| ObjectiveTo clarify the effect of CFSD(Compound Fritillaria Scutellaria Decoction)on the chemotherapy sensitivity through Wip1 pathway by the acute myeloid leukemia animal model.Methods1.Wip1 expression level in the pathogenesis of leukemia.We collected 3-5ml peripheral blood of healthy adults and acute myeloid leukemia patients to separate neutrophil and mononuclear cells,then the RNA were extracted to detect the Wipl and p53 gene expression level by Real-time PCR.Acute myeloid leukemia patients’ p53 gene mutation were detected.2.Construction of acute myeloid leukemia mice models.Four-five-week-old female SCID beige mice were transplanted by tail vein injection of different density HL60 or HL60/ADR cells after radiation of X-ray.The white blood cell count and the positive rate of promyelocytes in peripheral blood were dynamically monitored by detecting the cells that express CD33 using flow cytometry at the day before modeling and the 10th,18th and 28th day after injection.Bone marrow morphology was used to assess leukemia cell infiltration in bone marrow.Morphological examination and histopathological assay were employed to confirm promyelocytes infiltration in organs(liver,spleen and kidney).3.Treatment of leukemia mice models with CFSD combined with adriamycin.HL60 mice models were randomly divided into four groups:model group,CFSD group,adriamycin group and combination group,the model group was given the same amount of distilled water daily.We gavaged CFSD group CFSD once a day.Intraperitoneal injection of doxorubicin hydrochloride every other day for the adriamycin group.Combination group was intervened by CFSD and doxorubicin hydrochloride.for 14 days administration period.HL60/ADR mice models grouping and treatment methods are the same as above.Mice were observed survival status during the experiment,mice were sacrificed at the 15th day of administration or before dying,materials were taken for each index examination,including whole blood cell analysis by automatic blood cell analyzer.CD33 positive cell ratio by flow cytometer,weight of spleen.histopathological examination and Wipl and p53 expression by Real-time PCR.Results1.Wip1 expression of different stages of acute myeloid leukemia.Total 42 cases were collected to detect the expression of Wip1mRNA and wild type p53mRNA,including 10 cases in newly diagnosed/remission group,25 cases in relapse/refractory group,and 7 healthy adults were used as normal control group.The test results is as follows(normal distributions were shown as mean and standard deviation,for non-normal distributions were shown as median(range interquartile)):①Wip1mRNA:Wip1 gene expression compared with β-actin in neutrophil cells of normal control group,newly diagnosed/remission group and relapse/refractory group were 0.0021(0.0016,0.0116),0.0109(0.0050,0.0207),0.0092(0.0037,0.0278).Wip1 gene expression compared with β-actin in mononuclear cells of above-mentioned groups were 0.0024±0.0011,0.0080±0.0063,0.0042(0.0024,0.0100).Among them,Wip1 gene expression of relapse/refractory groups were raised significantly.②p53mRNA:P53 gene expression compared with β-actin in mononuclear cells of above-mentioned groups were 0.0056(0.0022,0.0063),0.0060(0.0041,0.0359),0.0018(0.0008,0.0045).P53 gene expression of relapse/refractory group expression was induced.③There were 8 acute myeloid leukemia patients’ peripheral blood had been detected,the p53 gene mutation rate was 25%,the mutational sites were in the exon 6 and 7.2.Acute myeloid leukemia mice model building complete situation.Four model groups had the abnormal behaviors of tremor retaredation piloerection.Acute myeloid leukemia progresses faster by injection higher density(1×107)HL60,HL60/ADR cells.Through comparing whole blood cells count,CD33 positive ratio of peripheral blood and histopathological examination,mice models were determined.Accompanied by experimental process,mice disease entered the final stage,were extremely wasted,and had a very poor state of survival.Bone marrow cells could not be blown out when the materials were taken and were not effectively produced.Infiltration of leukemia cell was more serious based on other results after injection of HL60 or HL60/ADR cells with 1 × 107 dose.3.Effect of CFSD on chemotherapy sensitivity of leukemia mice model through regulate Wip1.Each cell line mice models were divided into four groups to intervention.