降钙素在体内体外实验中对兔关节炎关节软骨退变及软骨下骨骨代谢的影响

[目的]研究降钙素(calcitonin, CT)对骨性关节炎关节软骨退变和软骨下骨骨代谢的影响.[方法]30只3个月龄雌性日本大耳白兔随机分为三组,其中两组行右膝关节前交叉韧带切断术(anterior cruciate ligament transaction,ACLT),分为ACLT+CT组和ACLT+NS组,第3组为Sham组.ACLT+CT给予每日1次皮下注射降钙素5 IU/(kg·d),持续8周,ACLT+NS组给予同样剂量生理盐水.术后8周后处死所有动物.取股骨髁制成切片行MMP-13和Ⅱ型胶原免疫组化染色.取胫骨近端制成硬组织切片行骨形态计量学检测.体外实验中,取兔膝关节软骨,经消化、培养,将第3代软骨细胞分三组:向IL-1β组加入人重组IL-1β(10 ng/ml). IL-1β+CT组加入人重组IL-1β (10 ng/ml)2 d后,再向培养液中加入CT(50 ng/ml).正常组不加任何诱导剂和干扰剂培养.然后行MMP-13、Ⅱ型胶原免疫组化检测和Realtime RT-PCR法检测.[结果]Sham组和ACLT+CT组软骨下骨骨小梁相对体积和厚度等均显著高于ACLT+NS组.Sham组和ACLT+CT组的Ⅱ型胶原的光密度值均显著高于ACLT+NS组,而MMP-13的光密度值显著低于ACLT+NS组(P<0.05).正常组和IL-1β+CT组的Ⅱ型胶原光密度值均显著高于IL-1β组而MMP-13的光密度值都显著低于IL-1β组(P<0.05).在正常组和IL-1β+CT组中Ⅱ型胶原的mRNA含量均显著高于IL-1β组而MMP-13的mRNA含量均显著低于IL-1β组(P<0.05).[结论]降钙素5 IU/(kg·d)皮下注射能够增加ACLT兔膝关节软骨Ⅱ型胶原的分泌和抑制MMP-13的表达,并可能通过调节软骨下骨的骨代谢和微结构来保护关节软骨; CT(50 ng/ml)能增加体外培养的含有IL-1β(10 ng/ml)的软骨细胞中Ⅱ型胶原的含量和抑制MMP-13分泌.

Effects of calcitonin on articular cartilage degeneration and subchondral bone metabolism of osteoarthritic rabbit knee in vivo and in vitro

[Objective] To access whether calcitonin (CT) exerts a protective action on the articular cartilage and sub-chondral bone of rabbit knees. [Methods] Thirty 3 - months Japanese white rabbits were divided randomly into three groups. The first two groups received anterior cruciate ligament transaction ( ACLT) on the right knee ( ACLT + CT group and ACLT + NS group) . The third group was sham group. ACLT + CT group received a subcutaneous injection of salmon calcitonin 5IU/ ( kg/d) for 8 weeks. ACLT + NS group received NS at the same dose. All rabbits were sacrificed at 8 weeks postoperativey. The samples were stained immunohistochemically for collagen type II and MMP-13 and stained for the bone histomorphometric measuring. In vitro, the chondrocytes from articular surface were cultured and divided into 3 groups at passage 3;IL-1βgroup, cells were cultured in 10% DMEM containing 10ng/ml IL -1β; IL-1β + CT group, cells were cultured in 10% DMEM containing lOng/ml IL-1β for 2 days, and the cultures were then exposed to medium containing 50ng/ml salmon calcitonin; and the normal group, cells were cultured in 10% DMEM.The cells was stained immunohistochemically for collagen type II and MMP-13, and mRNA expressions of collagen type II and MMP-13 were examined by realtime RT-PCR. [Results] Compared with the ACLT + NS group, the rabbits in sham group and ACLT + CT treated group markedly increased the trabecular bone relative volume (BV/TV) and trabecular bone thickness (Tb. Th) . Immunohistochemistry staining showed the integrated optical density (IOD) of type II collagen in ACLT + NS group was markedly lower than that of ACLT+CT group and sham group (P<0.05) . However the IOD of MMP - 13 in ACLT + NS group was higher (P<0.05) . The IOD of type II collagen in normal group and IL-1β+CT group were higher than that of IL-1β group, but that of MMP -13 in IL-1β group was higher (P< 0.05) . The IL-1β + CT group and the normal group expressed significantly higher type II collagen mRNA than that in the IL - 1β group. The normal group and the IL - 1β + CT group expressed lower MMP - 13 mRNA than that in IL - 1βgroup markedly. [Conclusion] Subcutaneous injection of calcitonin 5 IU/ (kg/d) probably protects cartilage of ACLT rabbit knee by increasing the expression levels of type II collagen, decreasing the expression levels of MMP - 13, modulating subchondral bone metablism and improving microarchitechture of subchondral bone. In vitro CT (50ng/ml) can incease the expression of type II collagen and inhibit the expression of MMP - 13 in chondro-cyte culture containing 10ng/ml IL -1β.