Immunohistochemical staining: Annexin II antibody was purchased from Abcam Reagent Company; Immunohistochemistry kit, DAB kit was purchased from Pikiton Reagent Company. Sliced was kept in high temperature and high pressure tissue antigen repair. The dyeing step is carried out according to the kit instructions. Positive plates were used as positive control, PBS instead of primary antibody as a negative control. The results showed that annexin II positive expression of brown matter in the cytoplasm appears brown matter. Each slice 5 representative of the high power field, Each field count 100 cells, The percentage of positive cells and the intensity of staining were scored separately. Specific criteria are as follows: (1) according to the degree of staining score: no staining for 0 points, Dyeing strength is 1 point; Medium dyeing intensity of 2 points; Strong staining intensity of 3 points. (2) Score according to the percentage of colored cells: The percentage of cells in the stained cells was < 5% for 0 points, 5%-25% for 1 points, 26%-50% for 2 points, 51%-75% for 3 points, > 75% for 4 points. (1) + (2) for the total points, The total score of 0-7 points. 0 is negative (-), 1-2 is divided into weak positive (+), 3-5 are divided into positive (++), 6-7 is divided into strong positive (+++).
Cell culture: Gastric cancer HGC-27 cells were cultured in RPMI1640 cell culture medium containing 10% fetal bovine serum and cultured in 5% CO2, 37 °C, saturated cell culture incubator.
Cell transfection: Gastric cancer HGC-27 cells (1 × 105 cells/mL) were seeded in 24-well plates, Cultured overnight at 37 °C, 5% CO2 and saturated humidity. Serum-free medium wash cells, 1 μL (20 pmol) of Annexin II siRNA was dissolved in 49 μL serum-free medium, 1 μL of liposomes was dissolved in 59 μL of serum-free medium, After keeping at room temperature for 10 min, Mix the two type of liquids, room temperature for 20 min. The mixture was then added to the 24-well plate of the cells to be transfected, Add serum-free medium to 500 μL, Placed in 37 °C, 5% CO2 cell incubator culture, 6 h later replaced with 10% newborn bovine serum RPMI1640 medium.
Real-time quantitative PCR: Use Trizol method to extract total RNA from cells, The total RNA samples of the extracted osteoblasts were reverse transcribed into cDNA, Using the following reaction system: 2 μg template RNA,1 μL oligo dT primer, 2 μL dNTP mixture,1 μL Ace reverse transcriptase,1 μL RNase inhibitor,4 μL 5 × RT buffer, 10 μL RNase-free water. The reaction conditions were as follows: first step 37 °C 30 min, The second step is 84 °C for 30 s. The reaction product was stored at 4 °C. And then real -time quantitative PCR reaction. Using the following reaction system: 12.5 μL Real Master Mix (SYBR I), 2 μL template cDNA, 1 μL forward primer, 1 μL reverse primer, 8.5 μL RNase-free water. The reaction conditions are: first step 94 °C 5 min; second step 94 °C 60 s, 57 °C 30 s, 72 °C 30 s, a total of 30 cycles; the third step 72 °C 5 min; the last 4 °C end of the reaction. The experimental data were analyzed by Option Monitor V3.1 software of real-time PCR.
Cell invasion experiment: A 1:3 diluted matrigel (RMPI 1640 dilution) was added to the matrigel invasion chamber, 37 °C for 2 h, 2 × 104 gastric cancer cells were resuspended in 0.1% fetal bovine serum culture medium and inoculated in the chamber, RMPI 1640 medium containing 10% fetal bovine serum was added to the 24-well plate of the lower chamber, 37 °C for 24 h. Remove the invasion chamber, With a cotton swab gently wipe the room did not pass through the cells, formaldehyde fixed for 10 min, Hematoxylin-eosin staining, Cut the filter, the neutral gum seal, light microscopy count the number of cells through the membrane. The number of cells in the membrane was calculated by taking 10 fields under light microscope. Each independent experiment was repeated three times.
Cell migration experiments: First with a marker pen in the 6-well plate even behind a horizontal line, Cross through the hole. About 2 × 104 gastric cancer cells were seeded in 6-well plates when the cells are completely fused, the tip of the nose is as far as possible to the horizontal line behind the horizontal scratches. Wash the cells three times with PBS, wash off the cells under the cells, and then add serum-free medium. And incubated in a CO2 incubator at 37 °C. 0 and 24 h under inverted microscope to observe and take pictures. The scribe width was measured with Image J software and the cell mobility was calculated. Cell mobility = (0 h scratch width - 24 h scratch width)/0 h scratch width × 100%. Each independent experiment was repeated three times.
Cell gelatin zymography experiments: Cells were treated with annexin II siRNA Digest the cells and count, the corresponding conditioned medium was collected, 1500 g low-speed centrifugation 10 min after removal of cell debris, Take the supernatant 4 °C spare. And then take the protein quality samples separately with non-reduction Loading Buffer Homogeneously mixed, incubated at 55 °C for 5 min. Prepare 5% concentrated gum and 8% separated gel containing 0.1% gelatin. Constant pressure electrophoresis until the edge of bromophenol blue separation from the front of the separation of about 0.5 cm. The gel was removed and transferred to the elution solution, eluting twice every 30 min. Rinse with gelatin incubation once, and then placed in gelatin incubation buffer at 37 °C for 18 h. Dyeing solution 4 h After the rinsing of distilled water, And then take a photo by a gel image analyzer.