The Effect Of Thalidomide On The Expression Of ?-Globin Gene And Related Regulatory Mechanism In Normal Human Erythroid Cells Cultured In Vitro

Objective:To investigate the effect of thalidomide on the expression of ?-globin gene related regulatory mechanism in human erythroid cells cultured in vitro,to further understand the relevant effects of thalidomide on the erythroid ?-globin gene.METHODS: Collect 10 mL of normal human bone marrow fluid mobilized by recombinant human granulocyte colony-stimulating factor(rhG-CSF).To obtain mononuclear cells using lymphocyte separation solution according to density gradient centrifugation,CD34 + hematopoietic stem progenitor cells were sort by using CD34 MicroBead Kit.Hematopoietic stem progenitor cells were inoculated into the established two-stage in vitro liquid erythroid culture system in the laboratory to promote the differentiation of CD34+ hematopoietic stem progenitor cells into mature red blood cells.The state of the cultured cells was observed by an inverted phase contrast microscope.Cell morphology was observed under a microscope using Giemsa staining.The differentiation ratio of CD235 a and CD71,which are specific markers of red blood cell differentiation,was detected periodically by flow cytometry during induction of differentiation to ensure smooth progress of in vitro culture of erythroid cells.The final concentration of thalidomide were 50 ?mol/L,100 ?mol/L,200 ?mol/,300 ?mol/L respectively as the experimental group.Thalidomide at a concentration of 0 ?mol/L was used as a negative control group,and hydroxyurea at a concentration of 100 ?mol/L was used as a positive control group.The erythroid cells were treated with different drug concentrations for 24 h,48h,72 h,and 96 h,respectively.The survival rate of erythroid cells under different concentrations and different time conditions was detected by CCK-8 kit;Real time-PCR was used to detect the expression level of ?-globin mRNA in erythroid cells.The optimal concentration and time of drug on erythroid cells were screened by the above two steps.According to the optimal concentration and time of the drug,real-time PCR was used to further detect the expression of ?-globin gene and related transcription factors,and Western-Bloting was used to detect the production of ?-globin.Results: 1.Culture and differentiation of erythroid cells in vitro.In the process of inducing normal human erythroid cell differentiation in vitro,the CD34+ hematopoietic stem progenitor cells obtained by CD34 MicroBead Kit were flow-detected and the cell purity was over 98%.After inoculation in the erythroid culture system,more orange colonies with erythroid differentiation characteristics were observed under the inverted microscope as the culture time was prolonged.Using Geimsa staining,we confirmed from the perspective of cell morphology that CD34+ hematopoietic stem progenitor cells gradually differentiated from pronormoblast to prorubricyte,rubricyte,and orthochromatic normoblast.With the prolongation of differentiation time,the expression of erythroid specific marker CD235 a and CD71 increased to 80%.2.Effect of CCK-8 on the survival rate of thalidomide and hydroxyurea.There was an interaction between the concentration and time of each drug on the survival of erythroid cells(F=10.725,P=0.000),and the effects of time and concentration were(F=92.092,P=0.000)and(F=309.847,P= 0.000)respectively The survival rate of erythroid cells decreased with the extension of cells induced by thalidomide.Under the induction of hydroxyurea,the survival rate of the cells decreased more significantly.3.Screening of the Concentration and Time of Thalidomide and Hydroxyurea.By screening the optimal time and concentration of thalidomide and hydroxyurea,the results showed that the expression level of ?-globin gene was affected by both time and concentration.The expression of ?-globin gene was significantly different at different time(F=15.087,P=0.000)and concentration(F=7.035,P=0.003),and there was an interaction effect between them(F=5.906,P=0.000).The expression of ?-globin gene in erythrocyte cells induced by thalidomide and hydroxyurea increased significantly 72 hours later(F=63.76,P=0.000)compared with the negative control group.The P values of thalidomide in 100 ?mol/L,200 ?mol/L,300 ?mol/L were(P=0.001),(P=0.002),(P=0.004)respectively,and hydroxyurea in 100 ?mol/L were(P=0.000).The difference was statistically significant.4.The result of Thalidomide and hydroxyurea induced the expression of erythroid cells ?-globin gene and protein after 72 hours.After induction of erythroid cells for 72 h,compared with negative control group(F=73.670,P=0.000),the concentrations of thalidomide 100 ?mol/L,200 ?mol/L,and 300 ?mol/L,the expression of ?-globin mRNA were increased.P value is(P = 0.000,P = 0.000,P = 0.005),the difference is statistically significant,the concentration of 100 ?mol /L hydroxyurea increased significantly(P = 0.000),the difference was statistically significant.In the detection of ?-globin production by western-bloting,compared with negative control group(F=8.559,P=0.001),200 ?mol/L of thalidomide and hydroxyurea expression of ?-globin was significantly increased.The P values of 200 ?mol/L thalidomide and hydroxyurea were(P=0.004)and(P=0.001),respectively,and the difference was statistically significant.5.Effects of thalidomide and hydroxyurea on transcription factors BCL11 A,KLF1,KLF2,GATA1,SOX6,TAL1 genes.When the concentration of thalidomide at 200 ?mol/L and hydroxyurea induced erythroid cells for 72 hours,respectively,the transcription factor BCL11 A decreased(F=6.278,P=0.034).Compared with the negative control group,the transcription factor KLF1 showed different degrees of decline(F=5.278,P=0.048),and the thalidomide group(P=0.043),the difference was statistically significant.Compared with the negative control group,the transcription factor KLF2 was down-regulated(F=9.767,P=0.013),thalidomide group(P=0.550),hydroxyurea group(P=0.013),the difference was statistically significant.Compared with the negative control group,the transcription factors SOX6(F=0.446,P=0.66),thalidomide group(P=0.954),and hydroxyurea group(P=0.430)were not statistically significant.Compared with the negative control group,the transcription factors TAL1(F=0.26,P=0.779),thalidomide group(P=0.541),and hydroxyurea group(P=0.962)were not statistically significant.Compared with the negative control group,the transcription factors GATA1(F=0.325,P=0.375),thalidomide group(P=0.409),and hydroxyurea group(P=0.801)were not statistically significant.Conclusion:1.This study demonstrates that thalidomide increases the expression of ?-globin gene and ?-globin production in erythroid cells cultured in vitro.The effect of thalidomide is similar to that hydroxyurea induced ?-globin gene elevation.2.Down-regulation of transcription factors BCL11 A and KLF1 may be one of the regulatory mechanisms of thalidomide-induced erythroid cell ?-globin gene expression.