The Study Based On NOD/SCID Mice To Investigate The Telomere Protein Of Realgar On THP-1 Leukemia Cells

Objective: To observe the effect on a single drug Realgar,explored the effects induced by POT1,TRF1,TRF2 of its regulatory role of telomere dynamics for the drug targeted in acute myeloid leukemia(AML),which seek to the supports for experiments.In addition,providing in a hope of a advantage of the clinical efficacy of leukemia and new ideas and methods.Methods: 1.In vitro:(1)Cultured the human acute myeloid leukemia THP-1.(2)Cellular viability was measure by CCK-8 assay, and the cells were incubated in the absence or presence of increasing concentration of realgar for different time,which determined the IC50.(3)Apoptosis and cell cycle distribution were monitored by FCM.(4)The protein expression levels of each group of POT1,TRF1,TRF2 were assessed by western-blot analysis.(5)The levels of POT1,TRF1,TRF2 mRNA were tested by realtime fluorescent quantitative PCR analysis.2.In vivo:(1)Establishment and evaluation of human acute myeloid Leukemia THP-1 Model in NOD / SCID Mice,and mice were treated of realgar.(2)We observed the survival status,then monitoring the changes of peripheral blood before and after treatment with daily i.g. of realgar,in addition we evaluated the dying mouse organization by microscopy,the application of G-banding method karyotype analysis of the bone marrow of mice in each group.(3)At the end of the experiment,the mice were killed by institutionally approved method after 3 weeks.The apoptosis and the cell cycle progression of spleen cells were examined using FCM.And thechanges of POT1,TRF1,TRF2 protein expression were detected using spleen.Results: 1.In vitro:(1)CCK-8 assay showed that THP-1 cell growth and decreased cell viability in a dose- and time- dependent manner, IC50 was 0.023?g/mL.Therefore,in our study,we choose with a medium concentration respectively was 0.015?g/mL(lower than IC50) at 48 h.(2)The study demonstrated that after treatment for 48 h, the apoptosis and the cell cycle progression of these THP-1 cell were compared with control group,obviously.(3)As shown by western blot analysis,as depicted showed that compared with controls,realgar group elevated POT1,TRF1 protein level and degraded TRF2 protein level(P<0.01),there were significant differences.(4)We evaluated the level of each gene mRNA expression by real-time fluorescent quantitative PCR analysis,respectively, and found that the level of POT1,TRF1,TRF2 mRNA were concordant with the protein levels.Overall,our results implied that after drugs treatments,the expression of the protein at the levels of gene transcription.2.In vivo:Transplantation was always successful,and results showed that the three groups treatments increased the average survival time(P<0.01).After drugs treatments,immunohistochemistry showed they were concordant with the vitro.Conclusion: These findings indicate that the study on single drug realgar.foetida in vitro and in vivo have certaining degrees of anti-cancer activity,which as a target of cancer treatment for experimental and theoretical basis.