动物造模,实体瘤局部治疗动物模 z-bio 2021-10-28 355

　　Background: Rabbit VX2 tumor models are commonly used in experimental oncology. Rabbit VX2 tumors are undifferentiated squamous cell carcinomas caused by viruses. It is worth noting that this model is used to simulate different types of cancer, including pancreas, kidney and liver. As mentioned above, VX2 cell transplantation can be achieved by transplanting solid tumor sections or injecting tumor cell suspensions (fresh or frozen). Various forms of neoadjuvant therapies and adjuvant therapies have been combined with surgery to improve local control of local tumors. The local recurrence rate after tumor resection includes rectal cancer, soft tissue sarcoma and pancreatic cancer. This study established a rabbit pararenal carcinoma model with locally transplanted VX2 frozen tumors. The tumor model can be used to evaluate the multimodal treatment of high risk of local recurrence after solid tumor surgery.

　　Method: Animal preparation: 25 New Zealand rabbits (weight 2.5-3.5 kg) can eat and drink at will, and adapt to the facility for one week. After the operation, the rabbit is placed in a temporarily heated cage to restore its vital functions. Then move it to a regular cage.

　　Preparation of VX2 tumor fragments: VX2 tumor fragments are stored in the refrigerator at -80°C until use. The bottle containing the tumor fragments was placed in a 37°C water bath. Although it was not completely melted, it melted quickly. Use sterile dissecting forceps to carefully remove the tumor mass from the cryopreservation tube and place it in 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, the debris was carefully cleaned three times. The tumor was placed in a 15ml cold HBSS sterile petri dish and placed on ice until transplanted into the animal. The development and harvest of VX2 hindlimb tumors: Four New Zealand white rabbits were used to replicate VX2 tumors of the erector spinae muscle. Before surgery, all medetomidine (0.2 mg/kg), ketamine (10 mg/kg), and bufenol (0.5 mg/kg) were anesthetized. General anesthesia was maintained with 2% sevoflurane and ketamine hydrochloride (30 mg/kg) through tracheal intubation. After anesthesia and preoperative skin preparation, a 2.5 cm incision was made from the right hind limb to the muscle abdomen, and two small tumor masses of 5 cubic mm were transplanted into the quadriceps femoris of each hind limb. Stop bleeding and suture the incision. .. 10-14 days after tumor transplantation, the surviving tumor appears as a palpable nodule measuring about 2.5 cm x 3 cm. After confirming tumor growth by ultrasound, the hind limb tumor is removed. Each rabbit was euthanized with 1 ml/kg tanax under general anesthesia. Subsequently, skin preparation and a 4-5 cm incision were made on the right hind limb in the area surrounding the tumor. Make two curved incisions in the muscle for mass removal. Divide the resected tumor into two parts and remove the necrotic nucleus to obtain a usable tumor. Place the tumor fragments in 30 ml PMI1640 medium containing 3% 10000 IU/mL penicillin, streptomycin 10000μg/ml and 1% amphotericin B (250μg/ml) for 30 minutes, and then place the tumor tissue in 100 mm Petri dish. -Scrape the tumor tissue. The tumor tissue is divided into several pieces (about 5 cubic millimeters each). Use about 20 ml HBSS, 20 ml plus 5% fetal bovine serum DMEM/F12 medium to quickly clean the tumor fragments 3 times. Half of the tumor tissue was used to establish a kidney tumor model, and the rest was frozen. Establishment of rabbit VX2 pararenal tumor model: In this experiment, 21 rabbits were used, 5 of which were transplanted with fresh tumor tissue, and 16 were frozen. Before sacrificing the animals, blood was drawn from the rabbit ears for testing. Centrifuge the blood (3000pm, 5 minutes) to separate the plasma fraction. Evaluation of liver enzymes (ALT, AST, GGT, alkaline phosphatase), bilirubin and complete blood count (CBC), repeated every 7 days, until the animal was sacrificed. Laparotomy of the xiphoid umbilical cord was performed on the rabbit and the tumor was transplanted into the kidney. The dull peeling will expose the peritoneum. Careful dissection of the peritoneum can reveal the abdominal cavity. Move the intestine and make an incision in the upper renal capsule in the left retroperitoneal space. Next, place a tumor in the pararenal space. Suture the incision. After confirming hemostasis, close the abdominal incision. After the operation, the rabbit is placed in a temporarily heated cage to restore its important functions, and then transferred to a normal cage. Observe appetite, feces, urine volume and concentration, mental state and activity level every day. Appropriate treatment measures will be taken to resolve abnormal situations found in daily observations, and appropriate animal welfare measures will be taken. In vivo ultrasound images assess tumor growth.

　　Invivo high-resolution ultrasound imaging: each rabbit lies on its own table. Monitor the body temperature with a rectal probe and heat the blanket to maintain 37±0.5°C. In the process of imaging all animals, all imaging settings will not be changed. The animals were examined for the surgical transplantation area and the development of liver tumors 7 and 14 days after the operation. For each rabbit, all abdominal solid organs and abdominal cavity were explored to assess whether there is free fluid in the solid disease or retroperitoneal cavity. Especially the size, shape, location of the kidney and the space around and beside the kidney. Histological examination: The animals were sacrificed under general anesthesia, and the kidney specimens were examined with naked eyes and a microscope. Cut each organ into 5 mm sections, and the consensus of the two observers determined the malignant lesion. A representative sample was frozen, sectioned with a cryostat, and inspected with a conventional HE staining optical microscope. Results: A rabbit VX2 limb tumor model was established: Four rabbits with a VX2 tumor model of the right lower right spinal cord muscle of the hind limbs were established. Frozen sections of two small tumors (previously thawed in warm water) were transplanted into the quadriceps of each rabbit's hind limbs. After 10-14 days, each rabbit had a tumor measuring about 2.5 cm x 3 cm. 7 and 14 days after the operation, the tumor was characterized by uneven and irregular infiltrating masses, and muscle tissue growth was detected. After sacrificing the rabbit, the hind limb tumors were removed to obtain the growth of the VX2 tumor cell line and the development of kidney tumors. Half of the tumor tissue is used to build kidney tumor models in a new way, and the rest is frozen. Histological analysis confirmed the presence of tumor cells in the tumor specimens.

　　Establishment of rabbit VX2 pararenal tumor model: The rabbit underwent open surgery on the xiphoid umbilical cord to create a rabbit model of kidney tumor VX2. The dull peeling reveals the peritoneum. The results are shown in the figure. It must be emphasized that the transplantation of tumor fragments can achieve 100% tumor growth. So far, this is the first report of the treatment of rabbit pararenal carcinoma model by surgical tumor transplantation of frozen VX2 tumor. Conclusion: In this study, a frozen section of VX2 tumor was first transplanted into the adrenal region of rabbits to establish a pararenal carcinoma model. This method minimizes the metastasis and development of non-necrotic tumors and optimizes the evaluation of experimental tumor response in local treatments.