Effects And Mechanism Of TLR9 Expression In Biological Behavior Of Pancreatic Carcinomal

PartⅠTLR9 expression in pancreatic cancer and its clinic significanceObjective:To detect the expression of Toll-like receptor 9 (TLR9) in pancreatic cancer, and to explore their clinical significance.Methods:The real-time RT-PCR technique, western blot method and immunohistochemical method were used to examine the expression of TLR9 in the pancreatic cancer, tissues near tumor, and normal pancreatic tissues which obtained during operation from 30 pancreatic carcinoma patients and 10 normal pancreatic tisssues. The relationships with pathological grade, clinical stage, and metastasis of pancreatic cancer were statistically analyzed.Results:The amplification value level of TLR9mRNA expression in human pancreatic tissues, paracancerous tissues were 2.47 (1.87～3.07) and 1.29 (1.14-1.44), respectively(t=10.49,p<0.01).The percentage of positive cells expressed TLR9 protein in human pancreatic tissues, paracancerous tissues and normal tissues were 73.3%,33.3% and 20.0%,the protein expression level of TLR9 was gradually descending(x2=13.99, p<0.01). The highly expressed TLR9 correlated significantly with the degree of tumor differentiation,an advanced TNM stage and lymph node metastasis. Similar result was obtained by Western blot(p<0.01).Conclusions:TLR9 has a higher expression in pancreatic cancer tissues. TLR9 expression has a close relationship with pathological grades of pancreatic cancer, suggesting that TLR9 play an important role in the development and progression of pancreatic cancer. PartⅡInfluence of CPG ODN down-regulation on biology of pancreas cell line Panc-1Objective:To investigate the effect of CPG ODN2216 on biological behavior of the pancreatic cell carcinoma, and to explore their clinical significance.Methods:The immunofluorescence staining test was used to examine the expression of TLR9 in the Panc-1 cell. And in vitro cell adhesion, Wound-healing scrape assay, transwell invasion assay and cell colony formation assay were performed to assess the effect of CPG ODN2216 on the invasive properties of Panc-1 cells.Results:TLR9 has a higher expression in pancreatic cell line Panc-1, and In vitro experiments as cell spreading assays,cell adhesion,colony formation assay and invasion assays showed the cell adhesion and cell motility properties of CPG ODN 2216 group were apparently weakened compared with the control group(P<0.01). The MTT assay showed cell proliferation ability in the CPG ODN group was notably decrease, and CPG ODN2216 had inhibitive effects on growth of panc-1 cells in a dose and time-dependent manner(P<0.01).Conclusions:TLR9 has a higher expression in pancreatic cell line Panc-1, and TLR9 gene is correlated with the invasive and metastatic potential of pancreatic carcinoma, and use of CPG ODN2216 can inhibit migration and invasion of Panc-1 cell line. Part IIIPancreatic tumor model established and CPG ODN2216 on the growth of pancreatic cancer in nude miceObjective:In the second phase of the study based on the results, through the establishment of human pancreatic cancer Panc-1 in animal models to further explore the CPG ODN on human pancreatic tumor growth in nude mice implanted subcutaneously inhibition.Methods:Through the establishment of nude mice model of pancreatic cancer Panc-1, to be the principle of growth after two weeks were randomly divided into three groups, control group, saline group and the CPG ODN2216 group. Draw the growth curve in nude mice, four weeks after treatment according to grouping mice were killed, compared three groups of tumor volume and quality of the different, and detected by immunohistochemistry in all groups TLR9 protein expression in growing tumors.Results:Nude mice in each group 6 weeks after inoculation of tumor cells were successfully induced tumor, tumor rate of 100%. Compared three groups of mice were killed after the tumor, CPG ODN2216 tumor volume and mass, respectively (0.29±0.07) cm3,(0.81±0.17) g, significantly less than saline group (0.69±0.09) cm3, (1.74±0.22) g and the control group (0.67±0.10) cm3, (1.72±0.25) g with statistical significance (P<0.01). Immunohistochemistry confirmed that CPG ODN2216 group of TLR9 protein expression in growing tumors was significantly lower than the other two groups.Conclusions:TLR9 gene in an important role in pancreatic cancer, CPG ODN2216 inhibited the subcutaneous human pancreatic tumor growth in nude mice.