Wu Hanqing Wang Bo Zhu Shikai Zhang Jianjun Wang Chunyou Wu Heshui* [Abstract] Objective To detect the expression of TLR9 in human pancreatic cancer and pancreatic cancer cells, to study the effect of CPG ODN2216 on the biological behavior of Panc-1 cells, and to explore its clinical significance. Methods The high expression of TLR9 protein in pancreatic cancer tissues was confirmed by immunohistochemistry, and its high expression in pancreatic cancer cell line Panc-1 was confirmed by immunofluorescence. The effects of CPG ODN2216 on cell adhesion, motility and proliferation were investigated by cell adhesion, scratch assay, invasion assay, cell cloning and MTT value-added assay. The results showed that both human pancreatic cancer specimens and human pancreatic cancer cell line Panc-1 were highly expressed TLR9, and the cell adhesion and motility of the CPG ODN2216 experimental group were significantly lower than those of the unsequenced control group as demonstrated by the scratch assay, in vitro adhesion assay, stromal gel invasion assay and cell cloning assay. dependence. Conclusion TLR9 gene is associated with the invasive metastatic potential of human pancreatic cancer, and the use of exogenous ligand CPG ODN2216 can significantly inhibit the invasive and migratory ability of human pancreatic cancer cells Panc-1. 武汉协和医院急诊科吴汉青【关键词】CPG ODN2216, 胰腺肿瘤, Toll样受体9,细胞生物学 Influence of CPG ODN down-regulation on biology of pancreas cell line PANC-1 and the expression of TLR9 in human pancreatic cancerHan-Qing Wu, Bo Wang, Shi-Kai Zhu, Jian-Jun Zhang, Chun-You Wang, He-Shui WuDepartment of Pancreatic Surgery Center ,Union Hospital ,Tongji Medical College ,Huazhong University of Science and Technology ,Wuhan Hubei 430022,ChinaCorrespondence to: Professor He-Shui Wu, Email:[email protected].[Abstract] Objective: To detect the expression of Toll-like receptor 9 (TLR9) in pancreatic cancer and investigate the effect of CPG ODN2216 on biological behavior of the pancreatic cell carcinoma, and to explore their clinical significance. Methods: The immunohistochemical method were used to examine the expression of TLR9 protein in the pancreatic cancer tissue and immunofluorescence staining was also performed to detect the TLR9 protein expression in pancreatic carcinoma cells．In vit While TLR9 is an important member of this family, it is able to stimulate the expression of a large number of cytokines and chemokines secreted by B cells and dendritic cells, such as interleukin (IL)-12, IL-6, interferon-γ, monocyte inhibitory protein and metalloplasmic proteases [2]. In order to investigate the role of TLR9 gene in pancreatic carcinogenesis, after confirming its high expression in pancreatic cancer, we used the synthetic oligodeoxynucleotide 2216 (CPG ODN2216), a specific ligand of TLR9, to stimulate pancreatic cancer cells Panc-1,and observed the growth and proliferation, tumorigenic capacity and The TLR9 gene was used in the pancreatic cancer cell growth and development to provide information for further study of the specific mechanism of TLR9 gene. 1. Materials and methods I. Experimental materials 1. Cells: Human pancreatic cancer cells Panc-1 were provided by the General Surgery Laboratory of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. 2. Tissue sections: 30 cases of human fresh pancreatic cancer tissues and the corresponding paracancerous tissues (1-2 cm according to the tumor margin) were taken from patients with pancreatic cancer, 10 cases of normal pancreatic tissue specimens were taken from non-tumor patients who needed partial pancreatectomy for other reasons, and all specimens were taken from patients with pancreatic cancer. All specimens were from pancreatic cancer patients who underwent surgical treatment at the Pancreatic Surgery Center of Union Medical College of Huazhong University of Science and Technology and were confirmed by postoperative pathology.3. Main reagents: CpG-ODN2216 (sequence GGGGACGATCGTCGGGGGG), synthesized by Shanghai Bioengineering Bioengineering Technology Service Co. monoclonal antibody (cell signaling company). Immunohistochemistry and fluorescence kits (Wuhan PhD Company). transwell chambers (Coring Company). matrigel gel (Sigma Company); MTT with DMSO (sigma Company). II. Experimental methods 1. Immunocytofluorescence chemistry: digestion of pancreatic cancer Panc-1 cells with trypsin to make a single cell suspension, and drop the single cell suspension onto the slide. According to the cell growth status, remove the crawling slides at the right time for 24 h or more. rinse three times with PBS for 5 min each time and fix with 4% paraformaldehyde for 15 min; rinse three times with PBS and perforate with 0.5% Triton for 20 min; rinse three times with PBS and close with 5% BSA for 30 min; after shaking off the liquid, add the diluted primary antibody directly, and replace the control group with PBS primary antibody. The sections were incubated overnight at 4°C; PBS was rinsed three times, diluted secondary antibody was added and incubated at 37°C for 1h; PBS was rinsed three times, diluted SABC-FITC was added and incubated at 37°C for 30min; PBS was rinsed three times, water-soluble blocker was used to seal the sections and observed by fluorescence microscopy. 