动物模型,胫骨骨癌痛模型 z-bo 2020-07-25 1644

      According to the latest research, about one-third of patients with advanced cancer will have bone metastases, making bone cancer pain the most common type of pain in cancer patients, seriously affecting their quality of life. Impact With the development of medicine, more and more bone cancer pain treatment methods are available, but due to short-term treatment effects and side effects, it is impossible to ideally control an important part of bone cancer pain. The key to limiting the emergence of new therapies is that the cause of bone cancer pain is unclear. The long-term lack of reliable animal models hinders the study of bone cancer pain mechanisms and the development of new analgesics. Until 1999, Schwei et al. reported the first mouse model of femoral bone pain. Then Medhurst waited. Established a globally recognized rat model of tibia cancer pain. The establishment of the model provides an excellent tool for understanding the etiology and treatment of human bone cancer pain. However, in China, due to the limitations of various laboratory conditions and cell lines used for modeling, the replication of bone cancer pain models is still a relevant research bottleneck. This chapter details the improved Medhurstrat tibial cancer pain modeling method. After cell inoculation, a self-control method was used to compare the changes in pain behavior before and after, X-ray and HE stained sections were used to confirm the occurrence of bone damage, and to evaluate whether the test model was successfully established. A series of surveys will provide the right tools.

　　The first section of laboratory equipment and reagents

  One. Experimental equipment The experimental equipment includes a light-heated tail pain tester, a plantar pain tester, an inverted microscope (DMI6000B), an automatic tissue embedding machine (ZMN-7803), an electric constant temperature water bath, a constant temperature drying box, a microwave oven, and a refrigerator (BCD-203) ), optical microscope (BX41TF), tissue slicer (Leica2135, 2145), micro sampler (5μl), pipette (1 ml), sterile console.

　　 2. Main reagents

　　 Gibco Fetal Bovine Serum, 75% ethanol, 0.01moL/LPBS, physiological saline, sterile bone wax, 4% paraformaldehyde, PBS fixative, 10%, 20%, 30% sucrose. , Frozen section embedding agent, section protection agent, 3.6% chloral hydrate, 0.25% trypsin, immunosuppressant, 2.5% cefotaxime sodium.

　　 3. Preparation of experimental reagents

　　 (1) Preparation of 0.01mol/LPBS

　　ACl8g

　　A2HPO4? 12H2O3.634g

　　KCl2g

　　KH2PO4g

　　KH2PO4g

　　KH2PO4g

　　KH2PO4g

　　KH2PO4

　　 (2) Prepare a beaker with a capacity of 1000 ml and hold about 800 ml of deionized water, and add the measured reagents in order.

　　 (3) Stir the glass rod to completely dissolve it, and finally dilute it to 1000 ml.

　　 (2) Prepare 0.25% trypsin

　　 (1) Prepare a clean, dry beaker with a capacity of 200 ml.

　　 (2) Weigh 0.25g trypsin with an electronic balance and dissolve it in 100ml 0.01mol/LPBS.

　　 (3) Filter with a 0.2μm filter membrane, package, and store in a refrigerator at -20°C for later use. Preparation of immunosuppressant 250mg 5ml cyclosporine diluted 10 times with normal saline to make 250mg 50ml, concentration 5mg/ml.

　　 (4) 3.6% chloral hydrate

　　 3.6 g of chloral hydrate, weigh out 100 ml of distilled water, mix well and seal for later use. (5) 4% paraformaldehyde-PBS fixative dissolve 40g paraformaldehyde in 800ml 0.01mol/L PBS, dissolve it in a constant temperature water bath at 60℃, and dilute to 1000ml. (6) 2.5% cefotaxime sodium 1g of cefotaxime sodium is made into a solution of 2.5% concentration in 40ml saline.

　　 4. Other equipment needed for the experiment

　　 (1) Experimental equipment

　　Inverted microscope, constant temperature water bath, CO2 incubator, 5ml syringe, 10ml syringe needle, tissue scissors, screw scissors, curved forceps, tooth-shaped forceps, toothless forceps, etc.

　　 cell culture device

　　 Sealing membrane, 75% alcohol cotton ball, alcohol lamp, 15 ml centrifuge tube, 6-well cell culture plate, 1 ml sterile pipette tip, 1 ml pipette, etc.

　　Section 2 Experimental Method

　　Ichi. In vitro culture of rat breast cancer cell line MRMT-1

　　 (1) MRMT-1 cell recovery

　　 (1) Put the pre-warmed culture medium on the stage.

　　 (2) Wear sterile surgical gowns, gloves, masks, caps and sterile gloves with 75% alcohol cotton balls.

