1.1 Experimental animal. 30 Japanese big ear white rabbits (hereinafter referred to as rabbits), half male and half female, 3 months old, weighing 2.0-2.5 kg, were purchased from Beijing Changyang Xishan farm [animal license No.: scxk (Beijing) 2016-0007]. The rabbits were fed at 25 ℃ and 50% humidity for 12 / 12 h light / dark cycle. Access to food and water is limited (2 times per day). This study was approved by animal ethics committee of Affiliated Hospital of Chengde Medical College (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 Experimental cells and instruments. X-ray machine (OEC9800, Shanghai Xianwei Optoelectronic Technology Co., Ltd.); Microplate reade (Multiskan FC) was provided by the Central Laboratory of the Affiliated Hospital of Chengde Medical College. Olympus-BX53 optical microscope (Olympus-BX53, Olympus company of Japan); Bone marrow aspiration biopsy needle [BM09/15, Demeter medical technology (Beijing) Co., Ltd.]. VX2 tumor cell line was purchased from Chongqing Mengbo Biotechnology Co., Ltd. PCNA, CD34 antibody kit (purchased from Hebei ruipat Biotechnology Co., Ltd.); The self-designed and improved channel built-in bone tumor pathological tissue extraction device for closed biopsy (Patent No.: zl201416055578.0) is made of titanium alloy (Fig. 1 and Fig. 2).
1.3 Preparation of VX2 tumor tissue. VX2 tumor cells were injected into the left lateral femoral muscle of rabbits for continuous passage culture, and the tumor size was evaluated by color Doppler ultrasound. Four weeks later, when a mass of 3 cm × 3 cm in size was palpable subcutaneously, anesthesia was performed by intravenous injection of pentobarbital sodium (35 mg/kg). The disappearance of corneal reflex was regarded as the successful sign of anesthesia. The rabbit hair was removed from the tumor area and fixed on the animal experimental table in prone position. The operation area was disinfected with Iodophor, covered with sterile pore towel, skin was cut, and subcutaneous tissue and tumor were separated. After the tumor was completely removed, the tumor was moved to a sterile worktable and washed in PBS at 37 ℃ for 3 times to remove the blood stain, necrotic tissue and fibrous tissue in the tumor. The tumor was cut into 1 mm × 1 mm × 1 mm tissue block with ophthalmic scissors.
1.4 Preparation of rabbit VX2 bone tumor model. 30 rabbits were anesthetized by intravenous injection of pentobarbital sodium (35 mg / kg). After depilation, the rabbit hind limbs were fixed on the operating table in supine position. The hind limbs were disinfected with Iodophor and covered with sterile hole sheet. A 1 cm incision was made in the inner skin of the knee joint. The subcutaneous tissue and fascia were incised layer by layer to expose the patellar ligament. The patellar ligament was separated longitudinally and the tibial plateau was exposed. Drill the tibial plateau with Kirschner wire (1 mm in diameter), insert a 2 cm long cannula into the bone hole, push 1 mm3 VX2 tumor tissue into the tibial medullary cavity along the cannula, pull out the cannula, and block the bone hole with bone wax. The tissue around the bone foramen was rinsed with absolute ethanol for 3 times, and then soaked for more than 1 min each time. After reconstruction of patellar ligament, the incision was sutured layer by layer. After the rabbits were anesthetized, they were put back into the cage to continue feeding. The left leg of all rabbits was set as the experimental group, and the modified closed biopsy technique was used for biopsy; the right leg was set as the control group, and the hollow core needle was used for biopsy. The rabbits in both groups were intramuscularly injected with penicillin 800 000 U / time, once a day, for 3 consecutive days
1.5 Application of closed biopsy technique in puncture. 7 days after modeling, rabbits were anesthetized by intravenous injection of pentobarbital sodium (35 mg / kg) through ear edge vein. Under the guidance of X-ray, a 0.5 cm longitudinal incision was made at 1 cm below the left knee joint and the lateral tibia as the puncture point. Install the built-in channel on the puncture handle, penetrate through the bone cortex and enter into the medullary cavity. Pull out the puncture handle. The built-in channel remains in the bone cortex to form an artificial channel. After extracting 2 ml of pulp cavity contents through the built-in channel with G14 syringe needle, the tail nail is screwed into the internal channel (Fig. 3) to achieve the effect of closing the puncture channel and suture the incision layer by layer. The same method was used in the diagnosis of bone tumor with hollow core needle aspiration biopsy in the right tibia. 1 cm below the right knee joint and the outside of the tibia was used as the puncture point. After exposing the bone surface, the bone marrow puncture needle was used for puncture biopsy. After obtaining 2 ml bone marrow sample, the puncture hole was not sealed, and the puncture hole was compressed to stop bleeding, and the incision was sutured layer by layer. After the puncture of bone tumor was completed, the rabbits were put back into the cage to continue feeding. The rabbits in both groups were intramuscularly injected with penicillin 800 000 U / time, once a day, for 3 consecutive days.
1.6 Imaging and pathological examination 14 days after modeling, X-ray examination of bilateral tibia, intraperitoneal injection anesthesia (pentobarbital sodium, 40 mg / kg), ear vein air embolism were performed to kill the rabbits, with the disappearance of palpable chest heartbeat as the death mark. The soft tissue within 2 cm around the puncture hole was resected. Hematoxylin eosin staining (HE staining) and microscopic examination were performed. The results of microscopic examination were performed by the same senior pathologist with blind method.
1.7 Elisa examination 0.1 mg tissue slice was transferred into a glass grinder on the sterile operating table. PBS (1 mg: 9 ml) was added to grind it fully, centrifuged at 4000R / min for 5 min. The supernatant was collected. The content of PCNA and CD34 in the tissue supernatant was determined by ELISA. The test process was carried out in strict accordance with the instructions of the kit.
1.8 Statistic analysis. SPSS 25.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Chi square test was used to analyze the results of imaging and pathological examination 14 days after modeling. The expression levels of PCNA and CD34 in the soft tissue around the puncture hole of VX2 bone tumor were analyzed by paired samples t test. P < 0.05 was statistically significant.