The Anti-pancreatic Cancer Research Of MUC1-VNTR DNA Vaccine In Vivo And In Vitro

The anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivo and in vitroPancreatic cancer is one of the most malignant tumors, and the cause of this disease is still unclear. At present surgical resection is still the major therapeutic method; however the surgery only could remove the visible mass not the invisible metastatic and minimal focus. And these invisible tumor cells are the major recourses of the recurrence of the tumor and induce the death of the patients. To find out ways to cure the pancreatic cancer has become one of the hot focuses of the international research groups. Based on the previous research, we furthered the research on the anti-pancreatic cancer effect of the DNA vaccine MUC1-VNTR in vivo and in vitro. The research is divided into two parts.Part oneThe anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vivoSection one The construction of the pancreatic cancer cell line panc02-MUC1Objective:To construct the pancreatic cancer cell line panc02-MUC1 that could express the MUC1 protein.Material and method:The murine pancreatic cancer cell line panc02 is presented by the American MD Anderson cancer center and the MUC1 plasmid which encoded the whole gene of the MUC1 protein was a gift from Dr. Finn, Pittsburg University. In vitro, we used the Lipofectamine2000 to transfect the plasmid into the pancreatic cancer cell and use the empty plasmid pcDNA3 transfected cell line as control. After transfection, the G418 was used to select the monoclonal pancreatic cancer cell line panc02-MUC1. Result:The monoclonal pancreatic cancer cell line panc02-MUC1 we had constructed could express MUC1 by the Western blot and by means of immunofluorescence we found that the MUC1 was expressed on the cell membrane. Then in the animal model, it could form tumors in the normal C57BL/6 mice and immunohistological examination showed that the cell line could express MUC1 in vivo.Conclusion:The cell line we constructed is the MUC1 expressing monoclonal and it could form tumor in the normal mice.Section two The research of preventing murine pancreatic cancer by the MUC1-VNTR DNA vaccineObjective:The normal C57BL/6 mice were immunized with the pcDNA3.1-VNTR plasmid i.m. three times, taken the empty plasmid pcDNA3.1 and PBS as control. To study whether the pcDNA3.1-VNTR plasmid immunized mice could prevent from the challenge of the panc02-MUC1 cells.Material and method:The normal female 6-7w C57BL/6 mice were randomly divided into 3 groups, 18 mice every group: PBS,Neo(pcDNA3.1),MUC1(pcDNA3.1-VNTR). We injected a total of 100μg plasmid DNA of pcDNA3.1-VNTR in 100μl of PBS into the anterior tibialis muscle of C57BL/6(every 2 weeks, 3 times). Mice inoculated with either the empty plasmid pcDNA3.1or PBS was used as control. 5 days after the third immunization, the blood of the mice was collected from the inner canthus vein which used for the ELISA assay of anti-VNTR antibodies. 7 days after the third immunization the spleen cells of each group(n=3) were collected and stimulated by the peptide of the MUC1 which used for the LDH cytotoxicity assay and at the same time the remaining mice were inoculated with the pancreatic cancer cell panc02-MUC1 at the interdermal of the left anterior leg armpit(1×10⁶/100μl/each mouse). 7 days after the tumor challenge, the length and width of the tumor was calculated with the caliper every 2-3 days, and the volume of the tumor was calculated by the formula Volume= length×width×width/2. When the tumor volume was over 1,000mm³, the mice was considered as dead. While the other 6 mice immunized with the pcDNA3.1-VNTR plasmid were inoculated with the pancreatic cancer cell line panc02-neo which did not express the MUC1 protein(1×10⁶/100μl/each mouse).Results:Anti-VNTR specific antibody was found significantly higher in the pcDNA3.1-VNTR immunized mice than the control group (P＜0.05). Cytotoxic assay showed that the intramuscular delivery of the recombinant plasmid into C57BL/6 mice resulted in more efficient induction of CTL lyses specific against VNTR polypeptide than the control group(P＜0.05). And this specific cytotoxity