| Part One Collecting, separating, identifying and cryopreservation of umbilical cordhematopoietic stem cellsAbstractObjective To separate hematopoietic stem cells, this will be identified and cryopreserved, and used for transplantation from umbilical cord blood.Methods:Separating nucleated cells (NC) and erythrocytes in umbilical cord blood by 6% hydroxyethyl starch (6%HES), counting NC by neubauer board. Flow cytometry to detect CD34 positive cells. Separated cells were preserved in cryoprotectant, which was consisted of DMSO, Bovine serum and HES, and in the-80℃circumstance. Trypan blue was used for counting survival rate of cells, before and after cryopreservation and recovery.Results:6%HES could collect 84.5% nucleated cells from umbilical cord blood, the rate of CD34 positive cells before and after separation was 1.3% and 1.2% respectively. After 4 weeks'cryopreservation, the survival rate of nucleated cells dropped to 92% from 100%.Conclusion:6%HES sedimentation is an effective method to separate nucleated cells and hematopoietic stem cells from umbilical cord blood. Cryoprotectant was composed of DMSO, Bovine serum and HES could preserve nucleated cells effectively in -80℃circumstance while survival rate of cells have no obvious decrease. Part TwoCultivating human hepatocellular carcinoma cell line MHCC97-H andestablishing implanted tumor animal model on BABL/c nude miceAbstractObjective:To establish implanted tumor animal models on nude mice by subcutaneous implantation after cultivating MHCC97-H cell line of human hepatocellular carcinoma in vitro.Methods:Hepatocellular carcinoma cells were cultivated in culture bottles and digested by Trypsin to passage generation then inoculated cells on 24-well plate for counting. Single cell suspension was prepared and adjusted to concentration of 2×106/0.2ml after acquiring sufficient number of cells. On the basis of volume ratio 1:1, mixed the single cell suspension and Matrivgel membrane matrix, used cells with concentration 1×106/0.2ml to implant on nude mice subcutaneously. Tumor latency and formation rate were calculated. Randomized 23 mice, which accepted tumor cells suspension implantation, into two groups:group A contained 12 mice, which would accept hematopoietic stem cells transplantation in follow-up experiment; group B, as controlled group, involved 12 mice.Results:Doubling time of MHCC97-H cell line was 35.12 hours. During two weeks, 23 of 24 nude mice formed tumor, the rate was 95.83%. All 12 mice in group A formed tumor, mean latency was 6.55±1.51 days; 11 mice in group B formed tumor, mean latency was 5.91±1.70 days. Difference of tumor formation latency between two groups had no statistical significance (P>0.05).Conclusion:MHCC97-H is a subset hepatoma cell line which has powerful activity and rapid metabolism. By mixture with Matrivgel membrane matrix, amount of cells which use to implant on single nude mice subcutaneously can be reduced while establishing implanted tumor animal models successfully. Part ThreeSuppressive effects of hematopoietic stem cells on hepatocellular implanted tumorAbstractObjective To explore effects of hematopoietic stem cells on implanted tumors of nude mice models.Methods:Nude mice in group A accepted hematopoietic stem cells transplantation every 7 days; group B, as controlled group, take no process. Every 24 hours after transplantations, measured and calculated tumor size, compare differences of tumor size between two groups.4 weeks later, executed all mice, removed all tumors then calculated sizes. Tail bloods of 6 mice were taken in each group to detect serum tumor markers DCP and AFP-L3 with ELISA kit, and then observed if there had any correlation between tumor marker and tumor size, and calculated differences between two groups. At the same time point, tail blood of remaining mice in group A were taken to detect differentiated CD4 positive and CD20 positive cells respectively used flow cytometry, judged whether had correlations between differentiated cells and tumor sizes.Results:Two weeks after first hematopoietic stem cells transplantation, mean tumor size of group A and group B was 387.75±25.77mm3 and 439.31±55.66mm3 respectively, P<0.05, differences of tumor sizes between two groups had significance; One week after transplantation, DCP level of group A and group B was 9.36±0.76ng/ml and 11.12±1.38ng/ml respectively, difference between groups was significant. The DCP level and tumor size were highly correlated (r=0.940, P<0.05), while difference of AFP-L3 between groups had no significance and no correlation with tumor size(r=0.254, P>0.05). The ratio of CD4positive between CD20 positive cells, which progressive increased, had moderate correlation with difference of tumor sizes between groups (r=0.642, r=0.707, P＜0.05)Conclusion:Umbilical cord blood hematopoietic stem cells have suppressive effect on the growth of implanted subcutaneously tumor on nude mice. Changes of tumor size and DCP level are highly correlated. The suppressive effect on the growth of tumors may be carried by the immune effector cells, CD4 positive and CD20 positive cells (T and B lymphocytes), which differentiated from hematopoietic stem cells after transplantation.
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