| Objective:Focuing on inhibiting the proliferation of leukemia cells with the compound Fritillaria Scutellaria Decoction(CFSD),taking CFSD and chemotherapy as interventiones,taking human acute myeloid leukemia cell kasumi-1 transplanted mice as the research object,through in vivo experimental system,exploring the CSFD promoting the recovery of p53 transcription regulation function by reducing wip1 expression playing a role in inhibiting the proliferation of leukemia cells and partially reversing drug resistance.Method:1.Establishment of NOD/SCID mouse models via subcutaneous transplantation of kasumi-1 cells.Twenty NOD/SCID mice aged 5-6 weeks were injected with 5×106(0.1ml)kasumi-1 cells in the left axilla,then observe the solid masses and the mass size in site of injection,If the volume reaches 50mm3,the model is determined to be successful.2.Grouping and administrating the tumor-bearing NOD/SCID mice:Twenty tumor-bearing mice were randomly divided into 4 groups,namely model group,chemotherapy group,traditional Chinese medicine group and combination group,with 5 mice in each group.The model group:0.3ml normal saline,gavage,d1-d14;The chemotherapy group:DNR 3mg/kg,tail vein injection,d1-d3 and Ara-C 40 mg/kg,tail vein injection,d1-d7;The traditional Chinese medicine group:CFSD 2.4g/kg,gavage,d1-d14;The combination group:DNR 3mg/kg,tail vein injection,d1-d3 and Ara-C 40 mg/kg,tail vein injection,d1-d7and CFSD 2g/kg,gavage,d1-d14.3.Observation indicators:The general status such as mental state,fur gloss and capture sensitivity,etc;Measuring the mice body weight and tumor volume every 3 days,and calculating the T/C%at the end of the experiment;Measuring the weight of the subcutaneous transplanted tumor and calculating the RI%;Taking blood for routine blood test and smear for peripheral blood cell morphology before modeling and after the end of the test;Making bone marrow cell and transplanted tumor single cell smear to test bone marrow and tumor cell morphology;Making pathological sections of organs and transplanted tumors,then observing structure and cell morphology of each tissue after HE staining;Detecting the relative expressions of wipl and p53 mRNA of tumor tissues by rt-PCR and investigating the relationship beteen the relative expressions of wipl and p53 mRNA and the anti-leukaemia effects of CFSD.Result:1.It has high tumor formation rate and rapid growth when injecting kasumi-1 cells in left axilla of NOD/SCID mice.Twenty NOD/SCID mice aged 5-6 weeks were subcutaneously injected with 5×106 kasumi-1 cells per mouse,and actual masses were appeared at the inoculation site on the 4th day of inoculation,and the volume of subcutaneous masses reached 50mm3 in all mice on the 7th day,the success rate of model building is 100%.2.CFSD can improves the survival status of mice,but it has adverse effects of the weight gain of mice.①With the increase in the size of the transplanted tumor and the advancement of treatment,the mice of each group have successively reduced activity,lethargy,lethargy and slow response to capture.The gloss of the fur of each group of mice decreased,model group>traditional Chinese medicine group>combination group>chemotherapy group.②The weight of the mice were measured every 3 days,the average initial weight was(22.98±0.30)g.In the experiment,the weight of the mice in each group were increased,and the growth rate was model group>chemotherapy group>traditional Chinese medicine group>combination group.On the 25th day of tumor-bearing,mice in each group weighed as follows:model group(27.95±1.21)g,chemotherapy group(27.06±1.90)g,traditional Chinese medicine group(24.67±0.73)g,and combination group(24.33±1.16)g.3.CFSD inhibited the proliferation of subcutaneous transplanted tumors in NOD/SCID mice bearing kasumi-1 cells.①Before the experiment,the average volume of subcutaneous transplanted tumors in 20 NOD/SCID mice was 197.79mm3.During the treatment,the transplanted tumor volume of each group of mice gradually increased,and the growth rate was model group>Chinese medicine group>chemotherapy group>combined group.The tumor volumes in the group were:1956.10±443.92 mm3 in the model group,606.23±172.54 mm3 in the chemotherapy group,1178.24±471.40 mm3in the Chinese medicine group,and 2434.51±125.10 mm3 in the combination group.Compared with the model group,each treatment group has a statistically significant difference(p<0.05).Compared with the chemotherapy group and the combination group,the traditional Chinese medicine group has a statistically significant difference(p<0.05).The chemotherapy group is compared with the combination group.The difference is meaningless(p>0.05).The relative tumor proliferation rate T/C%of each treatment group was 22.47%in the chemotherapy group,55.94%in the traditional Chinese medicine group,and 13.85%in the combination group.