ability could be suppressed by the VNTR-antibody VU3C6 (P＜0.05); meanwhile this cytotoxity was restricted to the MUC1 expressing cell line panc02-MUC1 not the MUCl-negative cell line panc02-Neo (P＜0.05). After the tumor challenging, the tumor growth rate in the pcDNA3.1-VNTR immunized mice was much slower than the control group (P＜0.05) and the survival curve showed that the life time of the pcDNA3.1-VNTR immunized mice was much longer than the control group (P＜0.05). Otherwise the life time of the pcDNA3.1-VNTR immunized mice which were challenged with the panc02-neo cell line was not affected (P＜0.05).Conclusion:The recombinant plasmid pcDNA3.1-VNTR could significantly induct VNTR specific CTL response and antibodies response. And the pcDNA3.1-VNTR immunized mice could suppress the growth of the MUC1-positive cell line panc02-MUC 1 not the panc02-Neo.Section three The research of treating murine pancreatic cancer by the MUC1-VNTR DNA vaccine combined with GMCSFObjective:Combining the pcDNA3.1-VNTR plasmid with the murine GMCSF to treat the murine inoculated with MUC1-expressing pancreatic cancer cell line panc02-MUC1.Material and method:On the day 0, 48 female C57BL/6 normal mice were inoculated with panc02-MUC1 cell line (1×10⁶/100μl/each mouse) in the interderm of the left anterior leg armpit. Then these mice were randomly divided into 5 groups as follows: G+M(GMCSF+MUC1) (n=10),M(MUC1) (n=10),G(GMCSF) (n=10),Neo(pcDNA3.1) (n=9) and the PBS group (n=9). And another 12 mice were inoculated with the MUCl-negative cell line panc02-neo, then were divided into 2 groups as follows: G+M(GMCSF+MUC1),M(MUC1) (n=6). On day 4, the treatment proposal of each group was as follows respectively: GMCSF50ng/100μl+pcDNA3.1-VNTR100μg/100μl,pcDNA3.1-VNTR 100μg/100μl,GMCSF50ng/100μl,pcDNA3.1 100μg/100μl, PBS100μl; And on the day 9 and 14 repeated it again. As the mice challenged with the cell line panc02-neo, each group was treated with GMCSF50ng/100μl+pcDNA3.1-VNTR100μg/100μl and pcDNA3.1-VNTR 100μg/100μl respectively; and repeated on the day 9 and day 14. After the inoculation of the tumor cells, the length and width of the tumor were measured from the day 6-7 every 2-3 day. The mice were considered dead when the volume of the tumor was over 1000 mm3. Eleven days after the last treatment, 3 mice were taken from each group and the splenic cells were isolated cultured for the VNTR specific CTL cytotoxity assay.Results:In the group G+M and M the panc02-MUC1 cell line growth rate was much slower than the group G, Neo and PBS (P＜0.05) and the group G+M was slower than the group M (P＜0.05). However in the mice inoculate with the cell line panc02-neo, neither of them showed treatment effect compared with the mice inoculated with panc02-MUC1 (P＜0.05). About the life time of the mice challenged with panc02-MUC1, the group G+M and M were much longer than the other 3 group (P＜0.05), and these two group had no difference in the survival time(P＞0.05). However after treated with G+M or M, the life time of the mice challenged with panc02-neo was much shorter than the life time of the mice challenged with panc02-MUC1(P＜0.05). The in vitro cytotoxity assay showed that only the splenic cells from the G+M treated group had significant cytotoxity ability(P＜0.05), meanwhile had no cytotoxity ability to the panc02-neo cell line(P＞0.05).Conclusion:Both MUC1-VNTR DNA vaccine and MUC1-VNTR DNA vaccine combined with GMCSF could suppress the growth of the panc02-MUC1 cell line not the panc02-neo cell line. And combined with GMCSF the tumor grew much slower than the group M. However the life time did not show much difference in these two groups. As for the cytotoxity assay, only the CTL from the G+M group showed cytotoxity ability to the the panc02-MUC1 cell line.Part twoThe anti-pancreatic cancer research of MUC1-VNTR DNA vaccine in vitroSection one In vitro induction and culture of the human dendritic cell and the research on the biological characteristicsObjective:The PBMC was isolated from the normal human peripheral blood and induced to the dendritic cell by the cytokine. And the biological characteristic of the dendritic cell was studied in vitro.Material and