1. The Therapeutic Effect Of IL3-LDM Targeting Fusion Protein On Human Acute Myeloid Leukemia NOD/SCID Mouse Model 2. Construction Of CD26 Lentiviral Expression Vector And Establishment Of Stable Transfected Cell Line

Objective:The aim of this study is to establish a steady murine model for human T-cell acute myelocytic leukemia by intravenous injection of the isolated human peripheral blood mononuclear cells into the NOD/SCID mice,and evaluate the model from the perspective of morphology,immunology etc.At the same time,the model was used to verify the therapeutic effect of IL3-LDM targeting fusion protein on leukemia mice.Methods:1.Human peripheral blood mononuclear cells were isolated and cultured,the immunophenotype were detected by flow cytometry,and the characteristics of AML stem cells were identified.2.Establishment of NOD/SCID mouse model of acute myeloid leukemia:NOD/SCID mice were randomly divided into experimental group and control group.Two groups of mice were irradiated by whole body X-ray irradiation of 300cGay.After 6-12 hours,1x107 human peripheral blood mononuclear cells with the highest rate of positive CD 123 were injected through the tail vein injection.The mice in the control group were injected with equal volume of PBS.3.Identification of acute myeloid leukemia NOD/SCID mouse model:observe the general situation of mice;The percentage of CD45 + cells in peripheral blood of mice was detected by flow cytometry;Mice were sacrificed 60 days after transplantation.Histopathological examination was used to detect the characteristics of leukemia in mice liver,spleen and lung.4.Use the established model to verify the therapeutic effect of IL3-LDM targeting fusion protein on leukemia mice,a number of treatment groups were set up and evaluated for efficacy.Results:1.Isolated and cultured human peripheral blood mononuclear cells,labeled with CD34-PE,CD38-FITC,CD123-PerCPcy5.5 antibodies,flow cytometry showed CD34 +CD3 8-cell population,and CD 123 + cells accounting for the proportion of AML stem cells are more than 90%.2.The model of NOD/SCID mice was successfully established in the model group.The mice in the model group showed typical leukemia and the mice in the control group had no tumor cells.CD45 + cells were detected in the tail blood of mice in the model group,while the mice in the control group had no.The liver,spleen and lung of NOD/SCID mice were infiltrated by tumor cells.The cells were agglomerated,surrounded by organs or infiltrating the organs,damaged by infiltration and tissue structure.The mice in the control group The corresponding tissue did not find significant tumor cell infiltration.3.The vivo experiments show that IL3-LDM targeting fusion protein has a targeted and cytotoxic activity against leukemia stem cells in leukemia mice and has a certain therapeutic effect on leukemia.ConcLusion:Acute myeloid leukemia murine model has been successfully established in the NOD/SCID mice by intravenous transplantation with the isolated human peripheral blood mononuclear cells by pretreatment of NOD/SCID mice whole body irradiation.It was a more convenient method for establishment of leukemia murine models.And it could provide a base for researching the mechanism of AML.The vivo experiments have further confirmed that IL3-LDM targeting fusion protein has a targeted and cytotoxic activity against leukemia stem cells.IL3-LDM fusion protein also provides a new idea for clinical treatment of AML.Background:CD26 is a kind of protease with many biological functions and plays an important role in the physiological and pathological processes such as inflammatory reaction,tissue repair,cell migration,tumor invasion,immunoregulation,signal transduction and cell apoptosis.It is clinically associated with a variety of tumor diseases,which can be used as a predictor of tumor or biomarkers.It has great significance to perform an in-depth study of CD26,which help to reveal the pathogenesis of related tumor diseases,to promote clinical diagnosis and identification analysis,to carry out molecular targeted therapy and determine the efficacy and prognosis,etc.CD26 is also expected to become a potential target for cancer disease treatment.Objective:To construct a human CD26 Lentivirus expression vector,transfect NIH/3T3 cells,and establish a NIH/3T3 cell line capable of stably expressing CD26.Methods:The pEZ-Lv105-CD26 pLasmid and the pLasmid which has pCDH1-MCS1-EF1-copGFP Lentiviral vector were extracted for futher experiments.After doubLe digestion with XBaI and BamHI,the CD26 target fragment recovered from the gel was cloned into vector pCDHl-MCS1-EF1-copGFP by T4 Ligase,and the pCDH-CD26 pLasmid was constructed.The competent products were transformed into DH5a.The positive clones were screened by colony PCR and identified by restriction enzyme digestion.Sequencing analysis dentemines the finaL results.293T cells were transfected with a three-plasmid Lentivirus system,Lentivirus was produced and the virus titer obtained was tested.NIH/3T3 cells were transfected with the obtain CD26 Lentivirus and the positive cells expressing CD26 were sorted by FACS AriaⅢ(BD)flow cytometer,further identified by FACS.The NIH/3T3 cell line capable of stably expressing CD26 was constructed.ResuLts:The pCDH-CD26 Lentiviral expression vector was successfully constructed and the Lentivirus was packaged and the titer of the virus suspension was 3×108 TU/mL.NIH/3T3 cells were successfuLLy infected with the virus.The positive rate of NIH/3T3 cells expressing CD26 was more than 70%after flow cytometry analysis and sorting.ConcLusion:Human CD26 Lentiviral expression vector and the stable transfected NIH/3T3 cell were successfully constructed,which laid a good experimental foundation for the further study of CD26 function.