　　 (3) Tear open the packaging of the sterile 6-well cell culture plate on the table, take out the 15 ml centrifuge tube from the lunch box and place it on the shelf.

　　 (4) Remove the medium sealing film, wipe the bottle mouth with a 75% alcohol cotton ball, heat and add 9 ml medium to the centrifuge tube.

　　 (5) Cover the mouth and stopper of the culture bottle because they are too hot.

　　(6)-Take out the cryotube containing the resuscitated cells from the refrigerator at -150°C, insert it into the bleaching plate, and then shake it at 37°C.

(7) Put the thawed cells on the table, disinfect your hands with 75% alcohol cotton ball, wipe the mouth of the cryostat with 75% alcohol cotton ball, heat, open the bottle cap, heat, freeze and freeze the cell culture medium , Put it in a 15 ml centrifuge tube containing 9 ml of medium, centrifuge immediately, and centrifuge at 1000/min for 10 minutes to remove the supernatant. (8) Next, add 12 ml of fresh medium (the serum is from Australia), mix and inoculate the culture plate. When observed with an inverted microscope, the cells float evenly.

　　(9) Incubate at 37°C in a 5% CO2 incubator.

　　 (2) Cells pass

　　(1) Observe the cell growth under an inverted microscope, and find that the cells occupy 80% to 90% of each well of the entire culture plate, start.

　　 (2) Prepare the ethanol and alcohol cotton balls needed for the experiment, and put the used culture medium in the incubator for preheating.

　　 (3) 30-minute ultraviolet lamp disinfection test table.

　　 (4) Put on a hat, mask, gown and gloves, and put the packed and sterilized pipette tip box and EP tube on an ultra-clean table for immediate delivery.

　　 (5) Take out the frozen cell culture plate and pre-warmed culture medium from the incubator, put them on a clean table just sprayed with alcohol, and wipe them with alcohol swabs for disinfection.

　　 (6) Remove the sealing film from the mouth of the bottle containing the culture solution, wipe the connection between the mouth of the bottle and the cap with an alcohol swab, and then place the alcohol lamp on the left side of the lamp.

(7) Remove the cover of the culture plate, put down the cover, lift the culture plate and make a 45° angle with the surface of the ultra-clean workbench, then fix it on the bottom of each culture well with a pipette, suck out the liquid and discard it, use PBS rinse 1 Next, remove the PBS, add 0.25% trypsin for digestion, cover the cells, close the lid, and digest for 2-3 minutes at room temperature.

　　 (8) Stop digestion with serum-containing medium, and gently pipette to suspend adherent cells.

　　 (9) Collect the cells in a centrifuge tube, and centrifuge the cells at 1000/min for 10 minutes. (10) Remove the medium, add 12 ml of new medium to make a cell suspension, and count the cells. (11) Dilute the cell suspension to a cell density of 2 x 100000 cells/ml for seeding.

　　(12) Incubate at 37°C in a 5% CO2 incubator.

　　 2. Bone cancer pain model replication

"See Medhurst et al. for the method of establishing a rat bone cancer pain model." (1) Twenty adult clean female SD rats were selected and randomly divided into two groups: sham operation group (control group) and bone cancer pain model group (experimental group).

　　 (2) When the anesthesia is effective, inject 1 ml/100 g of 3.6% chloral hydrate into the abdominal cavity and fix it in the supine position.

　　 (3) Trim the left hind leg, disinfect the skin with iodine, and spread the sterile drape.

　　(4) Make a small 1 cm incision in the thinnest part of the subcutaneous tissue above the tibia (about 5 mm below the knee) to clearly separate the subcutaneous tissue and carefully expose the tibia. (5) Separate the local periosteum with a 10 ml injection needle, make a hole perpendicular to the surface of the tibia at the surgical site, hold the tibia with the index finger against the force of the drill, and bend the knee joint of the hind limb. In the bone marrow cavity, you can maintain a sense of breakthrough in your body. Slowly inject 3μl containing

　　(6) 3 x 1000 cells of MRMT-1 rat breast cancer cells, and the needle hole was carefully sealed with sterile bone wax after injection. The sham operation group was injected with the same amount of saline.

　　(7) Wash the wound with saline and suture the skin. (8) Postoperative anti-infection (2.5% cefotaxime sodium) treatment.

　　3. Evaluation of bone cancer pain model

　　 Assessment of pain behavior in rats at different time periods (one day before surgery, one week after surgery, weekly temperature and pain check, four weeks after surgery). The Transitional Medicine Center of Sichuan University in Chengdu High-tech Zone tested the mechanical tenderness of the lower limbs and the thermal pain threshold of the buttocks in rats, keeping the room quiet during the experiment), local histology and image changes (4 after surgery) through weekly tibia X Radiographic inspection and HE staining confirm that the model has been copied) is successful.

