| Objective:Establishment the second human colon tumor SCID/Beige nude mice animal model,provide a reliable animal model for the study of the recurrence and metastasis of colon carcinoma,to research the inhibitory effect of specific DC vaccine derived from umbilical cord blood on second time tumor growth.Methods:Peripheral blood from healthy adult and umbilical cord blood from full-term normal delivery women were obtained,the mononucleocytes were collected by density gradient centrifugation,the DCs from different sources were harvested by attachment culturing,and the cell morphology and CD80,CD83,CD86 and HLA-DR expression at different culturing days of d1,d3,d5,and d7 were observed respectively.Cancer-specific DC vaccines were prepared by loading the tumor lysates of human SW-1116 colon cancer cell line to different source DCs.Healthy male SCID/Beige nude mice were randomly divided into 4 groups: blank group,negative group,experimental control group and experimental group.Human SW-1116 colon cancer cells were inoculates in the right alar of nude mice to construct the initial tumor bearing animal model.Blank group underwent no any treatment,while all the experimental group,experimental control group and negative group were experienced a tail intravenous injection of healthy PBMC to reconstruct the human immune system after the tumor growth identified.Third day after the immune reconstitution,umbilical cord blood derived DC vaccine(CB-DC-VAC)was given to the experimental group,peripheral blood derived DC vaccine(PB-DC-VAC),to the experimental control group,and peripheral blood DC with no antigen loading(PB-DC),to the negative group,respectively.Twenty-four hours after the vaccination,human SW-1116 colon cancer cells were inoculated again in the left alar of nude mice.The tumor formation time after the second inoculation in different groups were observed and exhibited by tumor growth curves.The animals were sacrificed 27 days after the first inoculation,the tumor weight were recorded,and tumor tissues were sampled and observed by HE staining.Results:1 Umbilical cord blood and peripheral blood DC's morphological change during maturationWhen at d0,both of them were round or oval shape;at d3,cells appear protruding and burr,peripheral blood DC have longer projection with fusiformis shape,Umbilical cord blood DC shorter but thorny;at d5,peripheral blood DC projection more obvious,narrow and straight,whlie Umbilical cord blood projection are more intensive;at d7,antigen loaded DCs were floated with no dendrite,round-like shape.2 Umbilical cord blood and peripheral blood DC's phenotype change during maturationWhen immature,the expression level of CD80,HLA-DR,CD86,CD83 in peripheral blood DC was gradually increased,but the expression of CD80 was significantly lower than that of the later three.The expression levels of CD80,CD83,CD86 and HLA-DR in umbilical cord blood were gradually increased with the prolongation of culture time.When the two sources of DC were mature,the expression levels of these four markers were significantly higher than those before their maturity(P<0.05).The expression in each stage of umbilical cord blood was significantly higher than that in the peripheral blood DC(P<0.05).3 Tumor bearing condition and general condition of miceWhen the first and second time tumor bearing succeed,the right and left alar of mice can touch a nodule about the size of mung bean slightly elevated skin.Kill the mice then take bilateral tumor to stained with HE,see tissue cells are arranged into irregular glands.No diarrhea and natural death was observed in any of four groups,with time going on,the reduce of spirit and activity were observed in the blank group,the performance of arching back and hair warp in the negative group and experimental control group were observed.There was no obvious abnormality in the spirit,food and activity of experimental group.4 The growth of tumor in miceThe right tumor growth curve shows,the tumor growth was rapid in the blank group,PB-DC group,PB-DC-VAC group and CB-DC-VAC group tumor growth were inhibited in different degrees,the tumor of CB-DC-VAC group grew most slowly,PB-DC group and PB-DC-VAC group had no significant difference.The left tumor growth curve shows,the blank group can't suppress tumor growth,tumor growth were inhibited in different degrees of PB-DC group?PB-DC-VAC group and CB-DC-VAC group,all of them compared with blank group have significant difference(P<0.05).In CB-DC-VAC group,the tumors growth were significantly inhibited,and tumor appeared delayed,grow tumor in the d13 after the second time tumor bearing.The CB-DC-VAC group was better than the PB-DC-VAC group and PB-DC group,the difference was statistically significant(P<0.05),PB-DC-VAC group grow tumor in the d10 after the second time tumor bearing,PB-DC group in the d8,PB-DC-VAC group compared with PB-DC group also had statistical difference(P<0.05).Blank group grow tumor in the d4 after the second time tumor bearing.After the end of experment,we weigh the heavy of each mice tumor,the right tumor weight of blank group,PB-DC group,PB-DC-VAC group,CB-DC-VAC group is respectively(1.07±0.38)g?(0.86±0.26)g?(0.67±0.17)g and(0.51 ± 0.13)g.The PB-DC group,PB-DC-VAC group and CB-DC-VAC group tumor weight were lower than the blank group,the CB-DC-VAC group was lower than PB-DC group and PB-DC-VAC group,the tumor weight of PB-DC-VAC group was also lower than that of PB-DC group.The left tumor weight of blank group,PB-DC group,PB-DC-VAC group,CB-DC-VAC group is respectively(2.56±1.36)g?(0.79±0.23)g?(0.52±0.15)g and(0.18±0.14)g.The tumor weight of PB-DC group,PB-DC-VAC group and CB-DC-VAC group were significantly lower than that of blank group,the difference was statistically significant(P<0.05).Both the PB-DC and the PB-DC-VAC groups were higher than the CB-DC-VAC group,and the difference was statistically significant(P<0.05),but there was no significant difference between PB-DC group and PB-DC-VAC group.Conclusions:1 This study established the first and second time tumor bearing animal model of human colon cancer in SCID/Beige nude mice,which provides a good experimental platform for the further study of colon cancer.2 Umbilical cord blood DC has stronger antigen presentation,lymphocyte proliferation and activate T cells ability than peripheral blood DC.3 Umbilical cord blood DC vaccine has a stronger tumor killing effect than the peripheral blood DC vaccine.4 Umbilical cord blood DC vaccine has a stronger effect on tumor recurrence and metastasis than the peripheral blood DC vaccine. |
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