| Object:Osteoarthritis (osteoarthritis, OA), is caused by various factors in the elderly with multiple chronic degenerative joint disease, which is the most common knee joint inflammation disease. Knee osteoarthritis is most common in the elderly population, especially in the obese elderly population. Both men and women both have the disease, more women than men. In the more than 60-year-old population,50% of people in the X-line has the performance of osteoarthritis, in which 35%-50% of clinical performance. With the progress of population aging, osteoarthritis is becoming a focus of common concern to doctors and patients, for which the World Health Organization (WHO) will be October 12th as "the world of osteoarthritis Day" and the first ten years of the 21st cencury as the "Decade of Bone and Joint Disease. " Clinical observation that patients with knee osteoarthritis has been extended to the general population over the age.of 35, seriously disturbing people's daily life and work. Osteoarthritis is a degeneration and loss of articular cartilage and joint marginal and subchondral bone regeneration, which is characterized by chronic arthritis of the disease. The site of origin of the disease is the cartilage. The etiology and pathogenesis of Osteoarthritis is not entirely clear.It is generally believed that it is the participation of systemic factors such as the existing age, sex, familial susceptibility, and local biomechanics, cartilage cell apoptosis, cytokines, and degradation of the role of enzymes.It is a multi-factor, multi-link caused by the disease. The literature has reported the chondrocyte apoptosis may be an important part of the pathogenesis of OA. At present, the cartilage cell apoptosis induced by means of signals (Fax, NO), signal factor of controlling chondrocyte apoptosis (Bcl-2 family, caspases protease) and cartilage signal to stimulate apoptosis are hot spots in the experimental studies. A large number of studies have confirmed, OA course of IL-1 (IL-1), tumor necrosis factor-α(TNF-α), matrix metalloproteinase-line (MMPs) [particular MMP-1, MMP-3, MMP-13 and tissue inhibitor-1 of metalloproteinase (TIMP-1)]have significant change. These indicators can be indirectly measured to understand the evolution of OA, not only suggesting the role of chondrocyte apoptosis in the pathogenesis mechanism of OA, but also is used to evaluate the role of OA intervention, s target and mechanism.Osteoarthritis in the Traditional Chinese Medicine is "Bi disease, " "Gubi" areas. Less than liver and kidney, Loss of nutrientsin the bones and tendons are important dystrophy pathogenesis. Drynariae is bitter taste, temperature, Huo xue continued injury, tonic kidney strong bones, as the important drugs of bone-setting, it is a certain improvement of cartilage cell function and it has a role of delaying degeneration of cartilage cells.The experiment uses mainly cell, molecular biology techniques, using reverse transcription polymerase chain reaction (RT-PCR), Western blotting (Western-blot) and other detection methods, through the cartilage cells of different apoptosis-related factor expression, further exploring the pathogenesis of OA, studing the mechanism Drynariae, observing on cartilage cell apoptosis,s influence of experimental osteoarthritis in rabbit knee, and the theoretical analysis. It provides experimental and clinical application of theory and practice basis oftraditional Chinese medicine for prevention andtreatment of knee osteoarthritis.Materials and Methods:1, experimental animals24 adult New Zealand rabbits, the general level, regardless of male and female, weight 2-3kg. Liaoning University of Traditional Chinese Medicine Center for animal experiments, animal permit number:SCXK (LU) 20030006.2,the experimental drug2.1 Drynariae Liaoning University of Traditional Chinese Medicine Pharmacy to provide. Condensed into a decoction with the liquid concentration lg/ml reserve, for a total of 630ml. 2.2 dimensional solid edge Ireland Rottapharm Ltd, batch number:H20040637, specifications:0.25g (glucosamine sulfate), Liaoning University of Traditional Chinese Medicine Pharmacy to provide3,experimental main reagents(1) Western Blot Detection of Anti-Ⅰantibody used:mouse monoclonal antibody anti-rabbit IL-1, goat anti-rabbit TNF-α, rabbit anti-mouse MMP3, rabbit anti-rat Bcl-2, goat anti-rabbit BAX, rabbit anti-rat Caspase-3, goat anti-rabbitβ-actin (the United States santa cruz biotechnology,(?) inc.). Anti-Ⅱantibodies:rabbit anti-mouse alkaline phosphatase marker, rabbit anti-sheep antibody (Beijing Zhongshan Biotechnology corporation).