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目的二膦酸盐类药物可抑制骨重塑,通过观察阿仑膦酸钠(alendronate,ALN)对IL-1β体外诱导培养的大鼠膝关节软骨细胞的影响,探讨ALN治疗骨性关节炎(osteoarthritis,OA)的可行性。方法取15只1月龄SPF级SD大鼠(雌雄不限,体重100～150g)膝关节软骨,体外培养软骨细胞并传至第3代。倒置相差显微镜下观察细胞形态并进行鉴定。将第3代软骨细胞分为3组:空白对照组(A组)软骨细胞用常规DMEM完全培养液培养5d;IL-1β诱导组(B组)软骨细胞以10ng/mL重组人IL-1β培养2d后更换为常规DMEM完全培养液培养3d;IL-1β诱导后加ALN培养组(C组)软骨细胞以10ng/mL重组人IL-1β培养2d后更换为浓度为1×10-6mol/L的ALN培养3d。培养后取各组细胞进行免疫细胞化学染色及实时PCR检测,观察细胞中Ⅱ型胶原(collagen typeⅡ,ColⅡ)、基质金属蛋白酶13(matrix metalloproteinase13,MMP-13)和β-连环蛋白(β-catenin)的表达水平。结果甲苯胺蓝染色示培养的细胞呈异染性,证实为软骨细胞。免疫细胞化学染色显示:A、B、C组ColⅡ表达积分吸光度(IA)值分别为15.3770±0.5718、5.4632±0.4504、10.2907±0.4992,C组高于B组,但低于A组,差异有统计学意义(P<0.05);MMP-13表达IA值分别为2.7775±0.1996、6.9981±0.3297、3.0686±0.2056,C组明显低于B组(P<0.05),但与A组比较差异无统计学意义(P>0.05);β-catenin表达IA值分别为4.3903±0.5519、11.7999±0.3487、6.6117±0.3818,C组低于B组,但高于A组,差异均有统计学意义(P<0.05)。实时PCR检测示,C组ColⅡmRNA表达高于B组,MMP-13及β-catenin的mRNA表达低于B组,比较差异均有统计学意义(P<0.05);ColⅡ和β-catenin mRNA表达高于A组,MMP-13mRNA表达低于A组,比较差异均有统计学意义(P<0.05)。结论 ALN可能通过抑制IL-1β诱导的大鼠膝关节软骨细胞中ColⅡ的降解并下调MMP-13及β-catenin因子的表达,对软骨细胞具有一定的保护作用。
Objective Bisphosphonates can inhibit bone remodeling by observing the effect of alendronate (ALN) on cultured rat knee joint chondrocytes induced by IL-1β and to investigate the effect of ALN on osteoarthritis osteoarthritis, OA) feasibility. Methods Fifteen 1-month-old SPF SD rats (both male and female, weighing 100-150 g) were used to culture chondrocytes and passaged into passage 3. Inverted phase contrast microscope observation of cell morphology and identification. The third generation of chondrocytes were divided into 3 groups: the blank control group (group A) chondrocytes were cultured with normal DMEM complete medium for 5 days; IL-1βinduced group (group B) chondrocytes were cultured with 10ng / mL recombinant human IL-1β After 2 days, the culture medium was changed to normal DMEM culture medium for 3 days. After the induction of IL-1β, the cultured chondrocytes of ALN culture group (C group) were cultured with 10ng / mL recombinant human IL-1β for 2 days and then changed to the concentration of 1 × 10-6mol / L ALN cultured for 3d. After culture, the cells in each group were harvested for immunocytochemical staining and real-time PCR. The expression of collagen typeⅡ (ColⅡ), matrix metalloproteinase13 (MMP-13) and β-catenin ) Expression level. Results Toluidine blue staining showed the cells were heterochromatic, confirmed as chondrocytes. The immunocytochemical staining showed that the integral absorbance (IA) values of Col Ⅱ in group A, B and C were 15.3770 ± 0.5718,5.4632 ± 0.4504,10.2907 ± 0.4992, respectively, which were higher in group C than in group B but lower than those in group A (P <0.05). The IA value of MMP-13 expression was 2.7775 ± 0.1996, 6.9981 ± 0.3297 and 3.0686 ± 0.2056, respectively, which was significantly lower in group C than that in group B (P <0.05), but there was no significant difference with group A (P <0.05). The IA value of β-catenin expression was 4.3903 ± 0.5519,11.7999 ± 0.3487,6.6117 ± 0.3818, respectively, which was lower in group C than in group B, but higher than that in group A (P <0.05) ). Real-time PCR results showed that the expression of ColⅡmRNA in group C was higher than that in group B, while the mRNA expression of MMP-13 and β-catenin was lower than that in group B (P <0.05). In group A, the expression of MMP-13 mRNA was lower than that in group A, and the difference was statistically significant (P <0.05). Conclusions ALN may have a protective effect on chondrocytes by inhibiting the degradation of ColⅡ in rat knee joint chondrocytes induced by IL-1β and down-regulating the expression of MMP-13 and β-catenin.