动物模型,兔VX2肾旁癌 z-bo 2020-12-06 331

　　Background: Rabbit VX2 tumor models are often used in experimental oncology. Rabbit VX2 tumor is an undifferentiated squamous cell carcinoma caused by a virus. It is worth noting that this model has been used to simulate various types of cancer, including pancreas, kidney and liver. VX2 cell transplantation can be achieved by implanting solid tumor slices or injecting tumor cell suspension (fresh or frozen), as described previously. Various forms of neoadjuvant therapy and adjuvant therapy have been used in combination with surgical operations to improve local control of local tumors. The local recurrence rate after tumor resection is high, such as rectal cancer, soft tissue sarcoma and pancreatic cancer. In this study, a rabbit pararenal carcinoma model with locally implanted VX2 frozen tumor was established. This tumor model may be used to evaluate multimodal treatments with high risk of local recurrence after solid tumor surgery.

　　 Method: Animal preparation: Twenty-five New Zealand rabbits (weight 2.5-3.5 kg) eat and drink freely, and acclimatize in the facility for one week. After the operation, the rabbit was placed in a temporarily heated cage to restore its vital functions. It was then transferred to a normal cage.

　　VX2 tumor fragment preparation: VX2 tumor fragments are stored in a refrigerator at -80 °C until use. The bottle containing tumor fragments was placed in a 37 °C water bath to thaw quickly but not completely. Use sterile dissecting surgical forceps to carefully remove the tumor mass from the cryotube and place it on a petri dish. The medium contains 25 ml of DMEM medium containing 5% fetal bovine serum. Subsequently, the fragments were washed with cold Hanks balanced salt solution. In the two cleaning processes, these fragments were carefully cleaned three times. The tumor is placed in a 15ml cold HBSS sterile petri dish and placed on ice until implanted in the animal.

　　 VX2 hindlimb tumor occurrence and harvest: Four New Zealand white rabbits were used to replicate VX2 tumors on the inferior spinal muscles. Before surgery, all rabbits were anesthetized jointly with metetomidine (0.2 mg/kg), ketamine (10 mg/kg) and butorphanol (0.5 mg/kg). General anesthesia was maintained by tracheal intubation with 2% sevoflurane and ketamine hydrochloride (30 mg/kg). After anesthesia and preoperative skin preparation, a 2.5 cm incision was made from the right hind limb to the muscle belly, and two small tumor masses of 5 cubic millimeters were implanted into each hind limb quadriceps femoris muscle to stop bleeding and suture the incision. 10–14 days after tumor implantation, a viable tumor appears as a palpable nodule of approximately 2.5 cm × 3 cm. Ultrasonography confirmed tumor growth, and then the hind limb tumor extraction was performed. Each rabbit received an injection of 1 ml/kg tanax under general anesthesia for euthanasia. Subsequently, skin preparation and 4–5 cm incision were performed on the right hind limb to the area surrounding the tumor. Two curved incisions were made in the muscle for mass tumor resection. The excised tumor is divided into two, and the necrotic nucleus is removed to obtain a usable tumor. The tumor fragments were placed in 30ml of RPMI1640 medium containing 3% 10000 IU/mL penicillin, streptomycin 10000μg/ml and 1% amphotericin B (250μg/ml) for 30min, and the tumor tissue was placed in a 100 mm In the petri dish, any visible non-tumor tissue is shaved off. The tumor tissue was broken into several pieces (about 5 mm3 each). Tumor fragments were quickly washed with about 20 ml HBSS, 20 ml with 5% fetal bovine serum DMEM/F12 culture medium, 3 times each. Half of the tumor tissue was used to develop a kidney tumor model, while the rest was frozen.

　　 Establishment of rabbit VX2 pararenal tumor model: 21 rabbits were used in this experiment, five of which were implanted with fresh tumor tissue and sixteen were frozen section. Before the animals were sacrificed, blood was obtained from rabbit ears for testing. Centrifuge the blood (3000 rpm, 5 min) to separate the plasma fraction. Liver enzymes (ALT, AST, GGT, alkaline phosphatase), bilirubin and complete blood count (CBC) were evaluated, and repeated every 7 days until the animal was sacrificed. In order to implant the tumor into the kidney, a xiphoid-umbilical laparotomy was performed on the rabbit. Blunt peeling exposes the peritoneum. Carefully dissecting the peritoneum can detect the peritoneal cavity. Move the intestine, left retroperitoneal space upper renal capsulotomy. Then place a tumor fragment in this pararenal space. Suture the incision. After confirming hemostasis, close the abdominal incision. After the operation, the rabbit was placed in a temporarily heated cage to restore its vital functions and then transferred to a normal cage. Observe the amount and concentration of appetite, feces and urine, mental state and activity level every day. Appropriate treatment measures are taken to resolve any abnormalities noted in daily observations and recorded in the appropriate animal welfare form. In vivo ultrasound imaging evaluates tumor growth.

　　 In vivo high-resolution ultrasound imaging: Each rabbit lies on a dedicated table. Body temperature is monitored by rectal probe and maintained at 37 ± 0.5°C by heating blanket. All imaging settings remain unchanged during imaging of all animals. The animals were tested on the surgical implantation area and the development of liver tumors on the seventh day and the fourteenth day after the operation. For each rabbit, explore all the abdominal solid organs and peritoneal cavity to assess the presence of free fluid in any solid lesions or retroperitoneum. In particular, the size, shape and location of the kidney, the space around and beside the kidney.

　　 Histological examination: The animals were sacrificed under general anesthesia, and the kidney specimens were studied macroscopically and microscopically. Each organ was cut into 5 mm sections, and the malignant lesion was determined by the consensus of two observers. Representative samples were frozen, sectioned with a cryostat, and examined under conventional HE staining light microscope.

　　Result: Establishment of rabbit VX2 limb tumor model: 4 rabbits with VX2 tumor model of right lower right spinal muscle of hind limb were established. Two frozen sections of small tumors (previously thawed in warm water) were implanted into the quadriceps femoris of each rabbit's hind limb. After 10-14 days, each rabbit acquired a tumor of approximately 2.5 cm × 3 cm. At 7 and 14 days after the operation, the tumor features uneven, irregular, infiltrating masses, growing in muscle tissues. In order to obtain the proliferation of the VX2 tumor cell line and the occurrence of kidney tumors, the hindlimb tumors were extracted after the rabbits were sacrificed. Half of the tumor tissue was used to establish a kidney tumor model in a fresh way, while the remaining part was frozen. Histological analysis confirmed the presence of tumor cells in tumor specimens.

　　 Establishment of rabbit VX2 pararenal tumor model: In order to create a rabbit model of kidney tumor VX2, the recipient rabbit underwent xiphoid-umbilical open surgery. Blunt peeling reveals the peritoneum. The result is shown in the figure. It must be emphasized that the implantation of tumor fragments can achieve 100% tumor growth. So far, this is the first report of a frozen VX2 tumor model for surgical tumor implantation to treat rabbit pararenal carcinoma.

　　 Conclusion: For the first time in this study, frozen sections of VX2 tumors were implanted into the accessory kidney area of rabbits, and a pararenal carcinoma model was established. This method minimizes non-necrotic tumor metastasis and development, and will optimize the evaluation of local area treatment experimental tumor response.