②After the treatment,the mice were sacrificed by dislodgement of the neck,and the tumor mass was removed and weighed.The weight of the transplanted tumor in each group was:model group 2.23±0.22g,chemotherapy group 0.77±0.14g,traditional Chinese medicine group 1.31±0.32g,and combination group 0.32±0.35g.The therapeutic effect of the traditional Chinese medicine group was not different from that of the chemotherapy group(p>0.05),and there was a significant difference compared with the combination group(p<0.01).The tumor weight of the chemotherapy group was significantly different from the combination group(p<0.05).Tumor inhibition rates in each treatment group were RI 65.47%in the chemotherapy group,41.26%in the traditional Chinese medicine group,and 85.65%in the combination group.4.CFSD can reduce tumor microenvironmental inflammatory factors such as neutrophils(NE),platelets(PLT)and average platelet volume(MPV),and improve the malignant proliferation of tumor cells.Pre-tumor NOD/SCID mice white blood cell count(2.44±0.88)x109/L,hemoglobin content(125.20±4.60)g/L,average platelet count(214.80±40.34)×109/L,average platelet volume(6.48±0.24)fL.① On the 25th day of tumor-bearing(after treatment),the white blood cells of the tumor-bearing mice in each group increased.The peripheral blood white blood cell counts of the model group,chemotherapy group,traditional Chinese medicine group and combination group were:(5.59±1.26)×109/L,(3.50±0.63)×109/L,(5.75±2.19)×109/L,(4.82±1.76)×109/L.Except for the chemotherapy group,they were all significant compared with those before tumor-bearing(p<0.05).②The peripheral blood hemoglobin content of mice in the model group,chemotherapy group,traditional Chinese medicine group and combination group were:(138.20±7.05)g/L,(14.80±5.68)g/L,(123.60±11.63)g/L,(117.80)±11.76)g/L.The model group was higher than before modeling,and the rest of the groups were lower than before modeling.Compared with the model group,the difference was statistically significant(p<0.05).③The peripheral blood platelet counts of mice in the model group,chemotherapy group,traditional Chinese medicine group and combination group were:(1009.8±101.42)×109/L,(396.00±232.27)×109/L,(772.00±144.30)×109/L,(186.00±43.60)×109/L.Except that the number of platelets in the combination group decreased compared with that before tumor-bearing,the platelet counts of the other three groups increased,and the platelet number of the model group increased the most,which was statistically different from the treatment groups(p<0.05).Compared with the chemotherapy group and the combination group,the traditional Chinese medicine group was statistically different(p<0.05).④The average peripheral blood platelet volume of mice in the model group,chemotherapy group,traditional Chinese medicine group and combination group were:(5.96±0.15)fL,(6.86±0.26)fL,(6.25±0.24)fL,(6.84±0.13)fL,Compared with the pre-tumor bearing,the chemotherapy group and the combination group were significantly higher(p<0.05),the model group was significantly lower(p<0.05),and the traditional Chinese medicine group was the same as before the tumor.5.CFSD destroys the integrity of the transplanted tumor cell membrane and reduces the infiltration of leukemia cells.① Observe the peripheral blood smears of NOD/SCID mice in each group under a microscope.In the peripheral blood of the mice in each group,there are different numbers of polychromatic red blood cells,a small number of mid-and late-myeloblasts,occasional blasts,and rare kasumi-1 Leukemia cells.②The histopathology of the transplanted tumor showed that the tumor cells in each group gathered in clusters,and there was focal necrosis and hemorrhage.The transplanted tumor cells in each treatment group were larger,with unclear boundaries,large nuclei,clear nuclear membrane,obvious nucleoli,located in the center,loose chromatin,and abnormal classification.The model group has smaller tumor cells,pyknosis nucleus and larger tissue necrosis area.The morphology of the spleen,liver and other tissues was basically normal,and there was no obvious leukemia cell infiltration.6.CFSD regulates wip1/p53 network-mediated inhibition of proliferation of subcutaneous transplanted tumor cells in NOD/SCID mice.The relative expression of Wip1mRNA in the transplanted tumors of mice in each group was:chemotherapy group>combination group>model group>Chinese medicine group.The expression of Wip1mRNA in the chemotherapy group was 4.34 times that of the model group,the difference was statistically significant,P<0.05.The expression of Wip1mRNA in the traditional Chinese medicine group was 0.17 times that of the model group,and the Wip1mRNA expression of the combination group was 1.7 times that of the model group,both of which were not statistically significant,P>0.05.The expression level of p53mRNA in the transplanted tumors of mice in each group relative to the model group was:Chinese medicine group>combination group>model group>chemotherapy group.The expression of p53mRNA in the traditional Chinese medicine group was 4.9 times that of the model group.Compared with the other three groups,the difference was statistically significant,P<0.01.Conclusion:CFSD regulates down-regulation of wipl and up-regulation of p53 to inhibit the proliferation of subcutaneous transplanted tumor cells in NOD/SCID mice. |
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