动物造模,人胃癌裸鼠转移模型 z-bio 2021-10-09 873

　　Background: Gastric cancer is the fourth most common malignant tumor in the world and the second leading cause of death after lung cancer. Current diagnosis and treatment have greatly prolonged the prognosis of patients with early gastric cancer, but the 5-year survival rate of gastric cancer patients at each stage after diagnosis is less than 50%. Metastasis is part of the reason for the high mortality rate from gastric cancer. About 50% of gastric cancer patients die from peritoneal metastasis. Therefore, metastasis has become a hot spot in many gastric cancer research. Transfer is a very complex process involving multiple consecutive steps. Genes involved in cell adhesion, motility, proliferation, survival, metabolism and signal transduction play an important role in tumor metastasis. How these proteins work synergistically to promote metastasis is not well understood. The previously reported mouse models of metastatic human gastric cancer present multiple challenges. Orthotopic transplantation of nude mice requires surgery, and the transplanted tumor tissue is derived from a human gastric cancer cell line, not a patient. As a result, the surgical procedure is very long and may cause massive bleeding and death in mice. The in situ tumor formation rate is close to 80-100%, but the metastasis rate is not high, the liver tumor metastasis rate is 45-60%, and the peritoneal metastasis rate is only 40%. Therefore, the establishment of these mouse models can benefit from improved methods that facilitate transplantation and lead to stronger metastasis. This report describes a mouse model of metastatic human gastric cancer that solves the problems of previous mouse models. By subcutaneously transplanting tumor tissue directly from patients with gastric cancer, a mouse model of metastatic gastric cancer was established. Compared with other mouse models previously described, this mouse model forms a higher proportion of tumors and, more importantly, exhibits a strong metastatic ability.

　　Method: 4-6 weeks old BALB/C nude mice, male and female weighing 16-18g, fresh tumor tissue was excised and transplanted immediately. Experimental study of subcutaneous transplantation of fresh tumor tissues under the skin of nude mice: The fresh tumor tissues removed from patients with gastric cancer were cut into 1 cubic millimeter, diluted with DMEM diluent, and then transplanted subcutaneously into the left and right armpits and and parts of nude mice. it. Sterile 16G needle. Each sample was transplanted into 4 mice at a dose of 5-6 tablets (0.8 ml) per mouse. When the nude mouse tumor grew to 1 cubic centimeter, the mouse was killed by cervical dislocation, and the tumor tissue was examined and excised under aseptic conditions. The tumor tissue of each mouse is divided into three parts, one part is used for another round of tumor transplanted into nude mice, and the other part is used for immunology, and is used for pathological examination and detection of CK8/18. Fix with 10% formaldehyde for histochemical (IHC) staining. Real-time PCR analysis of E-cadherin, VCAM-1 and ICAM-1, the third part is stored in 80℃ liquid nitrogen and CK8/18, E-cadherin, VCAM-1 and ICAM-1, dissected and inspected Tumor metastasis in mouse peritoneum, abdominal cavity, liver, spleen, stomach, intestine, kidney, lung and brain. The collected tissue was fixed with 10% formaldehyde for pathological examination. Establish and identify a mouse model of metastatic gastric cancer: Under aseptic conditions, 5-6 (0.8 mL) excised tumor tissues were subcutaneously transplanted into the left and right left of 5 nude mice under aseptic conditions. As described above, tumor tissues are removed and diluted, tumor growth in nude mice is inspected and removed, and transplanted tumor tissues and metastatic tumor tissues are inspected and treated. Effect on the metastasis rate of transplantation sites: As described above, human gastric cancer tissues transplanted from nude mice were transplanted subcutaneously under aseptic conditions to three groups of nude mice at different sites. On average, each mouse is transplanted with 5-6 mice. One group receives tissue in the left and right groin, the other group receives tissue in the left and right armpits, and the third group receives tissue in the back. They are divided into two groups. section. As mentioned above, tumor growth tests and excision were performed on nude mice. Further analysis included detection of tumor growth in various locations, peritoneal and abdominal metastases. Cryopreservation and passage of nude mouse transplantation and transplantation in human gastric cancer tissue: human gastric cancer tissue nude mice were transplanted into 4th and 8th generation nude mice, stored in liquid nitrogen, and subcutaneously transplanted as described above. Further analysis included tumor growth and metastasis to the peritoneum and abdomen in various locations. Pathological observation of nude mice transplantation and gastric cancer metastasis: 10% formaldehyde, paraffin embedding, sectioning, hematoxylin-eosin staining (HE), observation of the fixation of human gastric cancer transplantation and metastasis in nude mice. Immunohistochemical staining: Immunohistochemical methods were used to detect the expression levels of E-cadherin, VCAM-1, ICAM-1 and CK8/18 in nude mice transplanted and metastatic tumor tissues. The stained sections were taken from surgical specimens, transplanted metastatic tumor tissues and tissues containing metastatic tumors. The reagents used for staining include SP-9000 histidine plus kit, 3-3'-diaminobenzidine tetrachloride (DAB) kit, anti-E-cadherin primary mouse monoclonal antibody (1: 200 dilution), ICAM-1 (1:200 dilution). 500 dilution) included. And rabbit anti-VCAM-1 primary antibody polyclonal antibody (1:500 dilution). Two pathologists separately evaluated the IHC stained sections and determined that the difference in results can be resolved by consensus. The staining intensity was rated as negative, weak, medium or strong. Total RNA extraction and real-time PCR: Trizol is used to extract total RNA from transplanted and metastatic tumor tissues grown in nude mice and surgical specimens for transplantation. CDNA is synthesized by reverse transcriptase. SyBR premixed probes are used for real-time PCR. The 20 μL reaction solution contains 10 μL of LSyBr premix EXTaqTM, 1 μL of DNA template, 0.4 μL of each primer, and 8.2 μL of sterile water. The PCR cycle conditions were 37°C×5 minutes, 95°C×30 seconds, 95°C×5 seconds, 40 cycles, 60°C×30 seconds. Β-actin mRNA was used as an internal control, and the reaction mixture without template DNA was used as a negative control. All samples were tested separately for 3 times, and the quantitative PCR data was analyzed by contrast CT.

