动物造模,泡沫细胞模型 z-bio 2021-12-30 981

　　The pathological changes of atherosclerosis include lipid streaks, fibrous plaques, atherosclerotic plaques, compound lesions, etc., and foam cells are the early cytological characteristics of As lesions. Foam cells are made up of monocytes/macrophages and smooth muscle cells (SMC) that migrate from the arterial media to the intima and take up a large amount of lipids. The use of macrophages or SMC to replicate foam cell models is an important method commonly used in experimental research on atherosclerosis.

　　There are scavenger receptors on the surface of macrophages and vascular smooth muscle cells (VSMC), which can uptake large amounts of modified and denatured low density lipoproteins (LDL), especially oxidized LDL ( oxidized LDL, OX-LDL), which makes the cells filled with a large amount of lipids, and looks like a foam under the electron or light microscope, so it is called foam cell. After being stained with Oil Red O, it is red granular and contains a lot of cholesterol, and cholesteryl ester accounts for about 50% of it. Therefore, by first preparing OX-LDL and then making it act on macrophages or vascular smooth muscle cells, a foam cell model can be created.

　　One. Preparation and identification of ox-LDL

　　(1) Preparation of LDL

　　1. Take 100-200ml of fresh plasma and add PDB (a buffer containing 0.9% NaCl, 0.01% NaN3, 10 mmol/L Tris-HC1 and 1 mmol/L EDTA, pH 7.4) to prevent corrosion and oxidation.

　　2. Ultracentrifugation for 18 hours, 8°C, 42 000 r/min.

　　3. Aspirate the milky white liquid (VLDL) and the sublayer light yellow liquid (IDL) floating in the upper layer of the centrifuge tube.

　　4. Collect the liquid at the bottom of the centrifuge tube and add solid KBr to make the final density 1.063g/ml.

　　5. Ultracentrifugation was performed again for 20 hours at 8°C and 42 000 r/min.

　　6. Aspirate the upper orange liquid that is LDL.

　　7. The purified LDL was dialyzed in PBS containing 200 μmol/L EDTA for 48 hours, filtered and sterilized, and stored at 4°C.

　　8. It is best to use fresh plasma to prepare OX-LDL, and all reagents used should be above AR level. Careful operation should be performed during ultracentrifugation and sampling to minimize mechanical disturbance.

　　9. Serum density adjustment formula Vn=Vo(dm-do)/(dn-dm) where Vn is the volume of high-density liquid to be added (ml), dn is the density of high-density liquid to be added (g/ml), do is The density of the serum before density adjustment (g/ml), Vo is the volume of serum before density adjustment (ml), and dm is the density (g/ml) to be adjusted.

　　(2) Oxidative modification and identification of LDL

　　1. Before oxidation, LDL was dialyzed against EDTA-free PES solution for 24 hours to remove EDTA.

　　2. The LDL was oxidized in PBS containing 10μmol/L CuSO4 at 37°C for 12 hours; then dialyzed in PBS containing 100mmol/L EDTA at 4°C, changing the medium every 8 hours, dialyzed for 24 hours, filtered and sterilized, 4°C save.

　　3. The content of thiobarbituric acid reactive substance (TBARS) was used to identify ox-LDL. That is, 0.1ml of sample or standard malondialdehyde is added to 2.9ml composite solution (containing CCICOOH 0.92mol/L, C4H4N2O2S 0.026mol/L, HCl 0.25mol/L), placed in a water bath at 100℃ for 30 minutes, and measured after cooling. Calculate the TBARS content at the OD value of 532nm. Then LDL-C content was measured with the total cholesterol kit. The degree of oxidative modification of ox-LDL is expressed by the content of TBARS per gram of cholesterol.

　　One, cell culture

　　Replicating foam cell model cells generally use macrophages or vascular wall smooth muscle cells. Human umbilical vein endothelial cells (ECV304), human monocytes (U937), Chinese hamster ovary cells, mouse peritoneal macrophages, etc. can also be used.

　　(1) Collection and culture of macrophages Mouse peritoneal macrophages are the most commonly used. Take 8-week-old, non-infected mice, let them die by bleeding from the eyes or cervical dislocation, and disinfect the abdominal skin with 70% alcohol. Under aseptic conditions, use tweezers to lift the abdomen and cut off the 3~5cm long skin, expose the peeled abdomen, and then use a syringe to inject 3~4ml serum-free 1640 culture medium into the abdominal cavity; knead for a while and draw out the abdominal fluid, and Pour into the centrifuge tube and centrifuge at 1000r/min for 10 minutes at 4°C. Remove the supernatant and add the same culture medium to adjust the cell number to (1～5)×1000000/ml. If you need a large number of cells, you can collect cells from several mice. To obtain peritoneal macrophages in guinea pig rabbits, it is necessary to inject liquid paraffin into the peritoneal cavity a few days ago to induce macrophages to accumulate in the peritoneal cavity.

