A Study On The Reactivation Of γ;-globin Gene In K562 Cells By Hydroxyurea

OBJECTIVE: To treat K562 cells cultured in vitro with different concentrations of hydroxyurea(HU)drugs,observe the cells and detect the inhibition rate and apoptosis of the cells,and then detect the expression of γ-globin gene(abbreviated as HBG gene).To understand the cell inhibition and apoptosis of K562 cells expressing HUactivated gamma globin gene,and explores a new method for the treatment of severeβ-thalassemia by HU induced fetal hemoglobin(Hb F),provide a reliable basis for studying the safety and efficacy of HU for the treatment of new pathways for beta thalassemia.METHODS: Firstly,the effect of HU on the inhibition rate of K562 cells was detected by CCK-8.The cells were cultured at different concentrations of HU for 24 h,48h,and 72 h,according to the CCK-8 reagent instructions.Mix and incubate for a certain period of time and measure the light absorption value at 450 nm with an enzyme-linked immunosorbent detector,which can indirectly reflect the number of viable cells.Then,Flow cytometry was used to detect the apoptosis rate of K562 cells treated with different concentrations of HU for 24 h,48h and 72 h by using Annexin VFITC/PI double staining apoptosis assay kit.Next,Real-time Quantitative polymerase chain reaction(q PCR)was used to detect the expression of HBG genes,the total RNA was extracted by trizol method and c DNA was synthesized by reverse transcription.According to Genbank published HBG gene(Genbank: NM000184)and the internal reference gene GAPDH(Genbank: BC025925)m RNA full sequence,using Primer 6.0 designed primers,and then real-time fluorescent quantitative PCR to detect the corresponding m RNA expression.At last,it performs relative m RNA expression levels calculations.RESULTS: It was found from different concentrations of HU that when the HU concentration was greater than 100 um/L and less than 1000 um/L for 72 h,the cells were observed to be significantly inhibited.Therefore,the concentration gradients are set to 0nm/L,50 nm/L,100 nm/L,200 nm/L,300 nm/L,50 um/L,100 um/L,200um/L,and 24 h,48h,72 h.K562 cells were cultured for subsequent experiments in three time periods.Three time periods were determined by CCK8 kit,flow cytometry,real-time fluorescent quantitative PCR,and cells with different concentrations of drugs were found to be 0nm/L,50nm/L,100nm/L,200nm/L,300 nm.There was no significant difference in the inhibition rate,the apoptosis rate and HBG m RNA expression between the five concentrations of the three concentrations(P > 0.05),and 50um/L,100um/L,and 200um/L is only compared at 72 h.In the former control group and the concentrations of 50nm/L,100nm/L,200nm/L,300nm/L,there was a significant difference(P < 0.05).The higher concentration,the higher inhibition rate,and the higher expression level of cellular m RNA with the increase of concentration.Conclusion: Our experiments have shown that HU can promote the expression of HBG gene in K562 cells,and the optimal concentration of HU in K562 cells is100um/L and the optimal action time is 72 h,which provide basic research and judgment for the progress of subsequent projects.