| Osteoarthritis is a common disease of orthopedics, can cause severe physical disability and loss of function. World Health Organization to OA and cardiovascular diseases and cancer tied for the threat to human health "the three major killers". United Nations from 2000 to 2010 will also be designated as "Bone and Joint Decade", in order to arouse the attention of the international community to strengthen basic research, clinical prevention and treatment, thus fundamentally the conquest of OA. Aetiology of OA is still no definite conclusion, but recognized that the development of OA is not caused by a single factor, but the results due to a variety of factors, such as age, sex, obesity, joint trauma, genetic factors. After years of research, although currently not fully explain the pathophysiology of OA, but it has been recognized that the main pathological changes of the disease, including articular cartilage erosion, marginal bone hyperplasia, the formation of osteophytes, sclerosis, and intra-articular synovial subchondral generation and synovial inflammation callus.Carboxymethyl chitosan is a new derivative of chitosan, has good water solubility, and its structure and physicochemical properties are similar to hyaline cartilage proteoglycan outside of cells, such as hyaluronic acid; in our previous studies have carboxymethyl chitosan confirmed rabbit cartilage has a protective effect, but the exact mechanism is not yet fully clear. In recent years, a large number of studies suggest that regulation of bone metabolism OPG/RANKL/RANK system is the most important regulator of bone remodeling signaling pathways, osteosclerosis close this system and OA cartilage and plays an important role in the course of OA. Overseas study confirmed OA progression of bone sclerosis, subchondral joint osteophyte formation are closely related to the OPG/RANKL/RANK signaling pathway, abnormal bone rebuilding mode under OA cartilage, subchondral bone stress buffer weakened, leading to joint biomechanics change, further exacerbating the OA cartilage damage degeneration, subchondral bone reconstruction has become a hot research OA. Studies have shown that OPG/RANKL/RANK system may be a new target for the treatment of early OA, providing more options for OA treatment, but by the systematic study of OA's just getting started, how to use this entry pathways to treat OA still need deeper explore, so it has also become the subject of our research focus.According to the protective effect of this subject is relevant researches organize and speculate carboxymethyl chitosan osteoarthritis chondrocytes may be associated with OPG/RANKL/RANK signaling pathway, this study aims to further explore the carboxymethyl chitosan osteoarthritis affects sugar OPG/RANKL/RANK system.Part I:Effects of carboxymethylated chitosan on cell proliferation and secretion of extracellular matrix in cultured cartilage cells in vitroObjective:The purpose of this study was to observe the effects of CMCS on proliferation and extracellular matrix secretion of cartilage cells in vitro, and the probable mechanisms in those processes.Methods:Healthy 4-month-old New Zealand rabbits were used to gathering the cartilage cells. Remove bilateral knee cartilage were isolated under sterile conditions and cultured chondrocytes in vitro.Cultured cells were identified by immunohistoche-mistry of collagen type-2 and aggrecan. The CMCS was added into each cell group. The final concentrations were as following:0,100,200,300,500,800 and 1000?g/mL, after 24 hour treatment, the proliferation of cartilage cells were assessed by cell counting kit-8 (CCK-8). The growth and cell phenotype were observed by inverted microscope after addition of different concentrations of CMCS, and the messenger RNA (mRNA) expression of proliferating cell nuclear antigen (PCNA), collagen type-2 and aggrecan were detected by using real time polymerase chain reaction (eeal-time PCR) anaysis.Results:Chondrocytes in vitro ordinary adherent adherent cultured for 24 hours beginning; two days completely adherent cell growth, visible triangle, polygonal cells; 5 days cell growth, showing "pavement road" like appearance; seven days a large cell fusion, covered the entire bottom surface monolayer culture. Type II collagen and aggrecan immunohistochemical staining results of this study showed that cultured cells can secrete type ? collagen and aggrecan. CCK-8 test results showed that CMC within 100-1000?g/mL concentration range in vitro proliferation of chondrocytes can promote, the difference was significant (P<0.05), and when the concentration of 300?g/mL its most obvious effect of proliferation (P<0.05). RT-PCR and Western blot results showed that different concentrations of carboxymethyl chitosan can increase the expression of chondrocyte protein and PCNA mRNA, the difference was significant (P <0.05). In addition, by Real-time PCR detected at different concentrations of carboxymethyl chitosan can increase the expression of chondrocyte type ? collagen and aggrecan mRNA in comparison with the control group, a significant difference show carboxymethyl chitosan can promote cartilage cells in vitro synthesis and secretion of extracellular matrix (P<0.05).Conclusion:This study shows that CMCS has a stimulative