　　Culture method: Because of the strong adhesion of macrophages, the adhesion method is most commonly used for isolation. Add the suspension containing macrophages to a cover glass, culture flask or culture plate, and incubate at 37°C for 30 to 60 minutes. When macrophages begin to adhere to the wall, they are washed 5 times with Hanks solution to wash away other cells that are not adherent to obtain pure macrophages. Then, 1640 culture medium containing 10% calf serum was used to incubate in a 37°C incubator. Macrophages are terminal cells and cannot be subcultured for a long time.

　　(2) VSMC acquisition and cultivation VSMC is commonly obtained from the arteries of pigs, rats, mice and other animals.

　　1. Take the thoracic aorta under the sterile condition of primary culture, wash the artery several times with D-Hank balanced salt solution containing penicillin (100μg/ml) and streptomycin (100μg/ml), and then stick to the tissue when the material is washed away Blood clots and broken cells. Cut off the adventitia fat, cut the blood vessels longitudinally, carefully scrape off the intima, wash it with culture medium, and cut the remaining media into tissue pieces of about 1 mm3. After that, use enzyme digestion method or tissue block planting method for primary culture.

　　Culture solution preparation:

　　(1) RPMI-1640: Pour a bag of RPMI 1640 dry powder into 500ml of three-distilled water, mix well to dissolve it completely; add 2.1g NaHCO3, 5.95g HEPES, 110mg sodium pyruvate, penicillin and streptomycin 100,000 each U, make up three distilled water to 1000ml, mix thoroughly (place at 4°C, 4-6h), filter and sterilize with a pre-sterilized Zeiss filter, divide into sterile bottles, and store at 4°C.

　　(2) 0.125% pancreatin: Weigh 125 mg of trypsin, add it to 100 ml of D-Hanks solution, mix well, place it at 4°C for 4-6 hours, filter and sterilize through a 0.22 μm filter membrane, and store in aliquots and freeze in- 20°C.

　　Cell count and cell viability determination:

　　(1) Cell count: Drop the diluted cell suspension on the counting plate, make the suspension freely fill the gap under the cover slip, do not leave bubbles, wait for a while, observe under the microscope and calculate the number of cells in the four corners ; The line presser only counts the cells on the upper and right lines, and then calculates the cell concentration as follows:

　　(number of cells in the large grid/4) × 10000 × dilution factor = number of cells/ml

　　(2) Cell viability determination: Reagent preparation: ① 4% placental blue mother liquor: weigh 4g of placental blue, add a small amount of double distilled water to grind, and adjust the pH to 7.0-7.20. Add double distilled water to 100ml, centrifuge and take the supernatant. ②1.8% sodium chloride solution.

　　Operation steps: mix the first two liquids at a ratio of 1:1 to make 2% placental blue liquid. Take 1 drop of cell suspension and 1 drop of placental blue liquid to mix, take 1 drop of stained cell suspension, and carefully place it on a white blood cell counting plate. Live cells are not colored, and dead cell nuclei are blue. Count the number of dead and live cells in the four squares according to the white blood cell counting method. The viability is expressed as the percentage of live cells in the counted cells.

　　Live cell%=live cell number/(live cell number+dead cell number)×100%

　　1) Enzymatic digestion method: Put the cut tissue pieces in 0.1% collagenase, place them at 37°C for 1 hour, and then pour out the digestive juice. Then add 0.25% trypsin for digestion. Every 10 minutes of digestion, the digestion solution containing the cells is aspirated and replaced with fresh trypsin to redistribute the cells. Repeat this several times to collect all the digestive juice containing cells. The first two digestion solutions containing cells are often discarded, and the next few digestion solutions containing cells are aspirated and immediately added the same amount of D-Hank solution, or 10% calf serum can be added to the digestion solution containing cells . Centrifuge the collected cell-containing digestion solution (800～1000r/min, 10 minutes). The supernatant was discarded, the cells were collected, and 2ml of culture medium was added to make a cell suspension, which was counted and seeded into a culture flask. After being placed in a constant temperature incubator at 37°C for 1 to 2 days, the cells will naturally adhere to the wall.

