Articular Cartilage Injury Of Biomarkers Proteomics Preliminary Study

Objective: Improved Hulth made mould ways to create articular cartilage damage model, to observe the gross and microscopic cartilage injury condition characteristics, using proteomics plain: surface-enhanced laser desorption ionization time of flight mass spectrometry (MS) -- -- SELDI TOF technology screening articular cartilage injury serum, joint fluid biomarkers.Methods: Select New Zealand big white rabbit 20 for operation control group. Surgical control: aseptic conditions, along the rabbit knee medial longitudinal incision, long line type approximately 4 cm, exposing the knee, cut off before and after the cruciate ligaments and MCL ligament, complete resection within the lateral meniscus, retain articular cartilage surface. Postoperative free activities, not fixed injury segments. After seven days to 80 million units of penicillin to prevent infection. 30 min/d points 2 times, driven for 10 weeks. Not to do anything with 14 rabbit knee for normal controls. Build mode, 8,10 weeks after 4 do Mankin scores, the gross appearance and electron microscopy, light microscopy articular cartilage pathological feature, and with radiation X-ray articular cartilage injury. The operation control and normal control group rabbit executed, and extraction serum, joint fluid, 4℃at ambient temperature quiet place 1h, then the 4℃at ambient temperature 10 min, speed centrifugal serum 3000r/min, adjusted for joint fluid 1000 r/min, absorb serum, joint fluid 30ul, repacking 0.5 mlEP tube, place - 80℃cross-matched. With metal ion exchange chip (Cu) IMAC3 - and the weak cation exchange chip (CM10) combining surface-enhanced laser analytical/ionization time-of-flight mass spectrometry analysis of articular cartilage injury serum, joint fluid proteins in the spectrum of change, and seeks to biomarkers of articular cartilage injury.Results: 1 Articular cartilage damage model and the characteristics of each stage biology: (1) The normal control group, General histologic observation, smooth, glossy articular surface bright, no crack and soften the kitchen; Joint edge neat, no bone formation rules of vegetation; HE pictures showed maturity nucleus small, round and cartilage is 1 ~ 2 nucleoli, weak basophilic cytoplasm, size distribution uniformity, cartilage peripheral part is the naive in cartilage cells, smaller, often with a round, a single distribution. The closer cartilage cells, the more mature central, volume increase gradually, become round or oval; Electron microscopy ultrastructure, chondrocytes and the nucleus for the oval or nearly circular disk, cell surface have swelled and ruffle, nuclei is bigger. The cytoplasm of the visible rich rough endoplasmic reticulum and golgi complexes, mitochondria less, cells for translucent ring belt matrix collagen fibers, matrix, a fine and dense interwoven shape, proof of cartilage cells have the ability of secreted cartilage matrix . Mankin score of 0 points, radiation X-ray shows joint clearance is normal, no bone spur, bone supenllus generation, articular surface smooth, did not see the obvious exception. (2) Modeling 4 weeks compared with normal controls change: Performance for articular cartilage transparent appearance, colour and lustre is dim yellow; Mild coarse, have articular surface erosion, softening kitchen; A small synovial hyperemia hyperplasia, joint fluid increase; HE dyed cartilage surface is uneven, the hierarchical appear discontinuous, cartilage matrix destruction, matrix light dye, appear fibrosis, not hurt and cartilage lower level; Cell mild hypertrophy hyperplasia, with hyperchromatic nuclei, bigger, cartilage lacunes absorption phenomenon, visible parts necrosis cartilage cells; Mankin score for 3 points, transmission electron microscopic display: cell surface protuberant and creases did not decrease, nuclei increased. The cytoplasm of rough endoplasmic reticulum and golgi complexes, mitochondrial increased, cells for transparent substrate with that ring the cartilage cells secrete cartilage matrix ability strengthens, metabolic activity increases; Radiation X-ray observations saw no obvious exception.(3) Modeling 8 weeks of change: This is the gross appearance visible articular cartilage, expression is mid-term change colour and lustre dim articular cartilage tan; Articular surface coarse, surface erosion, softening oven; A synovial hyperemia hyperplasia, joint fluid increase, color turbidity, joint is neat edge rules, it is smaller, individual fractures formed bone vegetation can reach cartilage lower level; HE dyed cartilage tissue levels are not clear, cartilage cells are arranged disorder, appear fibrosis, cartilage cells moderate hypertrophy hyperplasia, with hyperchromatic nuclei, bigger, more appear ~ cell nuclear, matrix moderate light dye; Transmission electron microscopic display chondrocytes surface protuberant and ruffle reduce, fuzzy, the nucleus has swelled, show the polygons. The cytoplasm of rough endoplasmic reticulum and golgi complexes, mitochondrial increased, cell matrix was increased, indicating the transparency of cartilage cells of compensatory sex secretory activity are still; Mankin score for seven points, radiation X-ray observations visible osseous articular surface local disappear, joint bone structure change, joint space narrow, joint margin with bone supenllus generation, articular surface coarse, with a lip samples change, joint gap have free body. (4) Modeling 10 weeks change: This is the gross appearance visible articular cartilage injury late change, for the performance of the synovial nodular change, bone supenllus hyperplasia, little and turbidity, synovial fluid subchondral bone exposure, cracked; HE dyed cartilage fine flaw, appear cell vacuoles, mast cells, the apoptotic cells, matrix dyeing severe reduced; Transmission electron microscopic display chondrocytes and cell surface protuberant disappear, cell shape irregular corrugation, cell nuclei boundaries, moderate unclear; Mankin score of 10 points, radiation X-ray observations can be obviously articular cartilage damage, joint bone structure change, joint gap into ~ step narrow, appear worm articular surface corrosion samples change, coarse, bone change, joint edge sharp, snatching a bone spur, bone formation.2 CM10 include the differences between the protein:(1) serological sample: winning altogether 67 serum protein peak, 18 statistically Learning significance (P < 0.01), including qualitative nuclear than for 3475,6615,7558,16884, 33665 protein spectrum peaks significant difference; (2) Joint fluid samples from: get 104 joint fluid protein peak, 10 are statistically significant (P < 0.05), including qualitative nuclear 3475,3950,7558,16884,33175 protein spectrum peaks than for significant difference, and 3475,7558,16884 Da for serology and joint fluid samples from mutual difference protein spectrum peaks protein.Conclusion:1 Improved Hulth made mould ways to create the knee joint cartilage damage model, quite comprehensively reflect the articular cartilage injury from the early compensatory sex hyperplasia to decompensated after cartilage apoptosis and matrix decrease, cartilage softened into exfoliative missing feature of the whole process of late change.2 Articular cartilage injury of surgical treatment group and the normal control group serum and joint fluid protein expression spectral differences exist, indicating that the serum and joint fluid appears in the differences in proteins for articular cartilage injury may be part of the biological markers.