The establishment of an in situ gastric cancer transplantation model in Kunming mice can: (1) establish an ideal animal model that can realistically simulate clinical gastric cancer; (2) conduct in-depth research on the metastatic mechanism of gastric cancer and search for new therapeutic methods; and (3) provide a valuable tool for the metastatic mechanism and anti-metastatic treatment of human gastric cancer.
Operation method
Subcutaneous graft block in situ method
Principle
Two different tumor cell lines were used to establish gastric cancer models in situ in the stomach of Kunming mice, respectively. The growth rate of the in situ transplanted tumors was observed after modeling, and the expression of v EG F and M v D in the gastric cancer tissues were detected, and the results showed that the tumorigenicity rates of the models were both 100%. The rapid growth rate of in situ transplanted tumors in the stomach of Kunming rats shortens the period of gastric cancer modeling, and provides an effective and simple model for the screening of antigastric cancer drugs.
Materials and Instruments
Kunming Mouse Human Gastric Hypo-differentiated Adenocarcinoma Cell Line SGC7901 Move I. Subcutaneous Transplantation Tumor Transmission in Kunming Mice Caveat In light microscopy, the cells of gastric transplantation tumor were mostly oval, with large nuclei, deep staining, and pathologically divided phases. The tumor cells were arranged in nests or cords, without obvious glandular structures, and grew infiltratively in the gastric wall. Common Problems The nude mouse transplantation model of human gastric cancer underwent three representative stages: subcutaneous inoculation with cell suspension, inoculation with cell suspension at the gastric wall, and transplantation of intact tissue blocks into the gastric wall. The in situ transplantation model formed by injection of tumor cell suspension into the gastric wall of nude mice was most likely to undergo a metastatic process similar to that of human tumors. For more product details, please visit Aladdin Scientific website.
1640 medium VEGF antibody Anti-rabbit Envision antibody Rabbit anti-mouse CD31 antibody H2O2 PBS Anti-VEGF antibody Hematoxylin DAB color development solution Xylene Paraffin Formaldehyde Penicillin Streptomycin Sodium pentobarbital
OB glue Surgical scissors Disposable injection needles Microwave oven Optical microscope
S180 cells were injected into the peritoneal cavity of mice and cultured in vitro. After 5-7 days, the abdominal area of the mice was swollen with ascites, and the ascites was collected, counted, and adjusted to a cell density of 2 x 106-2x107 for injection into the axillary subcutaneous area of Kunming mice to form solid tumors. After the tumors grew to a diameter of 1.5-2.0 cm, the mice were executed, and the tumors were used as the source for in situ transplantation after repeated passaging for 3 generations as described above.
Establishment of in situ transplantation model
1. Take a Kunming mouse that has been growing for about 4 weeks in the logarithmic growth period after 3 generations of transmission, disinfect it routinely, and remove the tumor tissue from the axilla.
2. Remove the fibrous envelope and blood vessels, cut open and remove the necrotic tissue in the middle, and select the well-grown, light-red, fish-like tumor tissues, and cut them into small pieces of about 1-2 mm.
3. Kunming mice were fasted for 12 h before surgery, anesthetized with 0.5% sodium pentobarbital intraperitoneally (50 mg/kg), fixed mice, routinely disinfected the skin, and then incised with surgical scissors along the left side of the abdomen at the midline of the parietal line, with an incision of about 1.5 cm, carefully exposing the peritoneum and the gastric wall, and then using a disposable syringe needle to slightly injure the plasma layer in the middle part of the greater curvature of the stomach to the degree of bleeding, and then pushing in the damage with toothless forceps so as to form a localized gastric wall. Using toothless forceps, push the damaged area inward to form a concavity in the local gastric wall, implant a 1-2 mm3 tumor tissue block, and put 1 drop of OB bio-adhesive on the surface of the tumor to cover the surface of the tumor tissue, and then close the abdomen in layers.
4. After in situ implantation of human gastric cancer tissue blocks in Kunming mice, the nude mice were observed day by day for general condition, movement and abdominal signs. If the animal dies during the experiment, record the time and fully investigate the abdominal cavity to observe the in situ growth of gastric tumor.
III. Observation of executed animals and transplanted tumors
All nude mice were regularly observed for general condition, locomotion, abdominal signs, post-transplantation decapitation and dissection, and in situ tumors were taken. The specimens were fixed in 10% formaldehyde, embedded in paraffin, sectioned, and stained with HE for histopathological examination.
Immunohistochemical staining
1. VEGF protein expression was detected by EnVision immunohistochemistry, a new two-step immunohistochemistry technique that is simple, specific and sensitive.EnVision is a new two-step immunohistochemical technique, which is simple, specific and sensitive.
2. The procedure was as follows: thin (3-4 um) and flat tissue sections were selected, deparaffinized in xylene, hydrated with a gradient of alcohol, microwave-repaired with antigen, cooled at room temperature and washed with PBS for 5 min. 3.
3. The sections were incubated in H2O2 solution for 10 min, washed with PBS for 5 min, and then incubated with 1:100 diluted anti-VEGF antibody at 37 ℃ for 60 min, washed with PBS for 5 min. 4.
4. Dropwise add secondary antibody and incubate at 37 ℃ for 30 min, wash with PBS for 5 min.
5. DAB chromogenic solution was used to develop the color, and the staining time was about 8-20 min at room temperature, and the staining time was controlled according to the development of the color under the microscope.
6. After the color development, put PBS to wash for 5 min, re-stain with hematoxylin, and seal the plate.
7. Use PBS instead of primary antibody as negative control.
8. Judgment of VEGF positive results: 5 high magnification fields were taken randomly, and in terms of the percentage of positive cells, ≤5% was regarded as negative (+), 6%-25% was regarded as weakly positive (+), 26%-50% was regarded as moderately positive (+ + +), and ≥51% was regarded as strongly positive (+ + + +).V. Determination of microvessel density in transplanted tumors
1. EnVision method was used as in 4. The primary antibody was rabbit anti-mouse CD31 antibody at a dilution of 1:100. 2.
2. PBS was used as negative control instead of primary antibody. 3.
3. MvD of transplanted tumors were counted according to the criteria of Weidner et al. The stained capillaries and microvessels in the tumors were counted, and any single endothelial cell or endothelial cell cluster in brown color was counted as one blood vessel, but the blood vessels with thick muscular layer and lumen area larger than 8 erythrocyte diameters were not counted.
4. The area with the highest number of vessels is first searched for at 100x, and then the number of microvessels in each of the five areas is counted at 200x, and the mean is taken as representative.VI. Statistical processingThe MVD data of transplanted tumors were expressed as unscaled SD and t-test was used.
Reference: "Comparison of in situ gastric cancer models in Kunming mice and nude mice" Pharmaceutical Biotechnology, 2011, 18(5)