| Knee osteoarthritis(KOA) is a progressively degenerative knee joint disorder. It causes chronic disability in elderly people and becomes one of the major health problems worldwide. No rational medical therapy is currently available for KOA before patients require artificial joint replacement therapy. The reason is that the precise etiology of osteoarthritis(OA) is not still unknown.Knee articular cartilage is a kind of hyaline cartilage that comprises two main components: chondrocytes and extracellular matrix(ECM) synthesized and secreted by chondrocyte. Aggrecan and collagen II are the most characteristic cartilage-specific ECMs. In KOA, dysfunction of chondrocytes compromises synthesis of ECM and enhances degradation of ECM by matrix metalloproteinases(MMPs), aggrecanases(ADAMTSs) and other matrix proteases which lead to loss of ECM and cartilage degradation. Now it is believed that Calcineurin(Ca N) participates in the regulation of chondrogenesis and plays an important role in the occurrence and development of OA.Ion channels are the essential components that control ion movement in and out of the cell. As all living cell systems, articular chondrocytes are provided with a membrane potential. Although chondrocytes are non-excitable cells their plasma membranes contain different ion channels and porins, such as calcium activated potassium, ATP dependent potassium channel, voltage-gated potassium channel, epithelial sodium channels, voltage-gated sodium channel, aquaporin channel, chloride channel, NMDA receptor, transient receptor potential channel, voltage-gated calcium channels, and so on. Although the specific function of each channel is not entirely clear, all kinds of mechanical and biochemical stimulation can affect the function of chondrocytes through these channels. It was said that ion channel modulators caused changes of the membrane potential of chondrocytes and regulated the chondrocyte proliferation. Verapamil, the calcium ion channel blocker, results in a more negative membrane potential(RMP) by 18% compared to a control group. Now the function of voltage-gated calcium channel in normal cartilage has not been detected. A connection between these calcium channel activity and the osteoarthritis cartilage degeneration is far less well understood.In this study, by using performed patch clamp technique and molecular biology and immunology methods, m RNA expression of nine voltage-operated Ca2+channel α1 subunits were first detected in normal and KOA chondrocytes. Then we explored the possible role of Ca N in the regulation of matrix metabolism of chondrocytes via L-type calcium channel α1 subunit in rabbit KOA model so as to provide evidence for screening new KOA treatment concepts.Part 1 The rabbit KOA models making and identificationObjectives: Making and identifying rabbit models of osteoarthritis. Methods: Turn rabbit as specimen using Hulth method, making OA models on legs knees. 12 weeks after the surgery, there are multiple factors to identify the success of OA models through, basically is to observing the major changes of joints, such as HE staining, histopathological Markin rating and type II collagen immunohistochemical staining, biopsy toluidine blue stain and X ray film.Results: X-ray appears, KOA animals, knee femur and tibia marginal osteophyte generated 12 weeks after surgery, the gaps between joints surface are not the same, the inside gaps are narrower, bone sclerosis are visible on articular surface. The most specimens had synovial hyperplasia, along with synovial fluid turbid, surface defective and severe ulceration, subchondral bone exposed, cartilage softens the edges of cracks, rough surface, grayish yellow, surrounded by a large osteophyte emergence. HE staining showed that the cartilage damage is severe, a wide range of cartilage is fibrosing deeply, the number of cells considerably reduced, distribution uneven, the tide line is not continuous, calcified layer is difficult to distinguish; severe loss of stromal staining, Mankin score 13-14 points. Immunohistochemistry was found decreased expression of collagen type Ⅱ than the normal group. Tissue sections stained with toluidine blue discovery that OA cartilage lighter than the normal control group.Conclusion: Using the method of Hulth, successfully reproduced the pathological changes of knee joints in cases OA, then provided reliable animal samples for the experiment.Part 2 The selection of RT-q PCR reference gene after culturing rabbit cartilage cells in KOA Objective: To determine the best reference genes for RT-q PCR.Methods: Cultured normal and OA of rabbit articular cartilage cells, then passage for the first three generation, and then use the RT-q PCR technology to analyze the expression of the 15 housekeeping genes in the chondrocytes. The stability of housekeeping gene was sequenced by ge Norm and Norm Finder. Comprehensive the above stability of genes by Ref Finder to get the the most stable reference genes in severe OA.Results: There are difference among the expression of 15 housekeeping genes. The results by ge Norm and Norm Finder