1.1 Laboratory animals, tumor lines
Thirty Japanese rabbits were divided into two groups by the random number method: the experimental group and the control group, 15 in each group, aged 3 months, male, weighing 2.0-2.5Kg. They were purchased from Beijing Changyang Xishan farm where animal license number was SCXK (Beijing) 2016-0007. According to the relevant regulations of Chengde Medical College, all the experimental animals were kept in cages and fed with standard rabbit feed and sanitary water. VX2 tumor tissue was purchased from Chongqing Mengbo Biotechnology incorporated company. The process of animal use in this experiment has been reviewed and approved by the the affiliated Hospital of Chengde Medical College committee on animal care and use, which follow the requirements of medical ethics (Approval batch number: LL047). The study was conducted in strict accordance with the recommendations of the National Institutes of Health’s guidelines for the Care and use of Experimental Animals.
1.2 Main experimental equipment and reagents
X-ray machine (model: OEC9800, USA), optical microscope (Model: Olympus-BX53, Japan), microplate reader of the multi-wavelength measurement system (Model: Multiskan FC, USA), the self-designed and improved channel built-in bone tumor pathological tissue extraction device (Patent number: ZL20141065578.0, Affiliated Hospital of Chengde Medical College) which is made of titanium alloy, ELISA kit was purchased from Shanghai Yubo Biotechnology incorporated company. We used them to detect IFN- γ, IL-4 and PCNA.The other instruments were derived from our laboratory. For example, scissors, leather knives, cotton swabs, cotton balls, etc.
1.3 The establishment of VX2 bone tumor model in rabbit
1.3.1 The preparation of VX2 tumor tissue
Cryopreserved VX2 tumor tissue was resuscitated under -4℃ conditions, and then minced tissue into 1 mm×1 mm pieces under aseptic condition, and implanted the tissue piece into the lateral femoral muscle of the posterior limb of the rabbit to prepare the tumor-bearing rabbit. The tumor growth diameter was about 1 cm every week. The tumor-bearing rabbits were successfully prepared after 3 weeks of inoculation. The tumor-bearing rabbits were anesthetized by intravenous injection of 3% pentobarbital (35 mg/Kg) at the ear margin. The rabbits were fixed on the sterile operating table in supine position. We disinfected the skin of the tumor area and laid the sterile hole sheets. We cut the skin and subcutaneous tissue and completely removed the tumor tissue, which was placed in phosphate buffer solution. We also carefully removed the surrounding fibrous connective tissue and necrotic tissue and left the fish-like tissue. Under sterile conditions, the tumor tissue was minced into1mm×1mm×1mm pieces for spare.
1.3.2 The establishment of rabbit VX2 bone tumor model.
Thirty Japanese white rabbits were anesthetized by intravenous injection of 3% pentobarbital (35 mg/Kg) at the ear margin and then fixed on the operating table in supine position to remove the hair of the hind limbs of rabbits. After routine disinfection, we laid a sterile hole sheet. A 2cm long incision was made on the medial side of the upper tibia. The skin, subcutaneous tissue, and fascia were cut and separated layer by layer. The patellar ligament was split longitudinally to expose the medial tibial plateau. We used a 1.6 mm Kirschner wire to drill a hole parallel to the longitudinal axis of the tibia and perpendicular to the medial bone surface of the tibial plateau. The depth of the hole was about 2 cm. In the experimental group, we placed the prepared 2-3 pieces of tissue which size was 1mm×1mm×1mm, into the bone marrow cavity. In the control group, we took the above operation. But no tumor was implanted. We used bone wax to block the tumor graft hole to avoid tumor extravasation and medullary hemorrhage. We used distilled water to clean the surrounding tissue of the implant hole and reconstructed the patellar ligament. We used silk thread to suture the subcutaneous tissue and skin layer by layer. The incision was disinfected with Iodophor and bandaged with sterile gauze. The rabbits were sent back to the cage after complete recovery. The antibiotics were injected intramuscularly into rabbit three days after operation to prevent infection.