　　2) Tissue block planting method: ① Take 8-week-old male rats, decapitate them, disinfect the skin of the chest and abdomen with 3% iodine and 75% alcohol; ②Open the chest, expose the aorta, clamp the aortic arch, and separate the aorta downward Peripheral connective tissue: ③Cut off the aorta and place it in a sterile petri dish pre-filled with culture medium. After washing the blood clot, peel off the fibrous and fatty layer of the outer membrane: ④Using ophthalmic scissors to cut longitudinally along the outside of the blood vessel. With the intima face up, scrape from top to bottom with a blade for 1-2 times to remove endothelial cells; ⑤Use tweezers to carefully tear off the smooth base of the media near the intima surface, and soak in DMEM medium containing 10% fetal bovine serum , Use ophthalmological scissors to cut the blood vessel strips into small pieces of 1 to 2 mm; ⑥ Use a straw to suck out the small pieces into the culture bottle, place them evenly on the bottom wall, the distance between the small pieces is 0.5cm, and gently flip the culture bottle , The bottom of the bottle is up (be careful not to let the tissue mass flow out), add culture solution, 37℃, 95% air-5% CO2, incubate for 2 to 4 hours: ⑦Then turn the culture bottle slowly, so that the liquid slowly covers the tissues. Be careful not to wash off the small pieces from the bottle wall before continuing the cultivation. The movement in this process should be light, so that the liquid slowly covers the tissue block, and it is strictly forbidden to move too fast, the liquid produces impulse to make the pasted tissue block float up and cause the failure of the primary culture. The tissue block culture also does not need to be turned over, that is, after placing the tissue block, only a small amount of culture fluid is added to the culture bottle to keep the tissue block moist. Cover the bottle cap, put it in the incubator and incubate for 24 hours before adding culture fluid. ⑧After about 5 days, smooth muscle cells can be seen to grow out of the tissue mass, which can be subcultured when the bottom of the bottle is full.

　　It has been reported that the type I collagenase/elastase digestion method for culturing rat vascular smooth muscle cells can significantly shorten the digestion and culture time, and has the advantages of harvesting more cells and high cell purity. The method is as follows: ① Take materials. The 150-200g male SD rats were detached from their necks to death, the carotid artery was exsanguinated, the thoracic aorta was quickly taken out, immersed in a petri dish containing sterile Hanks, and transferred to a sterile operating table. ② Digestion. Digest in two times. First, rinse the artery with Hanks solution for 2 to 3 times, cut off the extravascular fat and connective tissue, and put in 2g/L collagenase. (1.5ml/artery) Digestion in digestive juice for 20-30 minutes. Then, clamp the outer membrane with No. 5 forceps, and gently pull in the opposite direction to remove the outer membrane. The artery was incubated in DMEM containing 10% NCS for 24 hours. Then take out the artery and put in the collagenase containing 2g/L. And 0.25g/L elastase compound digestion solution (1.5ml/arterial), incubate in a 5% CO2, 37°C incubator for about 2 hours. ③ Cultivation. After the blood vessel is completely dissolved, add DMEM containing 20% NCS to stop the digestion, centrifuge at 150g for 5 minutes, discard the supernatant, count the cells with a counting plate, and inoculate (1～3)×10000000/ml live cells in 20% NCS DMEM 25ml culture flask. Put it into a 5% CO2, 37°C incubator for culture. A small amount of adherent smooth muscle cells can be seen after 24 hours. Replace with fresh culture medium after 2 to 3 days. In about 5 days, when the cells reach 70% to 80% confluence, they can be passaged according to conventional methods.

　　2. Subculture After the growth and fusion of the primary cultured cells, trypsin and mechanical dispersion are used to digest and disperse the cells, and the cell count is quantitatively transferred to a new culture flask. The cells can continue to divide and grow continuously. Through passaging, other cell components in the original tissue block can be gradually eliminated, so as to obtain a purer cell line.

　　(3) Establish a foam cell model. Peritoneal macrophages or SMCs are passed to the 3 to 5 generations. After filling the bottle to change the culture medium, add the prepared ox-LDL to establish a foam cell model. The amount of ox-LDL added and the incubation time vary with the types of cells and the degree of modification of ox-LDL, which can be determined by observing the time-dose relationship and the dose-effect relationship. Existing data reports: U937 cells can successfully replicate U937 foam cells by incubating with 80mg/L ox-LDL for 48 hours; use phorbol esters (10-7 mol/L) to induce human monocytic THP-1 Cells differentiate into macrophages (72 hours), and then stimulate macrophages with ox-LDL50mg/L to form foam cells in 48 hours; C57BL/6J mouse peritoneal macrophages can be cultured for 72 hours with 10mg/L ox-LDL Promoting foam cells: porcine aortic smooth muscle cells cultured with 15mg/L ox-LDL for 72 hours can form smooth muscle cell-derived foam cells: rat aortic smooth muscle cells cultured with 50mg/L ox-LDL for 72 hours can also form Smooth muscle cells are derived from foam cells.

　　The established foam cells must be confirmed by cell morphology microscopic observation and determination of intracellular cholesterol content.

　　1. Observation of cell morphology Take out the cover glass attached with macrophages or VSMC, and stain with the oil red o method. There are many dyeing methods, with similarities and minor differences. Here is a method with few steps, short time, simple and effective dyeing, which can be selected first.

　　(1) Rinse 3 times with PBS solution, 5 minutes each time.

　　(2) Fix with 50% isopropanol for 1 minute.