method:The PBMC was isolated from the normal human leukocyte using Ficoll in vitro. Then after anchorage, the non-anchorage cells were washed away while the anchorage cells were cultured with the cytokine GMCSF and IL4, then induced to maturity by the cytokine TNFα. Using the FACS to analyze the phenotype of the PBMC and the mature dendritic cell. Using the mixed lymphocyte reaction to analyze the ability of the mature dendritic cell to stimulate the homologous lymphocyte proliferation.Result:Under the culture combined with the cytokine of GMCSF,IL4 and TNFα, the mature dendritic cell could be induced from the PBMC; and the surface marker of the mature dendritic cell HLA-DR, CD209 and CD86 was much higher than the PBMC(P＜0.05), while the lymphocyte surface marker CD14,CD3/CD4,CD3/CD8 was less than PBMC (P＜0.05). The mixed lymphocyte reaction showed that the mature dendritic cells had much higher ability to stimulate the homologous lymphocyte proliferation than the non-mature dendritic cells (P＜0.05).Conclusion:Combined with GMCSF,IL4 and TNFαculture, the dendritic cell could be induced from the PBMC and the mature dendritic cell had much higher antigen-presenting ability than the non-mature dendritic cell.Section two In vitro research on the function of the MUC1-VNTR DNA transfected dendritic cellObjective:To investigate whether the pcDNA3.1-VNTR plasmid transfected dendrtic cell could express the VNTR in vitro and stimulate the proliferation of the autologous T cells. Meanwhile using the Elispot to detect the IFNγand Granzyme B secreting CTL.Material and method:The dendritic cells were augmented from the normal human PBMC by the stimulation of the cytokine GMCSF and IL4. On the day 5, the immature dendritic cells were transfected with pcDNA3.1-VNTR plasmid in vitro by Lipofectamine2000, followed with the conculture of the cytokine TNFαto stimulate the maturation of the dendritic cells. 24 hours after transfection the expression of the VNTR was detected by Western blot. On the day 7, the mature dendritic cells were co-cultured with the autologous T cells to investigate wheather they could stimulate the autologous T cell proliferation. And using the Elispot to detect the number of the IFNy and Granzyme B secreting CTL stimulated by the mature pcDNA3.1-VNTR plasmid transfected dendritic cells.Result: The pcDNA3.1-VNTR plasmid transfected dendritic cells could express VNTR in vitro; and they could stimulate the autologous T cells to proliferate especially at the ratio of DC:T=1:10 (P＜0.05). The Elispt result showed that the number of the IFNγand Granzyme B secreting CTL in the group pcDNA3.1-VNTR were more than the group pcDNA3.1 and the group Lipofectamine (P＜0.05).Conclusion:The pcDNA3.1-VNTR plasmid could be expressed in the human dendritic cells and the pcDNA3.1-VNTR plasmid transfected dendritic cells could stimulate the proliferation of the autologous T cells and induce the IFNT and Granzyme B secreting CTL.Section three The research on the cytotoxity assay of the MUC1-VNTR specific CTL induced by the MUC1-VNTR DNA vaccine transfected dendritic cellObjective:To investigate whether the pcDNA3.1-VNTR plasmid transfected dendritic cells could induce the MUC1-VNTR specific CTL and their ability to kill the MUC1-positive pancreatic cancer cell lines.Material and method:The dendritic cells were induced from the healthy HLA-A₂+ human PBMC; then on the day 5, the dendritic cells were transfected with pcDNA3.1-VNTR plasmid by Lipofectamine2000, using the pcDNA3.1 transfected dendritic cells and the Lipofectamine2000 treated dendritic cells as control. After maturation, the dendritic cells were co-cultured with autologous T cells to induce the CTL and using the LDH method to detect the cytotoxity activity to the pancreatic cell line Capan-2(HLA-A2+MUC 1 +).Result:The CTL induced by the pcDNA3.1-VNTR plasmid transfected dendritic cells could specifically kill the Capan-2 cell line (HLA-A₂+MUCI+), and the cytotoxity ability could be suppressed by the MUCl-antibody VU3C6. However they could not kill the Aspc- 1 (HLA-A2-MUC 1 +).Conclusion:The pcDNA3.1-VNTR plasmid transfected dendritic cells could induce HLA-A₂ restricted VNTR specific CTL.