(2) endogenous peroxidase blocking agent:3% hydrogen peroxide solution(3) closed by normal bovine serum albumin working solution(4) alkaline phosphatase labeled anti-working solutionⅡ(5) concentrated PBS buffer solution(6) 3,3-Diaminobenzidine hydrochlorideⅣ(3,3-Diaminobenxidine tetrahydroehloride, DAB)(2)-(6) Fuzhoumaixin the new biotechnology development companies(7) total RNA extraction reagent guanidine isothiocyanate (Triozol Reagent): United States company Invitrogen Life technologies(8) reverse transcriptase (AMV)(9) RNA enzyme inhibitors(10) dNTP mixture(11) Oligo (dT) 15(12)γ-Taq DNA polymerase(13) target gene primers(8)-(13) Japan TaKaRa Company(14) agarose:The United States company Promaga(15) Coomassie brilliant blue protein quantitative kit:Nanjing established bio-engineering company(16) nitrocellulose membrane:purchased from the company Millopore (17) Other reagents:6×gel sample buffer, DNA electrophoresis buffer (TBE), sodium dodecyl sulfate (SDS), Tris (Tris), acrylamide (Acr),3% glutaraldehyde, methanol, developer, fixed liquid, acetone, xylene, ethanol, formaldehyde, skimmed milk powder bought in the company humei.4, the main experimental apparatus and equipment(1) OLYMPUS microscope (Japan, CH)(2) electric constant temperature water tank (Space Instrument Factory, Huanghua, Hebei Province, HH.W21.600 type)(3) slicer (Germany Leitz, Kryostat 1720)(4) The ice-making machine (Germany ZIGERA,2BE-70-25)(5) small centrifuge desktop (U.S. SIGMA,1-13)(6) PCR amplification instrument (Germany Biometra)(7) UV spectrophotometer (UK UV-visible Spectrometer, UV300).(8) Bio-Rad (USA BIO-RAD, PowerPac200)(9) Semi-transfer instrument (USA BIO-RAD Semidry Transfer System)(10) the level shaking bed (U.S. GFL)(11) vertical slab electrophoresis unit (U.S. BIO-RAD, Mini-Protein III)(12) Chemi Imager5500 gel electrophoresis image analysis system (United States Alphainnotech chemi Imager)(13) HITACHI-7500 type transmission electron microscope (HITACHI, Japan Corporation)5, the establishment of the rabbit knee OA Rabbit left hindlimb extension position fixed plaster cast for 6 weeks has been the model of rabbit knee OA.6, animal grouping and drug deliveryNew Zealand 24 rabbits are randomly divided into 4 groups.blank group 6, model not, do not use the normal diet.Model group 6, after the model, do not use the normal diet.Treatment group (Drynariae group) 6, gastric washing after the model,1 times/day, medication for 4 weeks. The control group (solid peacekeeping force group) 6, gastric washing after the model,1 times/day, medication for 4 weeks.7, animals killed and materialsAfter medication four weeks, the way of air ear vein embolization to kill animals in each group.Surgical operation to remove the left hindlimb of each rabbit femoral condyle (about lcm) and proximal tibia (about lcm), the fine separation of articular cartilage.Remove each of the articular cartilage in rabbits divided into three parts. A small part of the tibial plateau,3% glutaraldehyde solution fixed,4 C refrigerator for electron microscopy. Part of femur using Trizol soaking liquid, 4 C refrigerator for RT-PCR detection; another part in the plastic tube, the seal,-80 C frozen for Western blot detection.8, electron microscopyAt 4 C condition,-after the immersion in 3% glutaraldehyde fixative in 24 hours, Transparent liquid propylene oxide for decalcifying, with 0.1M phosphate buffer washing 3 times with 0.1% osmium tetroxide fixing for 60min, deionized water washing 3 times, the termination osmium tetroxide reaction, different concentrations of ethanol dehydration step by step, soaking using Epon812 epoxy resin, embedded, repair semi-thin slices after hardening, the ultra-thin slices after the positioning, slice thickness 60nm, dual-acetate uranium oxide and lead citrate double staining TEM and X-ray film.9, RT-PCR detection(1) TRIzol to extract total RNA(2) RNA concentration and purity determination(3) reverse transcriptase synthesis of cDNA(4) PCR amplification:Application Biometra PCR amplification instrument(5) RT-PCR product electrophoresis and analysis of quantitative 10, Western-blot detection(1) protein extraction (2) Determination of protein concentration(3) protein denaturation(4) with plastic(5) Electrophoresis(6) Transfer(7) closed(8) incubated with anti-first(9) incubated with anti-second(10) cleaning(11) Color(12) Western Blot results of quantitative analysis:The FlourChem V 2.0 gel image analysis software (America) analysis, the grayscale value of protein electrophoresis of each record and quantitative analysis.11, data processing and statisticsSPSS13.0 package used for RT-PCR and Western Blot results of variance analysis and Pearson correlation analysis, data indicated that the mean±standard deviation. The P<0.05 has statistical significance.Results:1, the general morphologyIn the blank group, Articular cartilage surface is smooth and a little blue. In the model group, Cartilage surface is a little white. Careful observation shows that there are scattered small punctate and linear depressions. Cartilage surface is also a little white or a little yellow in the Treatment group and control group, the surface is still smooth, but there are can be seen scattered punctate depressions.2, the ultrastructural changes under the electron microscope Under the electron microscope, the ultrastructure of the cartilage surface is relatively smooth in the blank group, we can see there is small pore symmetry, covering a little bit distribution of the white spherical gel-like