2. Immunohistochemistry: paraffin sections were dewaxed and hydrated with alcohol. 3% hydrogen peroxide was soaked at room temperature for 10 min. Sections were antigenically repaired in 0.01moL citrate buffer (pH 6.0). Sections were closed with 5% goat serum BSA at room temperature for 30 min and the closure solution was discarded. Add appropriately diluted primary antibody, control with PBS instead of primary antibody, overnight at 4°C. Rinse 3 times with PBS for 5 min each; add biotin-labeled secondary antibody and incubate for 30 min at room temperature, wash 3 times with PBS; add SABC and incubate for 30 min at room temperature. wash with PBS followed by DAB staining, hematoxylin re-staining, dehydration, transparency, blocking, and microscopic observation of results. 3. diphenyltetrazolium bromide Blue (MTT) method to detect the proliferation of pancreatic cancer cells: The digested pancreatic cancer cell suspension was inoculated in 96-well culture plates at 1×103/well, 100μl per well. 37℃, 5% CO2 and saturated humidity conditions were incubated, and after 24h cell apposition, the cells were incubated with different concentrations (0.1μg/ml, 0.5μg/ml, 1μg/ml, 5μg/ml, 10μg/ml ) of CPG ODN2216-acting cells Panc-1, 24 h, 48 h and 72 h after culture, respectively, 20 μl of MTT at a concentration of 5 mg/ml was added; 4 h of culture, supernatant was discarded, dimethyl sulfoxide was added, 150 μl/well, shaking and mixing, and the A value was determined at a wavelength of 490 nm using an enzyme standardization instrument. Inhibition rate = (A control – A drug group)/A control group × 100% [3]. The proliferation inhibition curve was plotted and the half inhibition concentration (IC50) was measured.4. Scratching experiment: Panc-1 cell suspension with cell concentration of 2×105/ml was added to 6-well plates at 3ml per well and incubated for 24h or longer, after the cell growth reached 80% full, the culture medium was discarded and the culture medium with concentrations of 1μg/ml and 10μg/ml of CPG ODN2216, respectively, was added. The supernatant was discarded, and a cell scraper was used to make a scratch on the cells in the center of the well, and four markers were made at equal intervals along the edge of the scratch as data measurement points, and the average value was taken when measuring. Cell adhesion experiment: 96-well plates were spread with glue, air-dried, blocked with BSA, and incubated for 60 min; the cell concentration of control and experimental groups (CPG ODN 1μg/ml and 10μg/ml for 24 h) was adjusted to (5×105)/ml, inoculated with 200μL/well, incubated for 1 h, washed with PBS, added 5mg/ml MTT, and incubated for 4 h. The cell adhesion inhibition rate (%)=(A control group-A drug group)/A control group×100％. Three sub-wells were set up for each group of cells, and the average value was taken. 6. Cell invasion assay: 30 μL of artificial basement membrane gel was added to the upper chamber side of the Transwell membrane and air-dried, 200 μL (containing 1×105 cells) of cells were added to starve culture for 24 h, and the concentration of CPG ODN was adjusted to 1 μg/ml and 10 μg/ml. 500 μL of the lower chamber of the invasion cell containing 10 After 24 h, the chambers were removed and the untransferred cells and excess fluid in the upper chamber were wiped off with a cotton swab. The chambers were fixed in 4% paraformaldehyde for 10 min and then stained with 1% crystal violet for 30 min. The membranes were washed well with deionized water. The cells were observed and counted under the microscope. Three secondary wells were set up for each group of cells and the average value was taken.7. Cell cloning experiment: Panc-1 cells were digested with trypsin, blown to make a single cell suspension and inoculated in a 6-well plate with about 200 cells/well, set up as no CPG group and CPG groups with concentrations of 1μg/ml and 10μg/ml, respectively, and 2 secondary wells were set up for each group of cells. The inoculated cells were continued to be cultured in the incubator until 14 d. The culture was terminated after the cell clones were visible to the naked eye, and the culture medium was removed, washed with PBS, stained with hematoxylin for 30 min, washed with PBS, and the rate of cell clone formation was counted and photographed by digital photography. The experiment was repeated three times.8. Statistical analysis: The experimental data were analyzed by SPSS13.0 software, and all data were expressed as mean ± standard deviation. t-test was performed to determine whether the differences between the experimental data of each group were statistically significant, and P