effect on proliferation of cultured cartilage cells. This effect is based on the alteration of PCNA systhesis. CMCS also could induce the synthesis and secretion of ECM such as collagen type-2 and aggrecan in cultured cartilage cells.Part ?:Protective effects of carboxymethylated chitosan on sodium nitroprusside induced apoptosis of cartilage cells and its mechanisms in vitroObjective:Apoptosis plays the important role in degeneration of nucleus pulposus. This study was designed to investigate the effect of CMCS on apoptosis in cultured cartilage cells in vitro. The cartilage cells apoptosis were induced by sodium nitroprusside (SNP) as the indicated methods. The protective effects of CMCS on SNP induced apoptosis in cultured cartilage cellswere investigated.Methods:Healthy 4-month-old New Zealand rabbits were used to gathering the cartilage cells. Remove bilateral knee cartilage were isolated and cultured chondrocytes in vitro under sterile conditions. The first choice ? chondrocytes grown in culture flasks, to be adherent cells with serum-free DMEM medium "hunger" cultured 24 h, the cells were synchronized.The cartilage cells were treated by SNP. The final concentrations of SNP were 0.5mM, 1mM and 1.5mM. Cartilage cells were stained by both Annexin-V and PI. The apoptotic rate of cartilage cells treated by SNP was assessed by flow cytometry (FCM) analysis. Meanwhile, the CMCS in concentrations of 100,200 and 300?g/mL was added into cartilage cells culture medium, the apoptotic model of cartilage cellswas identified by FCM assay, the effect of CMCS on apoptotic rate of SNP induced cartilage cells was also determined by FCM assay. Specific experiments included:1 CCK-8 method to detect the inhibition of cell proliferation; 2 FCM detected apoptosis in each group; 3 Hoechst33342 nucleus staining fluorescence microscopy analysis of changes in nuclear morphology of each group situation; 4 PCR detection of intracellular collagen ?, aggrecan, Caspase-3, Bcl-2, OPG and RANKL mRNA expression; 5 Western blot assay of intracellular protein Bcl-2, OPG and RANKL expression situation.Results:The FCM assay results indicated that SNP in range of 0.5 to 1.5mM could induce apoptosis of cartilage cells with concentration dependence. CMCS in concentrations of 100 to 300?g/mL has the protective effects on SNP induced apoptosis in cartilage cells. Hoechst33342 staining results showed that CMCS could decrease the nuclear fragment of apoptotic cartilage cells induced by SNP. Real-time PCR and Western blot analysis results indicated that CMCS could inhibit sodium nitroprusside-induced chondrocyte extracellular matrix secretion decreases.And can reduce the expression of caspase-3 and RANKL induced by SNP, and increase the expression of Bcl-2,OPG and OPG/RANKL induced by SNP.Conclusion:Those results indicated that CMCS has the protective effect on the SNP induced apoptosis in cartilage cells. Its anti-apoptotic effect is possible by changing the apoptotic kinase activity Caspase-3 and Bcl-2,and regulate the activity of OPG/RANKL/RANK system implementation.Part ?:Effects of Carboxymethyl-chitosan on OPG/RANKL/RANK expression in a rabbit osteoarthritis modelObjective:Osteoarthritis rational animal model for evaluation and research carboxymethyl chitosan treatment of osteoarthritis and to explore its degeneration of cartilage in the OPG/RANKL/RANK system, mechanism of action, for the treatment of osteoarthritis to find a new and effective method of treatment.Methods:Selection of 24 healthy adult New Zealand rabbits were randomly divided into two groups and given the following treatment:A group (CMCS group, n=12) and group B (OA group, n=12). Two groups of rabbits were cut off before the medial ligament of the left knee the way rabbit model of osteoarthritis, contralateral knee accept sham operation and were set to control. A group of four weeks after intra-articular injection of lmg/ml of carboxymethyl chitosan (CMCS) 0.3ml, repeated weekly for 5 weeks, the animals were killed after 11 weeks; group B received normal saline injection, after the same animals were killed after 11 weeks, general changes in each group within the articular cartilage of the femoral condyle, HE staining and pathological changes observed fast green check cartilage damage grade evaluation, using real-time quantitative polymerase chain reaction (Real-Time PCR) to detect cartilage RNA, proteins were detected by Western blot to analyze the expression of each group OPG,RANKL and RANK.Results:CMCS group cartilage damage (including the scope and level) lighter than the OA group (P<0.05). In normal cartilage, lower levels of OPG and RANKL expression, and the expression of OPG and RANKL are enhanced cartilage in OA model. After CMCS processing, OPG even highly regulated, and downregulation of RANKL. OA group OPG/RANK ratio decreased, while the treatment group increased CMCS; OA group RANKL/RANK CMCS ratio increased while the treatment group reduced RANKL/RANK ratio.Conclusion:CMCS articular injection may reduce the extent of cartilage degeneration, progression and treatment mechanism of OPG/RANKL involved in OA maintain homeostasis OPG/RANKL/RANK system may be one mechanism for the treatment of osteoarthritis. |
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