Separation,Cultivation And Identfication Of Rabbit Chondrocyte In Vitro

Background Osteoarthritis is a kind of the articular cartilage degeneration and secondary bone hyperplasia is characteristic of chronic joint disease, because the degradation of articu lar cartilage injury leading to joint edge and subchondral bone reactive hyperplasia. Is clini cal on multiple age-related diseases. With China’s aging population, the incidence of osteoa rthritis of increasingly high rates of the disease, pain and inconvenience caused serious infl uence old people’s lifestyle. Current treatment of this disease is still lack of medicine, may affect the cartilage cell destruction of cartilage, resulting in the secretion of inflammatory c ytokines in cartilage cells. Research shows that the regeneration of cartilage cell transplant ation can effectively solve the cartilage injury, cure osteoarthritis. The traditional method o f previous complicated cumbersome, and easy to pollution, caused cartilage cell death, low er survival rate and quality, prevention of osteoarthritis is very important in finding ways t o chondrocytes cultured in vitro of simple, so the purpose of this study is to explore the me thod of isolation and culture of chondrocytes in vitro, to obtain a large amount of cartilage cell division active with high activity, provides the theory basis for the cure osteoarthritis chondrocytes transplantation, success.Objective The isolation and culture of chondrocytes in vitro and the identification of chondrocytes cultured in vitro, and cultured in vitro, cultured the chondrocytes with high activity and high activity.. The study of the basic theory for the next step of artificial cartilage reconstruction and cartilage transplantation.Methods Take a 4 week old New Zealand white rabbits, ear marginal venous air embolism executed in New Zealand white rabbits, In the rabbit chest solution dig, exposed cardiac arteries, the most blood, application of centrifugal mechanism to prepare high concentrations of platelet plasma, refrigerator sealed, and to prevent pollution, for the late application. rabbit knee articular cartilage removed in aseptic operation, ultra clean table extraction separation joint tissue, cartilage tissue, cut into 1mm x lmm size fragments, PBS phosphate buffer solution washing three times, to join the mass fraction of 0.1% of ethylenediamine tetraacetic acid (EDTA) 5 ml stationary after 20 minutes under normal temperature centrifugal supernatant, join is three times the volume of 0.25% trypsin, placed 40 min digestion in 37℃temperature oscillator, blowing gently to refuse to go to clear liquid, PBS wash twice, according to the proportion of 1:1 to join the mass fraction of 0.2%Ⅱ collagenase and 5 ml of the mass fraction of 0.25% trypsin digestion in 20 min at 37℃temperature oscillator,4℃low temperature high speed centrifugal (1000 r. min-1), 5 min after centrifugal supernatant, add 6 ml containing 0.2% collagenase digestion liquid type Ⅱ at 37℃thermostat oscillator within the digestion, take qing 1 times every 90 minutes, remaining within the beaker replacement digestive juices continue to digest, take with volume fraction of 10% fetal bovine serum (FBS) DMEM/F12 culture based on the supernatant fluid and trypsin, high-speed centrifugal precipitation collecting cells, cells suspension collected in DMEM/F12 culture (containing 10% FBS, green streptomycin 100 each (including g/ml).Application of mechanical separation of rabbit chondrocytes double enzyme digestion method, the experimental group in vitro culture and PRP, the control group conventional culture, through microscopic morphology, two groups of cells using the Prism software, determined by MTT colorimetric method draw two groups of cell proliferation growth curve. Application of sappanwood eosin staining, collagen type Ⅱ immunohistochemical staining identified cartilage cells, compared two groups of the activity of cultured chondrocytes.The result Chondrocytes were isolated process when compared with the traditional method significantly shortened, in the training process of cartilage cells in vitro, join the activation of platelet rich plasma (PRP), can promote the proliferation of cartilage cells, with the passage number increased after still can have higher chondrocytes biological characteristics.Conclusion This study established cartilage cells is more simple and convenient separation method, in the training process, adding activated PRP can promote the expression of cartilage cell activity. This conclusion provides the theoretical basis for the future of artificial cartilage reconstruction and transplantation.