| Osteoarthritis is a degenerative disease of articular cartilage. The main pathological characteristic of osteoarthritis is cartilage destruction. Osteoarthritis seriously debased patient’s quality of life and brought enormous economic burden to the family and society. At present, NSAIDs are widely used to treat OA, which can relieve pain, but can not inhibit cartilage damage and delay the OA process. Chinese Medicine in treating osteoarthritis has the advantage of good therapy and little side-effect. In the earlier clinical research, the result has showed that tonifying the kidney to strengthen bone had significant curative effect in treating OA, total effective rate was90%, could improve remarkably pain, swelling and motor function of patients. The experimental study found that tonifying the kidney to strengthen bone can improve joint structure disorder in OA animals model and inhibit the decrease in the number of chondrocyte. Therefore, it is very necessary to further study Traditional Chinese Medicine in inhibiting cartilage damage to protect the cartilage and its possible molecular mechanism.This study is under the guidance of tonifying the kidney to strengthen bone in treatment of osteoarthritis, with the support from National Natural Science Foundation of China, this research using OA chondrocyte system in vitro as the object, and taking the advantage of herbal serum pharmacological method and with the help of modern molecular biology identification technique, aimed to study the effect of tonifying the kidney to strengthen bone in cell proliferation, apoptosis and matrix metabolism of chondrocytes.1Experiment OneObjective:To establish a finite cell line of human osteoarthritis chondrocytes, and observe the growth and morphology characteristic of chondrocytes, provide essential basis and experimental model for further studying molecular cell biology in the catabolism of chondrocytes and the protection function of decoction.Methods:The chondrocytes used in these researches were available with primary tissue culture by enzymatic digestion, cartilage tissue obtained during surgery for knee replacements from OA patients. Chondrocyte proliferation was detected by methyl thiazolyl tetrazolium (MTT); toluidine blue stain and immunohistochemistry of type Ⅱ collagen were used to identify the cell source; observed its morphologic changes under inverted microscope.Results:Successfully established a finite cell line of human osteoarthritis chondrocytes, which growth in good condition and had stable biological property. Toluidine blue stain and immunohistochemistry of type Ⅱ collagen identified that the cells are chondrocytes from mesodermal.Conclusion:Successfully established a finite cell line of human osteoarthritis chondrocytes with primary tissue culture by enzymatic digestion. The cells are chonrocytes from mesodermal. Provided a good vitro model for researching the pathogenesis of OA and the effect of decoction on interventing matrix metabolism.2Experiment TwoObjective:Normal human chondrocytes were taken as control group, observed the characteristics of OA chondrocytes in proliferation, the cell cycle and apoptosis, aimed to research the basic biological characteristics of OA chondrocytes.Methods:The chondrocytes used in these researches were available with primary tissue culture by enzymatic digestion, cartilage tissue obtained during surgery for knee replacements from those OA patients and those who needed acute amputation by accidence. Methyl thiazolyl tetrazolium (MTT) was used to detect the proliferation of OA chondrocytes and normal chondrocytes. Flow Cytometric analyzed the differences of cell cycle and apoptosis between OA chondrocytes and normal chondrocytes.Results:OA chondrocytes performanced excessive apoptosis (P<0.01), the number of cells in S phase was in lower proportion (P<0.05), and cell proliferation was significantly slow (P<0.01).Conclusion:The weaker proliferation ability and excessive apoptosis may be the important factors to accelerate cartilage damage in OA. 3Experiment ThreeObjective:Normal human chondrocytes were taken as control group, observed the the level of IL-1β in the supernatant of OA chondrocytes and the protein expression of IL-1β, aimed to research the role of IL-1β in the development of OA.Methods:Compared the level of IL-1β in the supernatant between OA chondrocytes and normal chondrocytes by ELISA. Compared the protein expression of IL-1β between OA chondrocytes and normal chondrocytes by Western blotting.Results:The level of IL-1β in the supernatant of OA chondrocytes is more than normal chondrocytes (P<0.05), and the protein expression increased(P<0.01).Conclusion:IL-1β plays an important role in the cartilage damage of OA. IL-1β can accelerate the catabolism by the abnormal regulation of MMPs; Meanwhile, IL-1β can promot the cartilage damage, maybe through inducing apoptosis and showing anti-inflammatory effect.4Experiment FourObjective:Normal human chondrocytes were taken as control group, observed the protein expression of MMPs, aimed to research the role of MMPs in the cartilage damage of OA.Methods:Compared the protein