount of liquid was changed in 48h,then every 2 days after the full amount of liquid exchange,inverted phase contrast microscope observation of cell morphological changes and growth.When the cell grows to 90%fusion,press 1:2.Continuous observation of MSC growth and morphological changes of P1,P2 and P3.2.Separation and identification of HSC of GFP mice:density gradient centrifugation to obtain single nucleus cell(MNC)in mouse bone marrow,MACS-CD117~+magnetic beads sorting HSC.The purity of CD117 positive cells was detected by flow cytometry(FACS).3.Study on the proliferation of hematopoietic stem cells by mesenchymal stem cells(MSCs):Set the control group:group HSC(inoculation of hematopoietic stem cells in 24 orifice plates)and group MSC(inoculation of mesenchymal stem cells in 24 orifice plates).The experimental group:Transwell co culture group(24 pore plates corresponding to Transwell specification,hematopoietic stem cells in the upper chamber,mesenchymal stem cells in the lower chamber),and 2D contact co culture group(24 orifice plates co inoculated with hematopoietic stem cells and mesenchymal stem cells).The growth of HSC,cell count and plot growth curve were observed in vitro for 7 days,and the Cell Counting Kit(cck-8)method was used to determine the expansion of HSC.The expression of SDF-1 and VFGF in the cell culture fluid of each group was also measured.Result:1.The isolation and culture of MSC of C57BL/6mice:Under the inverted microscope,the spindle shaped parietal cells began to appear on the 5 day of primary culture,approximately d12spindle cell fusion can be up to 90%-95%.After the transmission,the cell adherent wall grew faster,and the wall adhered to 24 h.It can grow up to80%and form a long fusiform dense arrangement between 6 and 7 days after the 3rd generation.The cell purity of mesenchymal stem cells was gradually increased.2.Isolation and identification of HSC in GFP mice:In the experiment,the marrow cells of each GFP mouse averaged about 5.5x 10~7 individual nuclear cells(MNC)after density gradient centrifugation,viable cells dyeing by trypan blue was 97%.After the separation of MNC MACS CD117 magnetic beads,about 2.6 x 10~5 CD117~+cells were obtained on average,and the rate of living cells was 98%.The content of CD117+cells in bone marrow MNC was about 0.47%.The fluorescence microscope shows that the HSC is round,and the green fluorescence can be excited and the size is uniform.The results of flow identification showed that the purity of CD117~+HSC after the separation of magnetic beads was 99.51%.3.Effect of mesenchymal stem cells on the proliferation of hematopoietic stem cells:(1)The effect of MSC on the proliferation of HSC was observed under the microscope:fluorescence microscopy showed that:the number of HSC cells in each group increased with the prolongation of co culture time,and the number of fluorescent cells in the 2D co culture group was significantly higher than that in the other groups.(2)The effect of MSC on the proliferation of HSC was detected by counting method:the count of cells was analyzed.On the first day of co culture,the number of HSC active cells in the 3 groups was compared with that at the time of inoculation(D0).There was no significant difference between the control group(the HSC group alone)and the inoculation time(P=0.151);the Transwell co culture group was compared with the inoculation,P=0.010;the 2D co culture group was compared with the inoculation;P=0.002.Coculture of first days,the number of HSC in the 3 groups,the control group compared with the Transwell group,the difference was not statistically significant(P=0.718);group 2D was higher than that of control group,P=0.000,and group 2D was higher than group Transwell,P=0.000.The number of HSC cells in 3groups was compared in 3 days.Transwell co-culture group was higher than the control group,P=0.004;The 2D coculture group was higher than the control group,P=0.002;The 2D coculture group was higher than the Transwell co-culture group,P=0.010.On the fifth day after co-culture,the numbers of HSC cells in three groups were compared,Transwell co-culture group was higher than the control group,P=0.039;2D co-culture group was higher than the control group,P=0.001;2D co-culture group was higher than Transwell co-culture group,P=0.000.On the seventh day of co culture,the number of HSC cells in the 3groups was higher than that in the control group,and the Transwell co culture group was higher than that in the control group,P=0.001 and 2D co culture group were higher than those in the control group,P=0.000and 2D co culture group were higher than those in Transwell co culture group,P=0.000.(3)CCK-8 assay was used to detect the proliferation of HSC:test OD value of HSC samples in 1,3,5 and 7 days,from the growth curve,it can be seen that the number of HSC in each group increases with the extension of incubation time,and the third day begins to enter the logarithmic growth period.The co-culture with MSC promotes the proliferation of HSC,and the 2D co-culture mode is more obvious than that of Transwell co-culture.On the first day,there was no statistically significant difference between the control group and the Transwell co-culture group(P=0.151);Comparison between the control group and the 2D co-culture group showed no statistical significance(P=0.054).The cell proliferation of the co-culture group was significantly higher than that in the control group,P<0.05.And the 2D coculture group was significantly higher than that of Transwell co-culture group,P<0.05.4.The possible mechanisms of mesenchymal stem cells to promote the proliferation of hematopoietic stem cells in different culture modes:(1)The content of SDF-1 in the culture medium was detected by double antibody sandwich ELISA method for seventh days.The results showed that:The 2D group was higher than that of HSC alone,P=0.000;Transwell group was higher than HSC alone,P=0.000;2D group was higher than MSC alone,P=0.000;Transwell group was higher than MSC alone,P=0.000;The 2D group was higher than the Transwell group,P=0.017.(2)In the 7th day,the VEGF content in the culture medium was detected by double-antibody sandwich ELISA,and the results showed that:The 2D group was higher than that of HSC alone,P=0.000;The Transwell group was higher than the HSC group,P=0.000;2D group was higher than MSC alone,P=0.000;Transwell group was higher than MSC alone,P=0.000;The 2D group was higher than the Transwell group,P=0.009.(3)The number of MSC in each group was detected by counting method,and the results shows:The number of MSC was increased with the increase of culture time.From the third day to the logarithmic growth phase,the numbers of MSC in 2D and Transwell co-culture group were higher than those in MSC alone group.The number of cells in 2D co-culture group was higher than that in Transwell co-culture group,The most obvious is at day 7.(4)The correlation analysis of HSC cell number,MSC cell number,SDF-1 content and VEGF content on the 7th day.The results showed that after 7 days of co culture,the contents of SDF-1 and VEGF in each group were positively correlated with the proliferation of HSC and MSC cells in each group,and the differences were statistically significant.Conclusion:1.Co-culture with MSC can significantly improve the proliferation ability of HSC,and the promotion of hematopoietic stem cell proliferation of mesenchymal stem cells is more obvious than non-contact co-culture under the mode of 2D contact co-culture.2.The mechanism by which 2D-contact co-culture promotes stronger hematopoietic stem cell proliferation may be as follows:(1)MSC and HSC secrete more angiogenic factor(VEGF)and chemokine(SDF-1)in 2D contact co-culture mode.(2)SDF-1 and VEGF have synergistic effects,which jointly promote the proliferation of HSC and MSC.(3)HSC promoted the proliferation of MSCs.The proliferation ability of MSCs was stronger than that of non-contact co-culture in contact co-culture mode.Proliferating MSCs further promoted the proliferation of HSC,forming a virtuous cycle and enhancing the proliferation ability of HSC.3.Compared to HSC alone and non-contact co-culture,the 2D contact co-culture mode mimics a bone marrow microenvironment that is closer to the natural state and therefore more conducive to the proliferation of hematopoietic stem cells in vitro.