| Objective(s):OCI-AML3(NPM1m A)cells with low expression of LncRNA XLOC＿109948 were constructed by lentivirus transfection,and then the model mice were injected into tail vein to study the role of XLOC＿109948 in NPM1 mutant acute myeloid leukemia(acute myeloid leukemia,AML)in vivo.Methods:1.NOD/SCID mouse leukemia model was established by injecting OCI-AML3 cells into tail vein.(1)Fifteen female NOD/SCID mice were randomly divided into three groups:control group,low dose group and high dose group.Cyclophosphamide(CTX)was injected intraperitoneally for two days before cell inoculation,and each mouse was injected with CTX with 200ul concentration of 10mg/ml every day to reduce the immunity of mice.Then OCI-AML3 cells were injected through tail vein.The injection doses of low dose group and high dose group were 5×10~6 and 1×10~7,respectively,and the control group was injected with the same volume of normal saline.(2)the mice were observed and recorded at the frequency of3 to 4 times a week,and the changes of body mass of mice in each group were compared.(3)the peripheral blood smears and nucleated cells of mice in each group were collected on the 10th,20th and 30th day after inoculation.(4)the mice in each group were killed on the 30th day,and the bone marrow,spleen and liver were taken for histopathological examination to comprehensively judge whether the mice were modeled or not.2.To verify the role of LncRNAXLOC＿109948 in NPM1 mutation AML in vivo.The main results were as follows:(1)the expression of XLOC＿109948 was interfered by lentivirus transfected OCI-AML3 cell line,and then the OCI-AML3 cell line with stable and low expression of XLOC＿109948 was screened by puromycin,and its interference efficiency was verified by RT-q PCR.(2)27 female NOD/SCID were randomly divided into three groups:control group,no-load group and interference group.Pretreatment before cell inoculation,as mentioned earlier,the no-load group and the interference group were injected with the corresponding cells,the number of cells was 1×10~7per mouse,and the control group was injected with the same volume of normal saline.(3)at 2 and 4 weeks after inoculation,the peripheral blood of mice was taken for flow anti-human CD45+detection.(4)at the 4th week after inoculation,3 mice in each group were randomly killed,and the expression levels of target genes in peripheral blood and bone marrow were detected by flow cytometry.(5)the survival time of the remaining mice was observed and recorded.results:1.On the 15th day and 21st day after cell inoculation,the mice in the two model groups began to show signs of lethargy,cuddling,circling,wrinkling and yellowing of fur,and even hair loss.Among them,the mice in the high-dose group developed paralysis of both lower limbs on the 28th day after inoculation of the cells,while the mice in the low-dose group and the control group did not.The weight of the mice in the two model groups gradually decreased from the 14th day after cell inoculation,and the weight of the mice in the two model groups was significantly lower than that of the normal control group on the 28th day,the difference was statistically significant(p<0.05).2.The number of white blood cells in the mice in the two model groups gradually increased after inoculation of cells,and the number of white blood cells in the mice in the two model groups was significantly higher than that in the control group on the 10th and 20th day after inoculation of cells,and the difference was statistically significant(p<0.05).After the 20th day,the number of white blood cells in the two model groups decreased gradually,and there was no significant difference compared with the control group(p>0.05).After cell inoculation,leukemia cells could be seen in the peripheral blood and bone marrow of mice in both model groups.3.On the 30th day after cell inoculation,the spleens of the mice in the two model groups were larger than those of the control group,especially the spleens of the mice in the high-dose group were significantly larger than those of the control group,and the difference was statistically significant(p<0.05).Leukemic cell infiltration was found in the spleen and liver of mice in the two model groups in tissue sections,and the expression of anti-human CD45 antigen could be detected in both spleen and liver by immunohistochemistry.4.The expression of XLOC＿109948 was interfered with by transfecting OCI-AML3 cells with lentivirus.RT-q PCR verified that the expression level of XLOC＿109948 in the interference group was significantly lower than that in the empty group,and the difference was statistically significant(p<0.01).5.The expression rate of anti-human CD45 in the peripheral blood of mice in the blank group and the interference group was compared by flow cytometry at the 2nd week and the 4th week after cell inoculation.The ratios of anti-human CD45~+cells in the peripheral blood of mice in the empty group were 53.39%±2.45%and 33.23%±1.44%,respectively.The percentages of anti-human CD45+cells in the peripheral blood of mice in the interference group were:61.16%±2.65%,41.97%±1.33%.No matter in the 2nd week or the 4th week,the proportion of anti-human CD45~+cells in the peripheral blood of mice in the interference group was significantly more than that in the empty group,and the difference was statistically significant(p<0.05).6.Whether in peripheral blood or bone marrow,the expression of XLOC＿109948 in the interference group was higher than that in the empty group,and the difference was statistically significant(p<0.05).7.The apoptosis rate of bone marrow cells and the expression rate of anti-human CD45 in splenic lymph were compared between the empty group and the interference group by flow cytometry.The apoptosis rate of bone marrow cells in the interference group was significantly higher than that in the empty group,and the difference was statistically significant(p<0.05).The anti-human CD45~+ratio in the spleens of mice in the empty vehicle group was 56.78%±1.28%,and the anti-human CD45~+ratio in the spleens of mice in the interference group was 53.46±2.03%,with no significant difference between the two groups(p>0.05).8.The survival time of the mice in the empty vehicle group and the interference group was compared.The survival time of the mice in the interference group was lower than that in the empty group.Conclusion(s):1.After cyclophosphamide pretreatment,the AML mouse model can be successfully established by injecting 5×10~6 and 1×10~7OCI-AML3 through the tail vein.Compared with the low-dose group,the high-dose group had a faster onset and a relatively shorter molding cycle.2.Downregulation of XLOC＿109948 can delay the progression of NPM1 mutant AML and prolong the survival of mice. |
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