expression of MMP-1,3,13between OA chondrocytes and normal chondrocytes by Western blotting.Results:The protein expression of MMP-1,3,13increased in OA chondrocytes (P<0.01).Conclusion:MMPs plays an important role in the development of OA, which can result in the cartilage damage of OA through degrading the extracellular matrix and accelerating the catabolism of extracellular matrix.5Experiment FiveObjective:Normal human chondrocytes were taken as control group, observed the effects of Niuxijianbu Particles on cell proliferation and apoptosis of OA chondrocytes.Methods:The chondrocytes were divided into normal chondrocyte group, OA chondrocyte group, OA chondrocyte+control serum group, OA chondrocyte+high-dose-decoction-containing serum group, OA chondrocyte+middle-dose-decoction-containing serum group, OA chondrocyte+low-dose-decoction-containing serum group. All the cells were treated according to the grouping for24h, Then cell proliferation was detected by CCK-8assay and the cell apoptosis by flow cytometry.Results:Niuxijianbu Particles can promote cell proliferation and inhibit cell apoptosis(P<0.01).Conclusion:Niuxijianbu Particles can promote cell proliferation and inhibit cell apoptosis, which may be the important mechanism to inhibit OA cartilage damage.6Experiment SixObjective:Normal human chondrocytes were taken as control group, observed the effect of Niuxijianbu Particles on the protein expression of IL-1β,aimed to research whether Niuxijianbu Particles can inhibit the cytokine IL-1β in OA chondrocytes.Methods:The chondrocytes were divided into normal chondrocyte group, OA chondrocyte group, OA chondrocyte+control serum group, OA chondrocyte+high-dose-decoction-containing serum group, OA chondrocyte+middle-dose-decoction-containing serum group, OA chondrocyte+low-dose-decoction-containing serum group. All the cells were treated according to the grouping for48h, then the level of IL-1β in the supernatant was detected by ELISA and the protein expression by Western blotting.Results:Niuxijianbu Particles can inhibit the level of IL-1β in the supernatant and the protein expression in OA chondrocytes(P<0.01).Conclusion:Niuxijianbu Particles can inhibit the secretion of IL-1β, then shows anti-inflammatory effect; Meanwhile, Niuxijianbu Particles can inhibit the degradation of extracellular matrix by inhibiting the abnormal regulation of MMPs, then to inhibit the OA cartilage damage. 7Experiment SevenObjective:Normal human chondrocytes were taken as control group, observed the effect of Niuxijianbu Particles on the protein expression of MMPs, aimed to research whether Niuxijianbu Particles can inhibit the MMPs in OA chonrocytes.Methods:The chondrocytes were divided into normal chondrocyte group, OA chondrocyte group, OA chondrocyte+control serum group, OA chondrocyte+high-dose-decoction-containing serum group, OA chondrocyte+middle-dose-decoction-containing serum group, OA chondrocyte+low-dose-decoction-containing serum group. All the cells were treated according to the grouping for48h, then the protein expression of MMP-1,3,13was detected by Western blotting.Results:Niuxijianbu Particles can inhibit the protein expression of MMP-1,3,13in OA chondrocytes (P<0.01).Conclusion:Niuxijianbu Particles can inhibit the protein expression of MMPs in OA chondrocytes, then can inhibit the degradation of extracellular matrix, which can balance the catabolism and anabolism to inhibit OA cartilage damage.8Experiment EightObjective:Normal human chondrocytes were taken as control group, observed the effects of Icariin on cell proliferation and apoptosis of OA chondrocytes.Methods:The chondrocytes were divided into normal chondrocyte group, OA chondrocyte group, OA chondrocyte+Icariin group. All the cells were treated according to the grouping for48h, then cell proliferation was detected by CCK-8assay and the cell apoptosis by flow cytometry.Results:Icariin can promote cell proliferation and inhibit cell apoptosis(P<0.01).Conclusion:Icariin can promote cell proliferation and inhibit cell apoptosis, which may be the important mechanism to inhibit OA cartilage damage. 9Experiment NineObjective:Normal human chondrocytes were taken as control group, observed the effects of Icariin on the protein expression of IL-1(3and MMPs, aimed to research whether Icariin can inhibit the catabolism in OA chondrocytes to inhibit OA cartilage damage.Methods:The chondrocytes were divided into normal chondrocyte group, OA chondrocyte group, OA chondrocyte+Icariin group. All the cells were treated according to the grouping for48h, then the level of IL-1β in the supernatant was detected by ELISA and the protein expression of IL-1p and MMP-1,3,13by Western blotting.Results:Icariin can inhibit the level of IL-1β in the supernatant (P<0.05)and the protein expression of IL-1β and MMP-1,3,13in OA chondrocytes(P<0.01).Conclusion:Icariin can inhibit the secretion of IL-1β,then shows anti-inflammatory effect; Meanwhile, Icariin can inhibit the degradation of extracellular matrix by inhibiting the abnormal regulation of MMPs, then to inhibit the OA cartilage damage. |
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