substance; In the model group, surface is rough and uneven,partial part has crack-like changes. In severe cases, the surface is loss, under which collagen fibers are exposed or cartilage surface appears fissures, collagen fibers have the rupture; In the treatment group, lesions is relatively light, the surface is still smooth and the surface is covered with large round gel-like material, it can be seen scattered clastic-like change, cracks and small holes are formed in the local place; In the control group, the cartilage surface is not smooth, we can see more of the cracks and small holes and different sizes of shallow ulcers, the local surface which is flake up is tear and endarterectomy. Under it there is collagen fibersChondrocytes are observed shows that the structure of cells is clear in the blank group, cell membrane is integral, outline is clear and the nuclear chromatin is evenly distributed. Extracellular collagen fibers can be seen criss-cross. In the model group, Membrane is dissolved, nuclear membrane is unclear, cytoplasm is disappeared. It is seen that cell is swelling and necrosis is increased significantly. Collagen fibers decrease with disorder around Cells, and some gathered into one another corporation or a bundle-like shape, and a staggering phenomenon, and some sparse. In the treatment group,there iskaryopyknosis,mitochondria dissolved and endoplasmic reticulum swollen; In the control group,there is nuclear enrichment, nuclear membrane unclear and the expansion of mitochondrial dissolved. The collagen fibers around Cells are arranged due to the rules in the treatment group and control group3, RT-PCR test resultsBAXmRNA expression levels increase more in the Model group than in the blank group there was a significant difference (P<0.05); BAXmRNA expression levels decreased more significantly in the control group than in the model group, there was a significant difference (P<0.05); BAXmRNA expression level decreased more significantly in the treatment group than in the model group, there was a significant difference (P<0.05); BAXmRNA expression of the difference was not significant in the treatment group than in the control group the expression level of bcl-2mRNA decreased more in the. Model group than in the blank group, there was a significant difference (P<0.05); The. expression level of bcl-2mRNA has increased more in the control group than in the model group, there was a significant difference (P<0.05); the expression levels of bcl-2mRNA has a more marked increase in the treatment group than in the model group, the difference was significant (P<0.05); The expression of bcl-2mRNA is no significant difference in the treatment group than in the control group.4, Western blot test results TNF-αprotein expression levels were more significantly increased in the Model group than in the blank group, there was a significant difference (P<0.05); TNF-αprotein expression level decreased more significantly in the control group than in the model group (P<0.05); TNF-αprotein expression levels decreased more significantly in the treatment group than in the model group (P<0.05), there was a significant difference; TNF-αprotein expression had no significant difference in the treatment group than in the control group. TGF-βprotein expression level decreased more significantly in the model group-than in the blank group (P<0.05); TGF-βprotein expression level increased more in the control group than in the model group, there was a significant difference (P<0.05); TGF-Bprotein expression was more significantly increased in the treatment group than in the model group of, there was a significant difference (P<0.05); the treatment group than in the control group of TGF-βprotein expression was no significant difference.Conclusion:1 TNF-α, TGF-β, BAX, bcl-2 and other factors are closely related to the course of osteoarthritis.2 The Study confirmed the existence of enhanced expression of the TNF-α, TGF-βand protein, weakened expression in the BAX, bal-2mRNA, in the OA articular cartilage, which may be part of the pathogenesis of OA. Therefore, the application of various methods of inhibiting TNF-α, BAX expression, and enhance the expression of TGF-β, bcl-2 and its biological effects may be an effective way of great significance to the treatment of OA. It is important.3 Through the RT-PCR, Western-blot detection, single herbs Drynaria better expresseS the regulation factor on the TNF-α, TGF-β, BAX, bcl-2, suggesting that its application plays an important role in the prevention and treatment of OA, it is necessary to further develop relevant clinical and basic research.4 Western medicine dimensional solid-edge (glucosamine sulfate) and single herbs Drynaria cartilage showed the same inhibition or enhance to the apoptosis-related factors and The data of this set showed no difference statistically, but There is a marked difference in the mRNA'and protein expression. It can be considered that a single herbs Drynaria has a comparative advantage of the prevention and treatment of OA. |
PDF Full Text Request