were not the exactly same. The GAPDH and β-actin, have poor stability, and cannot be used as the reference gene. The Ref Finder results showed that the best matched genes of the first generation were RP II and YWHAZ, the best matched one of the second generation were RPL13 a and PPIA, the best of the third generation were RPL13 a and RP II.Conclusion: The most stable cell reference genes of the first three generations are YWHAZ and RP II.Part 3 Voltage-gated calcium channel α1 subunit on the matrix metabolism of chondrocytes in rabbit knee osteoarthritisObjective: To examine the connection between the voltage-gated calcium channel α1 subunit and the matrix metabolism of chondrocytes in rabbit KOA.Methods: The changes of RMP were recorded from cultured chondrocytes of normal and KOA by performed patch clamp. By Anabolic and catabolic marker m RNA expressions, as well as nine voltage-gated calcium channel α1 subunit m RNA expressions in normal and KOA chondrocytes were measured by RT-q PCR.Results:(1) The changes of RMP: the average RMP in normal rabbit chondrocytes was-37 m V, and its in KOA chondrocytes was-28 m V. KOA chondrocytes were in a more positive membrane potential by 25.2% compared to a control group(P<0.01).(2) Calcium channel α1 subunit m RNA expressions: Cav1.2,Cav1.3,Cav1.4,Cav2.1, Cav2.2 and Cav2.3 m RNA expression, but no Cav1.1, Cav3.1 and Cav3.3 m RNA expression could be detected in normal chondrocytes. Compared to normal group, the m RNA expressions of Cav1.3, Cav1.4 and Cav2.1 induced 4.37, 8.96 and 3.63 fold up-regulation in KOA chondrocytes(P<0.05,P<0.01), whereas there are no significantly difference in the m RNA expression of Cav1.2, Cav2.2 and Cav2.3 between normal and KOA group.(3) Anabolic and catabolic marker m RNA expressions: Compared to normal group, the m RNA expressions in ColⅡ, aggrecan-1, SOX-9 and TGF-β were significantly decreased, and the number of m RNA expression of catabolic enzymes, especially MMP-1, ADAMTS-4 and ADAMTS-5 increased significantly in KOA chondrocytes(P<0.05).Conclusions: Ca2+ translocation through calcium channel may be suppress matrix anabolic markers and elevate catabolic markers in KOA chondrocytes.Part 4 The role of the Calcinuerin in L- type voltage-dependent calcium channel modulator of the experimental knee cartilage matrix metabolismObjective: To explore the possible role of Ca N in the regulation of matrix metabolism of chondrocytes via L-type calcium channel α1 subunit in rabbit KOA model.Methods: the changes of m RNA expression including Ca N and five members of NFATs were first observed in KOA chondrocytes measured by RT-q PCR. Next we observed whether L-type calcium channel α1 subunit could be regulated matrix metabolism of KOA chondrocytes via Ca N by adding L-type voltage-gated calcium channel blocker, nifedipine(10 μM) or Ca N inhibitor, CsA(1 μM).Results:(1) the mRNA expressions of Ca N and five members of NFATs: the m RNA expression of Cn Aα,Cn Aβ,Cn B, NFATc1 through NFATc4 and NFAT5 could be detected in normal and KOA chondrocytes. Compared with normal group, the m RNA expressions of Cn Aα and Cn Aβ induced 0.50 and 0.57 fold down-regulation in KOA chondrocytes(P<0.05), whereas there are no significantly difference in the m RNA expression of Cn B between normal and KOA group. Five members of NFATs m RNA expression changes were not synchronized. The expression of NFATc1 reduced most significantly(~0.34 fold, P<0.01), while the expression of NFATc2 obvious increased(~2.42 fold, P<0.01). NFATc3, NFATc4 and NFATc5 m RNA expressions were to reduce the trend, but there were no significance. So we select the most obvious change markers in KOA, Cn Aα, Cn Aβ, NFATc1 and NFATc2 for the following experiments.(2) The MTS method f on cell vitality: Compared with DMSO group of KOA, the vitality of KOA chondrocytes in the presence of nifedipine or Cs A for 72 h had no significantly difference(P>0.05).(3) Drug effects on KOA chondrocyte matrix metabolisms: Compared with DMSO group of KOA, nifedipine completely blocked the m RNA expressions of Cav1.2, Cav1.3 and Cav1.4. Nifedipine increased the chondrocyte matrix components ColⅡ(~7.48 fold, P<0.01), aggrecan-1(~5.16 fold, P<0.05) and TGF-β(~4.81 fold, P<0.01) m RNA expression. MMP-1, ADAMTS-4 and ADAMTS-5 m RNA expression were significantly reduced by nifedipine(~0.17 fold, 0.34 fold and 0.10 fold, P<0.01). Cs A inhibited ColⅡ(~0.25 fold, P<0.01) and increased MMP-1(~3.27 fold, P<0.01) m RNA expression.Compared with DMSO group of KOA, nifedipine significantly increased the Cn Aα m RNA expression(~3.28 fold, P<0.01), but remarkedly reduced the NFATc2 expression(~0.20 fold, P<0.01) m RNA expressions. Cs A inhibited Cn Aα(~0.63 fold, P<0.01) and increased NFATc2(~3.15 fold, P<0.01) m RNA expression. Both of them have no effects on the m RNA expressions of Cn Aβ and NFATc2.Conclusions: L-type calcium channel Cav1.3 and Cav1.4 could regulate matrix metabolism of KOA chondrocytes partly mediated via inhibition Ca N m RNA expression and increase NFATc2 m RNA expression. |
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