Effect Of Overexpression Of CD40L Gene On Bone Marrow Cell Apoptosis In Nude Mouse M5 Leukemia Model

Objective:The CD40L overexpressed THP-1 leukemia cell line was constructed by lentivirus transfection,and the M5 leukemia model in nude mice was constructed by tail vein injection.The effect of CD40L overexpression on apoptosis of bone marrow cells in nude mice was studied.Methods:1.Establishment and identification of M5 leukemia model in nude mice:(1)18 female NOD/SCID mice aged 3-4 weeks were randomly divided into three groups:control,model A and model B(6 mice per group).Each mouse was given intraperitoneal injection of cyclophosphamide 2 mg/(kg·d)for 2 consecutive days before inoculation,and cells were seeded within 24 hours after pretreatment.The model group was inoculated with THP-1 cell suspension in logarithmic growth phase1×10~7/head(group A)and 5×10~6/head(group B)in the tail vein,and mice in the control group were injected with the same amount of saline in the tail vein;(2)Observe the general situation,detect the blood routine and peripheral blood leukocyte classification before pretreatment,7,14,21 and 28 days after cell inoculation and at the time of execution;(3)Before dying,the specimens were taken and examined by histopathology.2.The effect of overexpression of CD40L gene on the apoptosis of bone marrow cells in the model of M5 leukemia in nude mice:(1)Lentiviral transfection was used to construct an over-expressing CD40L-THP-1 cell line.Puromycin was used to screen stable transfected cell lines.Flow cytometry was used to determine the purity after screening.Q-PCR was used to verify whether CD40L was overexpressed;(2)Forty NOD/SCID mice were randomly divided into 5 groups,8mice in each group,that is,blank control group(group A),THP-1 group(group B),idling group(group C),and overexpression CD40L group(Group D)and THP-1+DNR group(Group E).The pre-modeling treatment methods are the same as before.The control group mice were injected with 0.2ml saline through the tail vein.Phase corresponding cell suspension 1×10~7/head;(3)Samples were sacrificed before dying,flow cytometry was used to detect the proportion of human leukemia cells and bone marrow apoptosis in blood and bone marrow,and WB was used to detect PI3k/Akt pathway protein expression in spleen.Results:(1)The model group began to stand on the 7th and 10th days after inoculation of the cells,and there were vertical hair,atrophy,and less movement.Compared with the control group,the body weight of the model groups A and B decreased significantly after 21 days of inoculation(P<0.01),the number of white blood cells in the model group increased significantly after 28 days of modeling,and the difference was statistically significant(P<0.01).Among them,the group A inoculated with 1×10~7 per head was the most significant.Pathological tissue sections showed that diffuse leukemia infiltration was seen in the spleen of the model group.Immunohistochemical results showed that the leukemia cells had a positive anti-human CD13 result,which confirmed that the model was successfully established.(2)GFP green fluorescence was expressed almost at the same efficiency in the cells of idling group and over expressing CD40L genomic cells.Lentivirus was successfully transfected into THP-1 cells.The purity of GFP green fluorescence was more than 95%after puromycin screening.The results of Q-PCR showed that the expression of CD40L gene in CD40L-THP-1 overexpression group was significantly higher than that in THP-1 and idling group(P<0.05).(3)The bone marrow apoptotic rate of the control group was significantly lower than the other four groups(P<0.05);the bone marrow apoptotic rate of the mice in the THP-1+DNR group and the over-expressing CD40L-THP-1 group was significantly higher than that in the THP-1 group(P<0.05);there was no significant difference in the bone marrow apoptosis rate between the THP-1+DNR group and the over-expressed CD40L-THP-1 group(P>0.05);there was no bone marrow apoptosis rate between the THP-1 group and the idling group.Significant difference(P>0.05).Conclusion:(1)After the NOD/SCID mice were pretreated by intraperitoneal injection of CTX,each mouse was injected with THP-1 cells(1×10~7 or 5×10~6)via tail vein,which could successfully construct the animal model of acute myeloid leukemia,which was in line with the biological characteristics of acute myeloid leukemia,and the tumor formation was faster at high concentration.(2)Overexpression of CD40L gene can promote mouse bone marrow cell apoptosis.