| Objective: Osteoarthritis (OA) is one of the most common bone and joint diseasecharacterized by progressive cartilage degeneration. To this day, there's still rarecost-effective treatment for this disease. In this research, we take sixpetal clematisroot parenteral solution (WLX) as an interferential measure which we used in in-vitrochondrocyte culture, and OA animal model. After interference, we test the level ofcytokine interlukin-1β, and detect the chondrocyte and cartilage for alteration inhistology, ultra-microstructure, phenotype expression of collagen and proteoglycan.We discuss the probable mechanism of therapeutic effect of WLX on OA, so as to get adeep understand in the rationale of using WLX as a choice in the prevention andtreatment work of osteoarthritis in clinical work.Materials and methods:1.In-vitro: Rabbit chondrocytes were obtained and cultured in vitro, the secondarypassages were seeded in 25cm2 culture flask and 24-well plates prepared with slidesin each hole. The cells were then divided into three groups: Group I was incubatedwith different concentrations of WLX(0.00,0.075mg/mL,0.15mg/mL,0.30mg/mL,0.60mg/mL,1.25mg/mL,2.50mg/mL) for 72 hours; group Ⅱ was pretreated withdifferent concentrations of WLX for 24 hours and then stimulated by 10μg/mLlipopolysaccharide (LPS) for 72 hours; group ⅡI was stimulated by 10ug/mL LPS for24 hours and then incubated with different concentrations of WLX for 72 hours.Subsequently, the supernatant was collected and IL-1βlevel was tested byradioimmunoanalysis(RIA); chondrocytes in flasks were obtained, and RT-PCR wasused to test the expression level of IL-1βmRNA and the mRNA of pro-collagen I andpro-collagen Ⅱ. Cells in the 24-well plate were observed under theinverted-microscope, the slides were taken out when overspreaded with chondrocytes,fixed, rinsed, and the expression of type I collagen and type Ⅱ collagen of the threegroups of chondrocytes were detected by immunocytochemistry (ICC); toluidine bluestaining was performed to observe the expression of matrix proteoglycan of the treegroups under microscope.2.In-vivo: 45 healthy male adult New Zealand white rabbits were randomly divided 6into three groups (Group-S, Group-D, Group-K), take the right knee as experimentalside and produce OA model referring to Hulth-Talhag, calculate the dose of WLXaccording to the Meer-Rubner formula. The next day after operation, interferingmeasures were taken into practice every day and lasted for 4 weeks, animals ingroup-S were treated with WLX, those in group-D were treated with physiologicalsaline (NS) and those in group-K were treated as control. At the end of week 6, 12, 24after operation, 5 animals were sacrificed randomly in each group every time andspecimens were procured. The specimens received X-ray, articular cartilage wasobserved and scored under operative-microscope, IL-1βlevel in synovial fluid wastested by RIA, cartilage sections were made and stained with HE/SOFG for Mankinscore; the expression level of collagen I and Ⅱ in matrix was tested byimmunohistochemistry(IHC); the change of ultra-microstructure of cartilage wasobserved through TEM.Result:1 In vitro:Chondrocyte culture: Under the inverted-microscope, primary cultured chendrocyteswere seen to attach to culture flask 24 hours after inoculation, and attach completelyafter 72 hours, the shape of chondrocytes were rounded, ellipse or epithelioid, anddevelop into monolayer at about day 10-14, in some regions, chondrocytes formedlarge aggregate of rounded cells or multilayer. Subcultured chondrocytes attach toculture flask completely in 24-48 hours, and developed to subconfluence in 5-7 days.Fusiform cells were seen to increase in subcultured chondrocytes after three passages,and the ability of excretion and proliferation of chondrocytes were decreased, thepassage cycle was prolonged.Comp...
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