| Degenerative osteoarthritis (OA) are still not fully understood, currently considered many pathogenic factor, people adopt medical ozone (03) joint lumen injections treat early medium-term OA achieved good effect, but the mechanism of 03 and treatment are still not fully understood, this experiment used Hulth model in the rabbit knee lumen injection ozone (03), explore 03 inhibit chondrocytes degeneration mechanism, for medical 03 treatment early, middle OA provide experimental supportFirst, the pathology change of articular cartilageObjective:to observe the pathology change of articular cartilage by optical microscope. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date, muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur by razor-sharp blades, fixed cartilage in 10% formaldehyde solution, through a series of dehydration,10% EDTA demineralization processing, in all 10 days; paraffin embedded the Cartilage tissue; HE dyed, biological microscope observated. Results:in 03 group, the cells of cartilage surface is degeneration, arranging disorderly, visibled chondrocytes necrosis, fell off, a small amount of inflammatory exudated, erosion spot be visible on cartilage surface, fibroblasts and capillary hyperplasia is uncommon; in OA controls group, the cells of cartilage surface are arranged disorder, necrosis and degeneration is the apparent, ulcer can be see obviously, it is visibled obvious of the inflammatory cells and fibroblasts and newborn capillary hyperplasia, visibled fibre hyperplasia in ulcer bottom and ulcer cartilage cells of denaturation. Conclusion: The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.Objective: to observe the pathology change of articular cartilage by electron microscope. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date, muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; Fixed 24 hours in 3% glutaraldehyde; flushed in 0.1 M buffer of phosphoric, in all 3 times; fixed in 0.1% osmium tetroxide, in all 90min; flushed 3 times in deionized water; Dehydrated in different concentrations acetone; Saturated with 61G epoxy resin after demineralizated, embedded, hardened, repaired into semi-thin block; Positioned ultra-thin slices cut 0.05 mm, Electron microscopic observated after electronic dyeing. Results:In OA group, most of the cartilage cells is markedly swollen and form irregularly, the week's halo of cell is disappeared, rough endoplasmic reticulum apparent expansioned, microvilli fractured and shorten, rough endoplasmic reticulum retinal dissolved fractured, lipid drops exists can be see in the cytoplasm, the nucleus is irregularly; In 03 group, cartilage cells are almost normal, cells are elliptic or spindle completely. Conclusion:The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effect ively rel ieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.Second, to observe the IL-1βexpressed change of articular cartilage by DAB methodObjective:to observe the IL-1βexpressed change of articular cartilage by DAB method. Methods:36 rabbit randomly divided into 3 groups, respectively, the treatment group is 16, the model group is 16, the normal group is 4, Used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; Fixed .24 hours in 3% glutaraldehyde; tested IL1β. in cartilage cells by DAB method; Randomly selected vision in each section of the edge in cartilage damage, counts 200 cells in every vision, statisting, the number of positive dyeing cells with positive cells rate as IL1βexpression index. Results:the positive cells rate of normal group (group A) is 2.815±0.827%, the positive cells rate of model group (group B) is 47.159±4.431%, the positive cells rate of 03 group is 27.771±4.085%; Compared the model group (group B) and the normal control group (group A), p<0.01; Compared the treatment group (group C) and the model group (group B), p<0.05; with a statistical significance. Conclusion:The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.Three, to observe the nitric oxide (NO) expressed change in joint fluid by nitrate reductase.Objective: to observe the nitric oxide (NO) expressed change in joint fluid. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected 022ml to the left knee joint lumen of control group rabbit, continuous injected 7 days; the 8th day, anesthesiaed, iodine disinfection, and knee punctured, extracted joint fluid, in all 1 ml, Sealed and saved in -70℃; to observe the nitric oxide (NO) expressed change in joint fluid by nitrate reductase. Results:In control group, the content of NO is 145.57±12.17, In 03 group, the content of NO is 132.33±11.14, by statistical analysis, (p<0.05), with a statistical significance. Conclusion: The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2 - O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.Four, to observe the expression of iNOS in cartilage cells by RT-PCR.Objective: to observe the expression of iNOS in cartilage cells by RT-PCR. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; to observe the expression of iNOS in cartilage cells by RT - PCR. Results:In control group, the content of iNOS mRNA is 0.74±0.17, In 03 group, the content of iNOS mRNA is 0.83±0.21, by statistical analysis, (F=12.191, P< 0.01), with a statistical significance. Further statistical analysis shows that the expression of iNOS mRNA in 03 group is significantly below than the control group's. Conclusion:The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.Five, the semi-quantitative RT-PCR method in detecting articular cartilage matrix metalloproteinases-1 (matrix metalloproteti-nase-1, MMP-1) expressionObjective:to observe the expression of MMP-1 in cartilage cells by RT-PCR. Methods:32 rabbit randomly divided into two groups, respectively, the treatment group is 16, the model group is 16 used Hulth model, since the surgery date,muscle injected at 40 million u lignocaine penicillin, once daily for 3 consecutive days; injected 40mg/LO2 a O3 2ml mixed gas to the left knee joint lumen of treatment group rabbit, continuous injected seven days; injected O2 2ml to the left knee joint lumen of control group rabbit, continuous injected seven days; the 8th day, executed experimental rabbits by ear vein inject air, disinfected the skin of left knee joint by iodine and cuted left knee joint chamber, observated, cuted the condylar cartilage of left femur intol x 1 x 2mm little bar by razor-sharp blades; to observe the expression of MMP-1 in cartilage cells by RT-PCR. Results:In control group, the content of iNOS mRNA is 0.73±0.12, In 03 group, the content of iNOS mRNA is 0.67±0.13, by statistical analysis, (P<0.05), with a statistical significance. Conclusion: The 03 group more than the OA control groupis lighter in degeneration of articular cartilage, showing that 40mg/L O2-O3 mixture of gas can be effectively relieved the athological process of osteoarthritis cartilage degeneration in rabbit knee.
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