1.4 The device for removing pathological tissue of bone tumor
After the first week, all the instruments were sterilized. The suitable puncture needle was rotated into and through the tibial cortex of rabbit. We pulled out the core of the puncture needle. The hollow metal channel sleeve was screwed into the bone cortex with the channel implantation handle to keep the sleeve position still. The pathological needle was taken and rotated along the channel into the rabbit tibial bone marrow cavity to take out the bone tissue, and the plugging tail nail was screwed into the metal channel sleeve with a screwdriver to block the puncture hole. The puncture site was compressed for 5 minutes and then bandaged. After the completion of the puncture, we used tibial X-ray examination to evaluate the removal device(Figure 1A: hollow metal channel and blocking tailpiece; Figure 1B: adaptable piercing needle; Figure 1C: cross reamer and tailpiece; Figure 1D: channel implantation handle) .
1.5 The detection of peripheral blood and tissue supernatant
1.5.1 The specimen collection
The collection of peripheral blood: in the first and second weeks of tumor implantation, 3ml of ear vein blood was collected from the vacuum blood collection vessel containing coagulant. The collected blood was stood at room temperature for 1 hour, and then the serum was precipitated. After centrifugation at 2000r / min for 10min, the upper serum was collected at -80℃ which was preserved in refrigerator at low temperature. The collection of tumor tissue: after the first week of tumor implantation, the tumor tissue and surrounding soft tissue were collected by using the self-designed channel built-in bone tumor pathological tissue removal device. After the second week of tumor implantation, intraperitoneal injection anesthesia (pentobarbital sodium, 40 mg/kg), and ear vein air embolism were performed to kill the rabbits, with the disappearance of palpable chest heartbeat as the death mark. The tumor tissue and surrounding soft tissue were collected by tibial anatomy. After weighing all the collected tumor tissue, a certain proportion of phosphate buffer solution (1mg: 9ml) was added, and the tumor tissue was fully homogenized. After centrifugation at 2000 r / min for 10 min, the supernatant serum was collected at -80℃ which was preserved in refrigerator at low temperature.
1.5.2 The Elisa detection of collected specimens
The serum and tissue supernatant sample were take out of refrigerator and we put it in the refrigerator at 4 ℃ overnight. The kit, serum and supernatant sample were returned to room temperature 30 minutes before the experiment, and the test was carried out in strict accordance with the instructions of ELISA kit. We set up 7 standard solution tubes. Blank holes and sample holes to be tested were set on 96 well enzyme plate. 40ul of sample diluent was added to the sample hole to be tested, and then 10ul of sample to be tested was added. Two double holes were set for each standard and sample. After the enzyme plate label was mixed, the plate was sealed with plate sealing membrane and then incubated at 37 ℃(The detection of IFN-γ and IL-4 was incubated for 30min, and the detection of PCNA was incubated for 60min). Then the 30 fold concentrated washing solution was diluted with distilled water for 30 times and then was used for standby. After the completion of incubation, the sealing plate membrane was removed and the liquid was discarded and dried. Each hole was filled with washing solution, and then discarded after standing for 30s. After repeated washing for 5 times, the enzyme labeled reagent was added to each hole(the detection of IFN-γ and IL-4 was added 50ul, and the detection of PCNA was added 100ul ). The plate was sealed with plate sealing membrane and then incubated at 37 ℃ for 30min again. Then, we wash it repeatedly for 5 times, and then dry it. Then add A50ul of chromogenic agent and B50ul of chromogenic agent into each hole. After shaking and mixing, the color was developed at 37℃ for 15 min,and then we add 50ul of termination solution into each hole and mix well. Finally, we measure the absorbance value of each hole in 450nm wavelength and calculated the concentration of IFN-γ, IL-4 and PCNA.
1.6 The histopathological examination
The tumor tissue and surrounding soft tissue collected in two weeks were fixed with 10% formalin, dehydrated, embedded in paraffin, sectioned and stained with hematoxylin eosin(HE). The histopathological morphology was observed under microscope to understand the growth of tumor and verify the value of tumor biopsy. The results of microscopic examination were performed by the same senior pathologist with blind method.
1.7 Statistical data processing
The measurement data are expressed as mean±standard deviation and t-test is adopted. The counting data were tested by c2 tests. Pearson correlation analysis was used to test the correlation between IFN-γ and PCNA, IL-4 and PCNA. Using SPSS 25.0 (SPSS Inc., Chicago,IL, USA) for statistical analysis, the difference was statistically significant when P<0.05.