Objective:To discuss the anti-tumor effect of low dose of methotrexate(MTX)combined with sorafenib(SFN)on the human osteosarcoma(OS),and to clarify the possible mechanism.Methods:Four types of human OS cells(143B cells,HOS cells,U2OS cells,and MG63 cells)were cultured in vitro.Western blotting method was used to detect the expression levels of vascular endothelial growth factor(VEGF)and vascular endothelial growth factor receptor 2(VEGFR2)proteins in the above four kinds of cells.The human OS xenograft model was established in the nude mice,and 20 successfully modeled BALB/C nude mice were randomly divided into control group(given 2％dimethyl sulfoxide+98％corn oil),low dose of MTX group(given 2 mng·kg-1 MTX),SFN group(given 15 mng·kg-1 SFN),and combined drug group(given 2 mng·kg-1 MTX+15 mng·kg-1 SFN);there were 5 mice in each group.The tumor volumes of the mice in various groups were detected and tumor growth curves were plotted;HE staining was used to observe the morphology of tumor tissue of the mice in various groups;immunohistochemistry was used to detect the positive expression rates of VEGFR2,proliferation marker Ki-67,and hypoxia-inducible factor-1(HIF-1)proteins in tumor tissue of the mice in various groups.The human OS 143B cells were divided into 0,0.125,0.250,0.500,1.000,2.000,and 4.000 μmol·L-1 MTX groups(given 0,0.125,0.250,0.500,1.000,2.000,and 4.000 μmol·L-1 MTX,respectively).CCK-8 method was used to detect the proliferation rates of the 143B cells in various groups,and half inhibityory concentration(IC50)was calculated;the concentration of MTX that had no effect on 143B cell survival was selected as low dose of MTX.The human OS 143B cells were divided into control and low dose of MTX groups(given 0 and 0.250 μmol·L-1 MTX).ELISA method was used to detect the levels of VEGF in the 143B cells in various groups.Results:Compared with 143B cells,the expression levels of VEGF and VEGFR2 proteins in the HOS cells,U2OS cells,and MG63 cells were significantly increased(P＜0.001).In the xenograft model,compared with control group,the tumor volumes of the mice in SFN group,and combined drug group were decreased(P＜0.001);compared with low dose of MTX group and SFN group,the tumor volume of the mice in combined drug group was decreased(P＜0.01).The immunohistochemical results showed that compared with control group,the positive expression rates of Ki-67,VEGFR2,and HIF-1 proteins in tumor tissue of the mice in combined drug group were significantly decreased(P＜0.05).The CCK-8 results showed that there was no change in the proliferation of the 143B cells treated with 0.25 μmol·L-1 MTX.The ELISA results showed that compared with control group,the level of VEGF in the 143B cells in MTX group was signyicantly decreased(P＜0.05).Conclusion:Low dose of MTX enhances the anti-tumor effect of SFN on the human OS,which may be due to the inhibition of VEGF secretion by the OS cells,thereby enhancing the anti-tumor effect of SFN on the human OS.

Objective To explore the clinical significance of minimal residual disease (MRD) and WT1 in patients with acute myeloid leukemia (AML).Methods 349 bone marrow specimens of 42 patients with AML (except M3) before and after chemotherapy were collected.The expression level of MRD was determined by flow cytometry (FCM) and WT1 gene was detected by real time quantitative PCR (RQ-PCR).Results No correlation was observed between WT1 gene expression level of AML patients before chemotherapy with treatment response (x2 =0.166 3,P ＞ 0.5).34 AML patients achieved morphological complete remission (CR) after the first course of inductive chemotherapy.The recurrence rate was significantly higher in patients with high MRD level than that in those with low MRD level (63.6 ％ vs 21.7 ％) (x2 =5.729,P ＜ 0.025),and significantly higher in patients with high WT1 expression than that in those with low WT1 expression (87.5 ％ vs 23.1％) (x2 =10.749,P ＜ 0.005).WT1 expression level of patients with CR was significantly lower than that of patients without CR (t =4.669,P ＜ 0.001).The recurrence rate of patients with both low level of MRD and WT1 was significantly lower than those of either high level of MRD and WT1 (15.0 ％ vs 64.3 ％,P ＜ 0.05).The level of WT1 in patients with CR had positive correlation with the level of MRD (r =0.835,P ＜ 0.001).Conclusions MRD and WT1 have great importance in evaluating the therapeutic effects among patients with AML and increasing the positive rate of MRD.They also provide experimental basis for the individual and gradation treatment in the clinic.