Objective To investigate the feasibil ity of alendronate (ALN) in treating osteoarthritis (OA) by observing the effects of ALN on interleukin 1β (IL-1β) induced chondrocytes of rat in vitro. Methods The chondrocytes of knee articular surface from 15 SD rats (1-month-old, female or male, weighing 100-150 g) were cultured. The chondrocytes were observed by inverted phase contrast microscope and identified by toluidine blue staining and HE staining. The third passage chondrocytes were divided into 3 groups: the chondrocytes were cultured with DMEM for 5 days in group A, with 10 ng/mL IL-1β for 2 days and with DMEM for 3 days in group B, and with 10 ng/mL IL-1β for 2 days and with 1 × 10-6 mol/L ALN for 3 days in group C. Immunocytochemistry and real-time PCR were performed to determine the expression levels of collagen type II (Col II), matrix metalloproteinase 13 (MMP-13), and β-catenin. Results Toluidine blue staining proved that the cultured cells were chondrocytes. The integrated absorbency (IA) value of Col II in group C (10.290 7 ± 0.499 2) was lower than that in group A (15.377 0 ± 0.571 8) and higher than that in group B (5.463 2 ± 0.450 4), showing significant differences (P lt; 0.05). The IA value of MMP-13 in group C (3.068 6 ± 0.205 6) was significantly lower than that in group B (6.998 1 ± 0.329 7, P lt; 0.05), but there was no significant differenc when compared with group A (2.777 5 ± 0.199 6, P gt; 0.05). The IA value of β-catenin in group C (6.611 7 ± 0.381 8) was lower than that in group B (11.799 9 ± 0.348 7) and higher than that in group A (4.390 3 ± 0.551 9), showing significant differences (P lt; 0.05). The mRNA expression of Col II in group C was significantly higher than those in groups A and B (P lt; 0.05), the mRNA expression of MMP-13 in group C was significantly lower than that in group B (P lt; 0.05) but there was no significant difference when compared with group A (P gt; 0.05). The mRNA expression of β-catenin in group C was significantly lower than that in group B (P lt; 0.05) and higher than that in group A (P lt; 0.05). Conclusion ALN can protect rat chondrocyte from OA induced by IL-1β in vitro possibly by upregulating Col II and inhibiting the expression of MMP-13 and β-catenin in the chondrocytes.
Objective To investigate the role of transforming growth factor β(TGF-β)in the regulation of the gene expression of matrix metalloproteinase 13(MMP-13)in the human hyaline chondrocytes. Methods The human hyaline chondrocytes harvested enzymatically and cultured in DMEM supplemented with 20% fetus calf serum were divided into 7 groups. Group 1 was used as a contol, and 1 ng/ml TGF-β(group 2), 10 ng/ml TGF-β(group 3), 100 ng/ml TGF-β(group 4), 1 ng/ml TGF-β＋10 ng/ml IL-1β(group 5), 10 ng/ml TGF-β＋10 ng/ml IL-1β(group 6),and 100 ng/ml TGF-β＋10 ng/ml IL1β(group 7) were given for 12-hour coculture. The MMP-13 mRNA levels of passaged human hyaline chondrocytes were assessed by reverse transcriptionpolymerase chain reaction(RT-PCR) and real-time fluorescent quantitative PCR. Results TGF-β can increase the MMP-13 mRNA level respectively in the passagedhyaline chondrocytes. In the multifactor treated groups, TGF-β can decrease the MMP-13 mRNA level respectively and there was significant difference between groups (Plt;0.05).The level of MMP-13 mRNA expression had significant coherence withthe dosage of TGF-β. Conclusion The above results show that human chondrocytes express MMP-13 mRNA. TGF-β could cause a dosedependent stimulation on MMP-13 gene expression in human chondrocytes and have a potent effect of antagonizing IL-1β in osteoarthritis. TGF-β may play a crucial role in the occurrence anddevelopment of osteoarthritis through regulating MMP-13.
目的 检测基质金属蛋白酶13（MMP-13）和组织金属蛋白酶抑制因子1（TIMP-1）的血清含量，分析其在妇女绝经后骨质疏松发病中的作用。 方法 2009年3月－2012年9月选取武汉附近地区129例49～63岁绝经后妇女，根据双能X线吸收法检测的骨密度数值，分为正常组、低骨量组和骨质疏松组。采取酶联免疫吸附试验检测MMP-13、TIMP-1以及雌二醇（E2）、Ⅰ型原胶原N端前肽（PINP）和Ⅰ型胶原交联C末端肽（CTX）、骨保护蛋白（OPG）及其配体（OPGL）的含量，统计MMP-13/TIMP-1比值。 结果 ① 骨质疏松组中血清MMP-13水平[（44.25 ± 1.21） μg/L]高于正常组[（27.08 ± 1.41）μg/L]（P＜0.05）；② 骨质疏松组中血清MMP-13与骨密度、血清E2、OPGL水平存在明显负相关性 （P＜0.05），和OPG、PINP和CTX存在明显正相关性（P＜0.05）；③ 低骨量组中MMP-13略高于骨质疏松组，且两者差异无统计学意义（P＞0.05），但是明显高于正常组（P＜0.05），同时与骨密度和血清E2、OPG、OPGL、PINP和CTX存在明显相关性（P＜0.05）。 结论 血清MMP-13和MMP-13/TIMP-1比值与绝经后骨质疏松症妇女和绝经后低骨量组妇女骨代谢指标具有关联性。两者升高可能为绝经后妇女早期骨代谢尤其是胶原代谢过程增快的表现。
ObjectiveTo investigate the role of osteopontin (OPN) on the expressions of matrix metalloproteinase 13 (MMP-13) mRNA and protein in human knee osteoarthritic chondrocytes and to find the optimal time and concentration for OPN treatment. MethodsChondrocytes were isolated from articular cartilage tissues of patients with primary osteoarthritis (OA) and cultured using one step digestive treatment with collagenase typeⅡ. The chondrocytes were then identified using immunohistochemistry of collagen typeⅡ. The first generation of chondrocytes were stimulated with OPN at a concentration of 1μg/mL for 0, 24, 48, and 72 hours, and with OPN at the concentrations of 0, 0.5, 1, 2, and 4μg/mL for 48 hours. The levels of MMP-13 mRNA and protein expressions were measured with real-time fluorescent quantitative PCR and Western blot. ResultsThe immunohistochemical staining showed that first generation of chondrocytes expressed collagen typeⅡ. Both MMP-13 mRNA and protein expression levels in OA chondrocytes increased significantly in the presence of OPN (1μg/ mL) and peaked at 48 hours after incubation, showing significant difference between different time points (P < 0.05). The MMP-13 mRNA expression level in OA chondrocytes at the OPN concentration of 1μg/mL was significantly higher than those at the other concentrations (P < 0.05), and the MMP-13 protein expression level at the OPN concentration of 1μg/mL was significantly higher than that at 0μg/mL (P < 0.05). MMP-13 protein expression level at the OPN concentrations of 0.5, 2, and 4μg/mL were significantly higher than that at 0μg/mL (P < 0.05). ConclusionOPN induces up-regulation of MMP-13 mRNA and protein expressions in human knee osteoarthritic chondrocytes in time-and dose-dependent manners. The optimal time and concentration for OPN treatment are 48 hours and 1μg/mL, respectively.