Basic And Clinical Study On The Effect Of Recombinant Human Thrombopoietin On 32D Cells And Mesenchymal Stem Cells

Effects of Recombinant Human Thrombopoietin on 32D Cells and Mesenchymal Stem Cells from Mice in VitroObjectiveTo investigate the effect and possible mechanism of recombinant human thrombopoietin(rhTPO)on mouse 32D cells and rat bone marrow mesenchymal stem cells treated with serum from patients with aplastic anemia(AA)in vitro to elucidate the mechanism of rhthrombopoietin in the treatment of aplastic anemia.Methods(1)CCK8 method and trypan blue counting experiment were used to verify the effects of different concentrations of rhTPO(50u/ml,100u/ml,150u/ml and 200u/ml)on the proliferation of 32D cells stably transfected with rhTPO receptor(32D-MPL).The optimal concentration of rhTPO to promote the proliferation of 32D cells was obtained.(2)32D cells were divided into four groups:?blank control group(32D cells+normal FBS serum);?AA group(32D cells+normal FBS serum+aplastic serum);?rhTPO control group(32D cells+normal FBS serum+rhTPO);? rhTPO group(32D cells+normal FBS serum+aplastic serum+rhTPO);rat bone marrow mesenchymal stem cells(BMSC)were divided into three groups:?normal BMSC;?AA group(normal BMSC+AA serum);?rhTPO group(normal BMSC+AA serum+rhTPO).CCK8 method and trypan blue staining method were used to detect the proliferation ability and growth curve of cells under different culture conditions;(3)32D cells and BMSC cells were labeled with Annexin V-FITC,and the ratio of Annexin V+/PI-was detected by flow cytometry to understand the apoptotic status of each group.(4)Flow cytometry was used to detect the cell cycle of BMSC in logarithmic growth rats under different cell culture conditions.(5)Real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect the expression of anti-apoptotic genes(Survivin?BCL-2)and pro-apoptosis-related gene(BAX)and gene Bcl-xl,c-myc.(6)Western blotting was used to detect the expression of following gene:STAT3?STAT5?anti-apoptotic genes(Survivin?BCL-2)?pro-apoptosis-related genes(BAX)?Bcl-xl and c-myc.The same method was used to detect the protein expression of c-MPL in different groups of rat BMSCs.Results(1)After treatment with AA serum,the apoptotic rate of 32D cells was increased and the proliferation of 32D cells was significantly inhibited.rhTPO could reduce the apoptotic rate and promote the proliferation of 32D cells,but rhTPO could not restore the cell proliferation of AA serum group to the normal level.(2)The phosphorylation level of STAT3 protein in 32D cells of aplastic anemia serum group was higher than that in normal serum group,and the phosphorylation level of STAT5 protein was lower than that in normal serum group.After rhTPO treatment,the phosphorylation level of STAT3 protein in aplastic anemia serum group was down-regulated and the phosphorylation level of STAT5 protein was up-regulated,All these have statistical significance.(3)The levels of survivivin and BCL-2 antiapoptotic genes in 32D cells in AA serum group were significantly down-regulated,and the levels of their expression were significantly up-regulated after rhTPO treatment(P<0.05);The levels of BAX gene and protein in 32D cells in AA serum group were significantly up-regulated,and the levels of their expression were significantly down-regulated after rhTPO treatment(P<0.01).Compared with the normal serum group,the expression of BAX protein in 32D cells of the normal serum group after rhTPO treatment was significantly decreased(P<0.05),while the level of mRNA of BAX did not change significantly.(4)Rat BMSC cells cultured with aplastic anemia serum were stimulated by rhTPO,which promoted the proliferation of BMSC and the expression of c-MPL and inhibited the apoptosis of BMSC cells.Above these have statistical significance.Conclusion(1)After 32D cells were treated with aplastic anemia,the apoptotic rate was significantly increased.The addition of rhTPO could reverse the apoptotic rate of 32D cells and promote the proliferation of 32D cells,but could not restore the cells of aplastic anemia cells to normal serogroups.(2)The expression of anti-apoptosis gene(Survivin,BCL-2)mRNA and protein was significantly higher in T32 cells treated with rhTPO than in the aplastic anemia group.After rhTPO treatment,the expression of apoptosis-producing gene(BAX)mRNA and protein in the 32D cells of the aplastic anemia group was significantly down-regulated.Both of them were statistically significant.(3)rhTPO can promote the proliferation and c-MPL expression of rat bone marrow mesenchymal stem cells cultured with AA serum,and inhibit cell apoptosis.Impact of recombinant human thrombopoietin combined with hormonal and ciclosporin therapy on short-term response and security in patients with newly diagnosed immunorelated hemocytopeniaObjectiveTo evaluate the impact and security of recombinant human thrombopoietin(rhTPO)on short term response of hormonal and ciclosporin in patients with newly diagnosed immunorelated hemocytopenia.MethodsThe clinical data of 20 cases of adult IRH patients,who treated with hormonal and ciclosporin combined with rhTPO,was retrospectively compared with 14 patients by the hormonal and ciclosporin as control group during the same period.The hematological response,platelet recovery and safety evaluation were compared between the two groups.ResultsAfter 2 weeks of treatment,the platelet increased in the rhTPO group compared with the control group(p<0.05).After 6 weeks of treatment,the hemoglobin of rhTPO group was higher than that of control group(p<0.05).The time of platelet transfusion in group rhTPO was shorter than that of control group.After 4 weeks,the rhTPO group and the control group were free of platelet transfusion,but there was no statistical difference.There was no significant difference in hemoglobin infusion after rhTPO treatment 2 weeks,4 weeks,and 6 weeks.However there was significant difference in hemoglobin infusion at 8 weeks after treatment.There was no statistically significant difference in hematological response between the rhTPO group and the control group at 3 and 6 months.ConclusionRhTPO increased the early hematologic response rate of IRH,accelerated platelet recovery,and had high safety.