①Lifetime(day):lifetime of HL60 model,CFSD,adriamycin,combination groups were 17.83±6.88,20.00±6.23,22.00±4.43,23.00±2.00.Lifetime of HL60/ADR model,CFSD,adriamycin,combination groups were 20.83±5.00,21.33±4.55,23.17±2.04,24.00±0.00.CFSD extended the lifetime of mice models,and the survival rate of the combination group was the highest.②The weight of mice(g):the weight of HL60 model.CFSD,adriamycin,combination groups were 9.80±2.60,10.24±1.59.11.03±1.31,11.62±1.96.The.weight of HL60/ADR model,CFSD.adriamycin,combination groups were 10.94±0.92,11.67±2.03.13.52±1.65.1 5.56±0.87.Only HL60/ADR combination group have gained weight compared with the beginning(13.16±1.04g).the other three groups all lost weight.③Peripheral white blood cell count(×109/L):white blood cell count of HL60 model,CFSD,adriamycin,combination groups were 3.36±1.08,1.39±0.39,0.45±0.24,0.45±0.23,adriamycin and combination groups were declined significantly,P<0.05.White blood cell count of HL60/ADR model,CFSD,adriamycin,combination groups were 0.96±0.40,1.24±1.04,0.66±0.44,0.81±0.72,the difference was not statistically significant.④The CD33 positive ratio of peripheral blood(%):the CD33 positive ratio of HL60 model,CFSD.adriamycin,combination groups were 55.21 ±7.29,39.24±2.94,31.49±2.36,25.52±0.81.CFSD induced apoptosis of leukemic cells and increased chemotherapy sensitivity.The CD33 positive ratio of HL60/ADR model,CFSD,adriamycin,combination groups were 77.42±0.73,75.65±0.18,72.74±1.18,67.03±3.83.Experimental groups of HL60/ADR mice models were chemotherapy resistance,CFSD combined with adriamycin can reversed multidrug resistance.⑤Spleen weight(g):The spleen is the main site of proliferation and infiltration of extramedullary leukemia cells.The weight of the spleen can reflect the leukemia burden in vivo.The spleen weight of HL60 model,CFSD.adriamycin,combination groups were 0.1637±0.0359,0.1132±0.0075,0.0662±0.0152,0.0854±0.0008.The spleen weight of HL60/ADR model,CFSD,adriamycin,combination groups were 0.1768±0.0226,0.1765±0.0191,0.0828±0.0074,0.0968±0.0052,spleen weight of HL60/ADR experimental mice models were higher than HL60 mice models.CFSD combined with adriamycin induced infiltration range.⑥Pathological examination and HE staining of spleen:pathological examination and HE staining of HL60 model groups’ spleen biopsies demonstrated the normal organizational structure was completely destoryed.and a large number of promyelocyte in the spleen tissue.CFSD combined with adriamycin induced infiltration range significantly.HL60/ADR mice models’infiltration range were more serious than HL60 corresponding experimental groups.⑦Relative expression of Wip1mRNA:Wip1mRNA expression of HL60 model,CFSD,adriamycin.combination groups were 1.3733±0.4347,0.4525±0.2298,0.4700±0.3220,0.1825±0.1410.Wip1mRN A expression of HL60/ADR model,CFSD,adriamycin,combination groups were 0.8166±0.6886,0.7193±0.3412,0.1061±0.1074,0.1011 ±0.0976.Wip1mRNA expression was down-regulated with the disease condition controlled.CFSD combined with adriamycin induced Wip1 expression further.ConclusionRelapse resistance of leukemia is associated with high expression of Wip1.CFSD has the effect of inducing apoptosis of leukemia cells,which can enhance the chemosensitivity of acute myeloid leukemia mice models and partially reverse drug resistance. |
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