　　Conclusion: Tumor formation and metastasis: In the first surgical specimen on the 76th day, only one of the four mice developed a tumor at the transplantation site. After 25 days, the mice developed cervical dislocation and the tumor was analyzed. The average size of the tumor tissue is 1 cubic centimeter, showing a perfect envelope and a hard texture. Visual inspection revealed retroperitoneal metastasis. There was no metastasis to the peritoneum, abdominal cavity, liver, spleen, stomach, intestine, kidney, lung and brain. Pathological analysis showed that the transplanted tumor and metastatic tumor tissue were composed of poorly differentiated cancer cells with only a small amount of mesenchyme and blood vessels. Similar results were obtained in a parallel study involving transplanting tumor tissue into four mice. On the twenty-sixth day after transplantation, only one mouse developed a tumor. No metastases were found in the peritoneum, abdominal cavity, liver, spleen, stomach, intestines, kidneys, lungs or brain. None of the other mice transplanted with the second and fourth surgical specimens showed tumor growth. Transplanted tumor stability after multiple generations: The first surgical specimen was inherited for 10 generations. Regardless of whether the original tissue is fresh or frozen, tumor growth is 100%, and retroperitoneal and visceral metastasis is 80-100% (average 94%). Metastases were found in the lymph nodes of the esophagus, gastric submucosa, serosal membrane, spleen, hilar area, central vein and sine wave, liver parenchyma, liver capsule, renal hilar, renal parenchyma, adrenal gland, intestinal serosa, pancreas and blood vessels.

　　Tumor metastasis rate at different sites: the effect of transplantation at different sites on the tumor metastasis rate, not on the tumor growth rate. Inguinal implants cause 94% of retroperitoneal and visceral metastases, and back implants cause 10% of retroperitoneal and visceral metastases. After axillary transplantation, there is no retroperitoneal metastasis, and 20% of organs are metastasized. Occurrence time: 16 days for inguinal tumor transplantation, 20 days for back tumor transplantation, and 14 days for axillary tumor transplantation. Metastatic organs include liver (50%), kidney (44%), intestine (28%), esophagus (12%), pancreas (12%), stomach (6%), spleen (6%) and blood vessels. Including 6%). )).

　　"Characteristics of transplantation and metastatic tumors: IHC and real-time PCR results show that ICAM-1, VCAM-1 and CK8/18 are mainly expressed in primary and first-generation transplantable tumors and E-cadherin. will not occur. The primary and first-generation tumors stained positively for VCAM-1 and CK8/18, but the offspring stained these proteins weakly: VCAM-1 staining was second. The first generation (++) was moderately positive/18, and the signal was weak (+), and CK8 was stained as the first generation weak signal (+). The E-cadherin staining of tumors at each stage was negative, while the E-cadherin, ICAM-1, VCAM-1 and CK8/18 staining of each generation of metastatic tumors were all negative.

　　Conclusion: Tumor metastasis is a complex multi-step process. Many genes and factors are related to tumor metastasis, but the exact molecular mechanism is still unclear. This study established a mouse model of metastatic human gastric cancer with a strong metastatic phenotype, which helps to understand the molecular mechanism of this process.