The Influence And Treatment Of Fibroblast Activation Protein (FAP) To Mouse Pancreatic Cancer Cells

Objective:To construct an eukaryotic expression plasmid system of fibroblast activation protein (FAP) gene and detect both the efficiency of its expression and the efficiency of its capacity of killing and inhibiting the mouse pancreatic cancer cells and tumor associated fibroblasts by tranfecting it into cells cultured in vitro and tumor-bearing mice in vivo. In order to provide a novel method and idea for pancreatic cancer treatment and prevention.Methods:Firstly, the PCR primer has been designed based on the full sequence of mouse FAP in GenBank (NM₀07986). The FAP gene has been inserted into the eukaryotic expression vector pcDNA6/myc-His-B, then transcripted into the E. coli competent cells and screened recombinants. The IF and Western Blotting were used to detect whether the exogenous FAP gene could be expressed in HEK293 cell lines or not. Secondary, the mouse primary pancreatic cancer cells and tumor associated fibroblasts were isolated and cultured together, the siRNA which targeting the endogenous FAP gene was used to silence the FAP expression. The IF, RTQ-PCR and Western Blotting assay were used to detect the effect of interference by siRNA. At the same time, the growth inhibitor and apoptosis of co-cultured model of "primary pancreatic cancer cells-tumor associated fibroblasts" were tested by MTT and FCM assay in order to identify whether the relationship among FAP protein expression and the growth and proliferation of mouse pancreatic cancer cells and tumor associated fibroblasts exists or not. Thirdly, after the FAP expression plasmid was inoculated into C57BL/6 mice, the FAP expression can be detected by ICH and RTQ-PCR. Finally, the mouse pancreatic cancer cell lines were inoculated under mice subcutaneous (including FAP inoculated group, blank plasmid inoculated group and blank control group), then whether the FAP DNA vaccine can be used to prevent and control the proliferation and growth of mouse pancreatic cancer cells or not were estimated by calculating tumor tissue volume and tumor-bearing mice's life cycle.Results:Firstly, the results of digestion with restriction endonuclease and sequencing revealed that the pcDNA6-mFAP recombinants which we have screened were correct positive clones, and it could express FAP protein in HEK293 cell lines. The results of IF and Western Blotting assay indicated that the exogenous FAP protein can be tested and which was showed green and red fluorescence by combined the anti-FAP and anti-Myc antibody. In comparison with the blank plasmid transfected group and untransfected group, there are not any fluorescence on it. Secondary, we isolated and cultured mouse primary pancreatic cancer cells and tumor associated fibroblasts, and constructed a co-cultured model of them. The result of HE stain indicated that two kinds of cells were cultured in one environment. The result of RTQ-PCR revealed that the level of FAP mRNA expression in siRNA-FAP transfected group (0.52±0.02, n= 3) was much lower than in the siRNA-MOCK group (0.99±0.07, n= 3, P< 0.05, t test) or untransfected group (1.01±0.10, n= 3, P < 0.05, t test). The same as the result of Western blotting, which showed that the level of FAP protein expression in siRNA-FAP group (27.18%±3.23%) was much lower than in the siRNA-MOCK group (61.58%±4.72%, P=0.0317, t test) or untransfected group (65.29%±4.78%, P= 0.0389, t test). The results above indicated that the siRNA can knockdown the endogenous FAP gene expression. Then, in comparison with the siRNA-MOCK group and untransfected group, the MTT assay indicated that the speed of cells growth in siRNA-FAP transfected group was remarkablely slower than in other two groups. And, the FCM assay showed that the apoptosis cell subpopulations could be found in the siRNA-FAP transfected group (42.31%±5.34%) but not in other two groups. These results indicated that mouse primary pancreatic cancer cells and tumor associated fibroblasts would be led to apoptosis and growth and proliferation inhibited by knockdowning the expression of FAP protein. Finally, the FAP expression plasmid could express FAP protein in tumor-bearing mice successfully by IF and RTQ-PCR assay. Using the tumor tissue volume and tumor-bearing mice's life cycle calculated, we found that not only the speed of tumor tissue growth in FAP inoculated group were much slower that in the blank plasmid inoculated group and blank control group, but also the life cycles of mice in FAP inoculated group were obviously longer than in the the blank plasmid inoculated group and blank control group. Lastly, the result of ICH revealed that the levels of expression of FAP,Vimentin and CK8 proteins in tumor tissue of FAP inoculated group were much lower than in other two groups.Conclusion:The FAP expression plasmid expressed successful the exogenous FAP protein product in the eukaryotic cell lines and mice which was constructed by genetic engineering. And, the FAP DNA vaccine could be used to prevent and control the proliferation and growth of mouse pancreatic cancer cells.