| Osteoarthritis which is affected by multiple factors is a chronic progressive joint disease,and many factors play an important role in its occurrence and development process.Studies have shown that VCAM-1may play an important role in the pathogenesis ofosteoarthritis, but its exact mechanism is not yet clear.ObjectiveIn this study, firstly, by enzyme-linked immunoadsorbent assay and VCAM-1monoclonal antibody immunohistochemical staining, VCAM-1in the serum, synovial andcartilage tissue of the normal and patients with osteoarthritis is determined and compared.Secondly, we study the methods for isolation, culture and identification of articularchondrocytes from normal human and osteoarthritis patients in vitro, compare thebiological characteristics and evaluate the biological activity between normal cartilagecells and osteoarthritis cartilage cells, which provides the experimental foundation forfurther study of the other biological characteristics in the cartilage cells and pathogenesisof osteoarthritis. Finally, by RT-PCR and the Western blot test, the expression ofVCAM-1is compared in gene and protein level between normal cartilage cells and osteoarthritis cartilage cells that we obtain, and the expression of VCAM-1in the normalcartilage cells from human is compared under diffirent concentration IL-1β, whichprimarily approaches VCAM-1’s mechanism in the pathogeneses of osteoarthritis andprovides an experimental evidence for discovering VCAM-1′s mechanism in thepathogenesis of osteoarthritis.Methods1．The expression of VCAM-1is measured by using ELISA in serum of healthy humanand patients with osteoarthritis. Normal and osteoarthritis synovial and cartilage tissue arestained by VCAM-1monoclonal antibody immunohistochemical staining.2．Cartilage is harvested under sterile conditions from human traumatic knee joints andosteoarthritis knee joints in Total Knee Arthroplasty. Human articular chondrocytes areisolated by using two-step enzymatic digestion and the cells are cultured and subculturedrespectively in vitro. The cartilage cells from healthy human and osteoarthritis patients areidentified by observing cell morphology under phase-contrast microscope, drawinggrowth curve, measuring the cell proliferation, toluidine blue staining and type II collagenimmunohistochemistry staining.3．By RT-PCR detection of VCAM-1mRNA and the Western blot analysis of VCAM-1protein, the expression of VCAM-1is compared between normal cartilage cells andosteoarthritis cartilage cells that we obtained from human.4．By RT-PCR and the Western blot essay, the expression of VCAM-1in the normalcartilage cells from human is compared under diffirent concentration IL-1β in gene andprotein level.Results1．The expression of VCAM-1in the serum of patients with osteoarthritis at all gradesis significantly higher than that in the normal control group (P<0.05), and increased withthe severity of osteoarthritis.2． The synovial and cartilage tissue in Osteoarthritis is positive by VCAM-1monoclonal antibody immunohistochemical staining, and with the severity of lesions,color deepened. The expression of VCAM-1in the cartilage tissue of patients with osteoarthritis at all grades is higher than that in the normal control group (P<0.05), andhad statistically significant among the grades of osteoarthritis patients (P<0.05), whichindicated that the expression of VCAM-1is increased with the severity of osteoarthritis.3．The normal cartilage cells are round after the two-step enzymatic digestion, cellsshowed adhesion, deformation, triangle or multi-angle after two-three days cultured. Thecells proliferated significantly after one week, and formed a cohesive multiplayer onplastic surface about two weeks. The adherent time of the first generation after passagecells are shorter than the primary cells, and the proliferation rate of them are faster thanthe primary cells. However, with the increase in the number of passages, the proliferationrate gradually slowed down, and the cells shape gradually conversed to the "fiber-like"cells. But the morphology of osteoarthritis cartilage cells is similar to fibroblasts, and thegrowth rate is significantly more slowly than that of normal cartilage cells.4．MTT assay results showed the growth curve of the normal chondrocytes in "S" shapewithin five generations. The proliferation rate of osteoarthritis cartilage cells is lower thanthat of normal cartilage cells, and the biological characteristics of cartilage cellsdisappeared in the fourth generation.5．Toluidine blue staining showed that the blue-violet metachromatic granules are seenin the normal cartilage cells, and a small number of metachromatic granules appearedaround the cells. After repeatedly subcultured, the cells are stained in light blue withtoluidine blue staining. The osteoarthritis cartilage cells are stained in blue, and are stainednon-blue after several passages.6． Type II Collagen immunohistochemical staining showed that primary normalchondrocytes are colored in positive tan, yellow-brown around the nucleus, and seenyellow-brown granules around the cells. The tan gradually weakened after subcultured,and the chondrocytes are fibrosis, prominence increased and staining faded after fivegenerations. But primary osteoarthritis chondrocytes are colored in tan, and showednon-yellow-brown after several passages.7．The quantity of mRNA and protein expression of VCAM-1in osteoarthritis cartilagecells is significantly higher than that in normal human cartilage cells. 8．The quantity of mRNA and protein expression of VCAM-1in the normal cartilagecells from human in the intervention group is significantly higher than that in the blankcontrol group. With the increase of IL-1β concentration, it also has the tendency toincrease.Conclusion1．The expression of VCAM-1in the serum, synovial and cartilage tissue is closelyrelated to the osteoarthritis, and increased with the lesion increased.2．Articular chondrocytes from normal human and osteoarthritis patients are obtained byusing trypsin and type II collagenase digestion. Within five generations, the normalcartilage cells grew well with obviously biological characteristics, suitable forexperimental study, and turned to dedifferentiation after five generations. Theosteoarthritis chondrocytes with slow cell proliferation and early degeneration ofbiological characteristics are in accordance with the performance of degeneration ofcartilage cells, and provided experimental foundation for study of the osteoarthritis in thecellular level.3．The expression of VCAM-1in osteoarthritis cartilage cells is significantly higherthan that in normal cartilage cells from human, which confirmed the representative of thecell lines obtained in this study. The cells can be used as model cells in the follow-upstudy and provides an experimental evidence for discovering VCAM-1′s mechanism in thepathogenesis of osteoarthritis.4．The expression of VCAM-1in the normal cartilage cells from human in the IL-1βintervention group is significantly higher than that in the blank control group, and with theincrease of IL-1β concentration, it also has the tendency to increase, which confirmed thatthe expression of VCAM-1is correlated with cytokines that lead to osteoarthritis,confirmed the reliability of VCAM-1as the risk predictors of the osteoarthritis, andprimarily approaches VCAM-1’s mechanism in the pathogeneses of osteoarthritis. |
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