　　(3) Oil red o working solution for dyeing for 10 minutes.

　　(4) Rinse 3 times with single distilled water, 1 minute each time.

　　(5) Stain with hematoxylin for 5 minutes.

　　(6) Rinse gently under running water for 30 minutes.

　　(7) After drying, the tablets were sealed with 10% povidone.

　　After staining, the film was observed under an optical microscope, the lipid droplets were stained red and the nucleus was blue.

　　2. Determination of intracellular cholesterol (CH) and cholesterol ester (CHE)

　　(1) Enzymatic method: Digest with 0.25% trypsin for 1 minute, collect the cells, centrifuge at 1000 r/min for 10 minutes, discard the supernatant, rinse once with PBS solution, add 0.5 ml of isopropanol, and place on an ultrasonic cleaner. Shake 10s/min for 3 times, and then centrifuge at 1000r/min for 15 minutes. TC reagent kit for aspirating supernatant and

　　The FC reagent kit was used to determine the total cholesterol (TC) and free cholesterol (FC), and the centrifuged pellets were lysed with 0.1mol/L NaOH 0.5ml, and the cell protein content was determined by the Lowry method. The cholesterol content in the cell is expressed as TC mg/g cell protein and FC mg/g cell protein, and the difference between the two is the CHE mg/g cell protein content.

　　(2) High performance liquid chromatography: cell culture and processing: culture cells and establish foam cell models according to the aforementioned methods. ①Cholesterol standard preparation. Weigh 1.5g of standard cholesterol first dissolved in a little ethanol, gradually increase the temperature to 20℃, and fill the volume of ethanol to 150ml, the working concentration of standard cholesterol solution are 0.29(50), 0.258(100), 0.516(200), 1.032 (400), 2.064 (800) mmol/L (mg/L), stored at 4°C for later use. ② Extraction of cholesterol and cholesterol esters in cells. Collect each 10ml of cells into a 10ml plastic tube with a lid, centrifuge at 1500r/min and 4°C for 15 minutes, remove the supernatant, re-dilute the cells with 1ml 0.9% NaCl solution, and ultrasonically break the cells in an ice bath. The working condition is 600 weeks, the working time is 4 seconds, the intermittent time is 8 seconds, and the number of timings is 6 times. The protein content was determined by Lowry method. Add an equal volume of freshly prepared 15% alcohol-soluble KOH (-20°C) to the cell lysate, vortex until the cell lysate is clear, add 6% trichloroacetic acid to remove protein, and then add an equal volume of n-hexane: Isopropanol 4:1 solution (V/V), vortex the mixture for 5 minutes, then centrifuge at 3500r/min, 15°C for 5 minutes, collect the upper organic phase, and extract the lower aqueous phase twice as described above , Transfer the mixed organic phase to a test tube with a lid, and then dry it in a vacuum freeze dryer at 65°C. After cooling at room temperature, add 100μl of isopropanol: n-heptane: acetonitrile with a ratio of 35:12:52 ( V/V) mixed solution, dissolve the sample, depigment with activated carbon, ultrasonic degassing for 5 minutes, centrifuge at 1500r/min for 5 minutes, collect the supernatant, take 10μl into the sample, and perform HPLC analysis. ③Analyze intracellular cholesterol. Using Waters 991 chromatographic system, including 510 pump, U-6K sampler, 991 photodiode matrix detector, TCM column thermostat box, using Gen-PAK FAX column produced by Waters company, with n-heptane: isopropyl Alcohol: Acetonitrile is the mobile phase for non-gradient elution, the flow rate is 1ml/min, the column temperature is maintained at 4°C (with ice cubes), the UV detection time is 12 minutes, and the detection is at 226nm. The Gen-PAK FAX column was rinsed with deionized water for 10 minutes (flow rate 0.5ml/min) before use to remove alcohols in the column. ④Quantification of intracellular cholesterol and cholesterol esters. The quantification of cholesterol was carried out with reference to the method of Wang Zuo et al. The quantification was based on the peak area and the unit was mg/g cell protein. The quantification of cholesterol ester refers to the method of Zhou Xin et al. The total cholesterol is determined by HPLC with cholesterol esterase hydrolysis. The total cholesterol amount minus the free cholesterol amount represents the amount of cholesterol ester, and the unit is mg/g cell protein.

　　Human disease animal models are all designed according to research purposes, and the animal models used for different research purposes can be very different. Special attention should be paid to the researcher to choose the appropriate animal model according to the research objectives and research content of the subject. In addition, due to the great differences between animals and humans, even if the replicated As animal model reflects one or several aspects of the pathogenesis of human As relatively truly and objectively, the animal disease model cannot be compared with human disease. Exactly the same. Therefore, the limitations of animal models should be fully considered when analyzing experimental results, especially when using animal disease models to study drug efficacy.