| Objective:1.The biological characteristics of CD34+CD38-KG1a leukemia stem cells(LSCs),which are KG1a stem cells sorted from KG1a cell line,were studied.2.To study the effect of CFSD on the activity of KG la stem cells.To study the effect of CFSD on phosphoenzyme gene deleted on chromosome 10 homologous to tensin(PTEN),phosphatidylinositol 3-kinase(PI3K)and rapamycin target gene(mTOR)expression in KGla cells.To explore the mechanism of CDSD affecting the activity of KGla stem cells by regulating the expression of PTEN,PI3K and mTOR.3.To study the effect of CFSD on the expression of PTEN and PI3K in peripheral blood neutrophils of patients with acute myeloid leukemia(AML)Method:1.Flow cytometry was used to sort CD34+CD38-KG1a stem cells from KG1a cells and compare the ratio of CD34+CD38-cells before and after stem cell sorting,identifing the purity of cells after sorting.Flow cytometry for detection of KG1a stem cell cycle.The ability of cell colony formation after KD1a cell line and KG1a stem cell culture for 14 days was observed and compared,and the colony formation rate was calculated.2.Effect of CFSD on the activity of CD34+CD38-KG1a cells:?The effect of different concentrations of CFSD lyophilized powder on the proliferation of CD34+CD38-KG1a cells was detected by CCK-8 method.?CCK-8 method was used to detect the inhibition of cell growth after blank group,DNR group,CFSD group,CFSD+DNR group effect on CD34+CD38-KG-la cells for 24h,48h and 72h.? The cell cycle distribution of KGla stem cells treated with KGla stem cells was analyzed by flow cytometry with PI single staining.?The apoptosis of KGla stem cells was detected by flow cytometry with Annexin V-FITC/PI double staining.The apoptosis rate and mortality were calculated after 24,48 and 72 hours.3.The expressions of PTEN,PI3K and mTOR mRNA in KGlas tem cells were detected by Real Time PCR for 48h4.Twenty-one patients with AML were randomly divided into the control group(10 patients treated with chemotherapy alone)and the treatment group(11 patients treated with CFSDcombined with chemotherapy)to compare the efficacy of the control group and the treatment group.The neutrophils were extracted from human neutrophil isolation solution The expressions of PTEN and PI3K mRNA in neutrophils were compared between the control group and the treatment group by Real Time PCR.Results1.Before the flow cytometry,the proportion of CD34+CD38-cells accounted for(38.83±3.57)%in KG1a cell line,and the proportion of CD34+CD38-cells accounted for(96.33±2.80)%after sorting.CD34+CD38-The purity of the cells was over 95%;the KG1a stem cells were sorted,(55.72±4.33)%were in the G0 phase of the cell cycle;the KG1a stem cell clone formation ability was strong,and the clone formation rate was(56.20±7.74)%,which was higher than that of the KGla cell line.The colony formation rate was(38.62±5.31)%.Sorted KG1a stem cells conform to the biological characteristics of LSCs and can be used in subsequent experiments.2.Effect of CFSD on the activity of CD34+CD38-KG1a cells:?The effect of different concentrations of CFSD on the proliferation of CD34+CD38-KGla cells:12.5,25,50,100ug/ml CFSD can significantly inhibit the proliferation of CD34+CD38-KG1a cells,and the IC50 value is greater than 25 ug/ml.(g)The effect of each group on the proliferation of CD34+CD38-KGla cells:24h in each group,compared with the blank group,the inhibition of KGla stem cell proliferation was not significant in each drug group,and the difference was not statistically significant(P>0.05).48h and72h in each group,compared with the blank group,the inhibition of KG1a stem cell proliferation was enhanced by time,and the difference was statistically significant(P<0.05).Compared with the three groups,CFSD+DNR group had the most significant inhibitory effect on KG1a stem cell proliferation,followed by DNR group.The effect of CFSD group was not obvious,the difference was statistically significant(P<0.05).?The effect of each group on the cell cycle of CD34+CD38-KGla:Compared with the blank group and DNR group,the proportion of G0/G1 phase in CFSD group,CFSD+DNR was significantly decreased,the proportion of cells G2/M phase,S phase increased,and the difference was statistically significant(P<0.05).?After 24,48,and 72 hours of treatment,the apoptotic rate and mortality of KG1a cells in each drug group were increased compared with the blank group,which was correlated with time(P<0.05).In comparison,the apoptosis rate and mortality of CFSD+DNR group were the highest,followed by CFSDgroup and DNR group,the difference was statistically significant(P<0.05).3.The expressions of PTEN,PI3K and mTOR mRNA in KG 1a stem cells were significantly higher than those in the blank group.The expressions of PTEN,PI3K and mTOR mRNA were significantly different(P<0.05).The expression of PTEN mRNA was significantly up-regulated and the expression of PI3K and mTOR mRNA was significantly down-regulated in CFSD group.The difference was statistically significant(P<0.05).4.There was no significant difference in the ratio of CR,NR and total efficiency between the treatment group and the control group(P>0.05).The control group OR was slightly lower than the treatment group.Before treatment,the expressions of PTEN and PI3K mRNA in the control group and the treatment group were not significantly different(P>0.05).After treatment,the expression of PTEN mRNA in the treatment group was significantly higher than that in the control group,and the expression of PI3K mRNA was significantly lower than that in the control group.Statistically significant(P<0.05).Conclusion1.The leukemia stem cells sorted by flow cytometry are of high purity,and the sorted cells conform to the biological characteristics of leukemia stem cells.2.CFSD can promote the entry of KG1a stem cells into the cell cycle,enhance the proliferation inhibition and induce apoptosis of KGla stem cells by DNR.The mechanism may up-regulate PTEN mRNA and down-regulate PI3K mRNA and mTOR mRNA expression.3.CFSD combined with chemotherapy can up-regulate the expression of PTEN mRNA in peripheral blood neutrophils and down-regulate PI3K mRNA in AML patients. |
PDF Full Text Request