　　 (1) Mechanical pain detection in rats: The weight of the plantar pain meter is 20g, the scale is 0-25, and the paw weight = scale x 20g. The left and right lower limbs of the rat are measured respectively, and the lower limbs are measured. Place the center part on the platform and start measuring. Move the test rod slowly until the pressure reaches a certain threshold. The mouse retracted its calf. Record the ratio at this time. A total of 4 measurements were taken, with each test interval exceeding 1 minute. Do not calculate the data, and average the last three data.

　　 (2) Rat heat pain test: Set the infrared intensity of the photothermal tail pain tester to 80°C, and the time threshold is 22g. After placing each rat on the immobilizer, the heat pain test cannot be performed immediately. Wait for about 1 minute, and then start the test again. During the measurement, the rear third of the rat's tail was placed in infrared light. After pressing the start button, the time starts. When the thermal stimulation reaches a certain threshold, the rat's tail will shake and the infrared light disappears. The timing stops immediately, and the time at this time is read. A total of 4 tests. The test interval exceeds 1 minute, the first data is not calculated, and the last three data are averaged.

　　 Fourth, use SPSS 16.0 statistical software to analyze statistical methods

　　. The data is expressed as x-±s, the comparison between groups uses one-way analysis of variance, and the comparison of different time points within the group uses repeated measures analysis of variance P\u003c. 0.05 means that the difference is statistically significant.

　　 Section III Experimental Results

  One. Changes in pain behavior

As shown in Figure 23-1, there is no difference in the preoperative mechanical analgesia threshold between the sham operation group and the bone cancer pain group (P\→0.05)) compared with 7 days, 14 days, 21 days and 28 days after surgery, The preoperative mechanical paw withdrawal threshold of this group of rats showed a slight downward trend, but it was not statistically significant (P\→0.05). Compared with the sham operation group, rats in the bone cancer pain group were significantly reduced at 7d, 14d, 21d, 28d after surgery (p\u003c0.05), while compared with the sham operation group, it was significantly reduced (p\u003c0. 05), statistical differences (P\u003c0.05).

　　二. X-ray results The X-ray photo of the rat tibia is shown in Figure 23-3. There was obvious bone destruction in the tibia on the side where the rat tumor cells were injected, and the degree of bone destruction was positively correlated with the modeling time. The longer the time, the greater the damage. By the 28th day, bone destruction intensified, and the cortical bone lacked worm feeding. X-rays of the tibia in the sham operation group showed no obvious bone destruction.

　　 3. Histological observation

　　 Section 4 Experience and Precautions

　　 (1) The cell culture process must be strictly sterile, and the experimental work must be completed in an ultra-clean workbench. This is very important for cell recovery and passage. Hair loss during operation in rats, aseptic operation and postoperative anti-infective disease are essential for establishing

　　(2) The model is very important. It is necessary to absolutely prevent infection of the surgical site after surgery, and when an inflammatory infection occurs at the surgical site, inflammatory pain will occur and it will seriously hinder normal results. (3) When making a hole in the tibia, please choose the part with less subcutaneous tissue and muscle, and completely separate the subcutaneous tissue, periosteum and the bone surface of the puncture area, so as not to trap the soft tissue with the drill. Excessive subcutaneous tissues and muscles can cause severe damage during separation and exposure, cause pain and prevent normal results.

　　 (4) The bone surface must be perpendicular to the puncture site to minimize tibial injury. Otherwise, the compressibility of the tibia will be impaired. Fractures at the puncture site are more likely to occur. (5) After surgery, sterile bone wax must be carefully sealed to prevent tumor cells from leaking through the perforation. Increased pressure in the bone marrow cavity is one of the causes of bone cancer pain. The outgrowth of the tumor reduces the pressure in the medullary cavity and relieves pain. This will not lead to experimental detection.

　　 (6) Pain threshold level detection belongs to sensory detection, which is easily interfered by external factors. Therefore, when performing a behavioral pain test, the rats should be allowed a certain amount of time to keep the environment quiet and adapt to the environment in which the test is placed for testing. (7) The quality of tissue fixation is an important factor affecting the results of immunohistochemistry. Tissue specimens should be placed in a fixative or stored at low temperature within 30 minutes after separation. Neutral formaldehyde solution is the best fixative, and the fixation time is 12-24 hours. The neutral formaldehyde solution has strong penetrating power, uniform fixation and small tissue shrinkage, making it an ideal fixative for HE sectioning or immunohistochemistry.