The establishment of an in situ transplantation model of human gastric cancer tissue block nude mice gastric cancer can: (1) provide an ideal animal model for the study of the pathogenesis of gastric cancer; (2) provide an ideal animal model for the study of experimental treatment of gastric cancer; and (3) establish an ideal animal model that can best represent the invasion and metastasis of human gastric cancer.
Operation method
Fibrinogen-thrombin method
Materials and Instruments
BALB C nu nu nude mice Human gastric cancer cell line SGC-7901 Move I. Preparation of experimental materials Common Problems I. Experimental discussion The establishment of an ideal model of invasion and metastasis that conforms to the natural growth state of human gastric cancer tissues is the key to the study of gastric cancer pathogenesis and preventive measures. Although the initial subcutaneous transplantation tumor of human gastric cancer in nude mice was very similar to human gastric cancer in terms of morphology, biochemical characteristics and function, the encapsulation of connective tissues around the tumor tissue restricted the metastasis of the tumor, thus failing to simulate the onset and development of gastric cancer more realistically in human body. With the continuous improvement of experimental methods, the in situ transplantation model can overcome the deficiencies of the subcutaneous transplantation model, which is closer to the basic rules of clinical gastric cancer development in terms of tumor infiltration and metastasis, and has a significant superiority compared with the subcutaneous transplantation model. Since then, it has been found that the in situ transplantation model of gastric cancer tissue block has a higher transplantation success rate and metastasis rate compared with the in situ transplantation model of cancer cell suspension, especially OB adhesive is used in the in situ transplantation model of gastric cancer tissue, which makes the experimental operation easier and the success rate of the modeling is higher. At present, OB glue has been widely used in the construction of in situ transplantation model simulating the clinical metastasis of human gastric cancer. It has the advantages of in situ transplantation, which not only maintains the structural integrity of the cancerous tissues, but also makes the modeling operation relatively simple, with less intraoperative bleeding and quicker postoperative recovery, and obviously improves the survival rate of the animals, which provides an ideal model for the research on the growth, metastatic mechanism and treatment of human gastric cancer. For more product details, please visit Aladdin Scientific website.
Human fibrinogen lyophilized powder Human thrombin lyophilized powder Thrombin
Needles Surgical scissors Ultra-clean table Sutures
Twelve BALB /C nu /nu nude mice, both male and female, 4～6 weeks old, body mass 18～20 g, were raised under SPF grade conditions. The SGC-7901 human gastric cancer cell line was passaged subcutaneously in nude mice.
Establishment and Transmission of Subcutaneous Transplantation Tumors in Nude Mice
1. Collect SGC-7901 cells cultured in vitro, make the cell content of 5 x107 /ml, make cell suspension and inject 0.2 ml into the subcutaneous of the back of the neck of nude mice.
2. When the subcutaneous graft tumor grows to about 1 cm in diameter, remove the graft tumor aseptically, remove the necrotic tissue, rinse the tumor tissue, cut the tumor tissue into small pieces of about 1~2 mm, grind and filter the cells into single-cell suspension and inject them into the subcutaneous area of the back of the neck of the nude mice, and then pass the tumor on to the nude mice repeatedly for 5 generations.
Establishment of in situ transplantation model
1. The 5th generation of tumorigenic nude mice were removed from the 5th generation of subcutaneous transplantation tumors by aseptic operation after cervical dissection and execution, and the fibrous envelope and necrotic tissues were removed, and the well-grown tumor tissues in the form of light-red fish-like tissues were selected and cut into 1 mmx1 mmx2 mm pieces with ophthalmology, and then immersed in the prepared fibrinogen solution and placed in an ice bag for spare.
2. The experimental nude mice were fasted before the operation, anesthetized with 1 ml of ketamine ( 5 g/ml) intraperitoneally, the limbs were fixed and the skin was disinfected routinely, and the skin was incised 1.5 cm from the left side of the abdomen, the stomach was gradually entered into the abdomen, and the gastric wall was revealed and then the stomach was pulled out, and the plasma musculus layer of the gastric wall was scratched with a 1 ml sterile empty needle at the greater curvature of the stomach near the gastric sinus, and 3～4 tumor tissue blocks were implanted, and the surface of the tumour was dropped with about 10 & & ml of coagulation solution. mu;l thrombin to cover the surface of the tumor tissue, about 10 s or so to be thrombin and fibrinogen reaction to form a gel, the tumor tissue block adhesion in the broken gastric wall, and then the mouth of the gastric wall into the abdominal cavity.
3. 6-0 suture was used to close the peritoneum (including the muscle layer), 3-0 suture was used to close the abdominal wall, and the abdomen was closed to end the operation, and all the above operations were carried out in an ultra-clean table, and a total of 6 models were constructed.
IV. Observation of tumorigenicity rate of in situ transplantation and invasion and metastasis of transplanted tumors1. After transplantation, all nude mice were carefully kept and regularly observed for general condition, feeding, exercise and abdominal signs. 2.
2. when the tumor-bearing mice showed signs of general exhaustion such as emaciation, depression and arching of the back, they were decapitated and killed, and the abdominal cavity was examined for the growth of the tumor, the formation of peritoneal fluid and the involvement of the surrounding organs.
3. Then, the in situ tumor tissue, the important organs in chest and abdominal cavity, such as stomach, lung, liver, peritoneum, etc., shall be fixed with 10% formaldehyde, embedded in paraffin, sectioned, and stained with HE, and then subjected to histopathological examination.
4. Immunohistochemical staining (SP method) was used for the detection of proliferating cell nuclear antigen (PCNA) in the in situ tumor tissues, and the cells with nuclei stained brownish-yellow were considered as positive cells. The criteria for judging the positive results were: 5 high magnification fields were taken randomly, and the percentage of the positive cells was judged by their percentage, ≤5% ( - ), 6%～25% ( + ), 26%～50% ( + + + ), and ≥51% ( + + + + ).