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目的探讨体外培养情况下脂多糖(lipopolysaccharides,LPS)刺激对小鼠骨骼发育和矿化的影响及其机制。方法分离E18.5 d小鼠跖骨和胫骨,分为空白对照组(Blank组)及LPS组(10μg/m L LPS)。分别于培养后第1、7天照相,计算跖骨和胫骨全长增长率(TL-rate)、矿化带增长率(CZ-rate)、第7天矿化带占骨全长的比例(CZ%)。培养7 d后固定、石蜡包埋切片,臧红固绿和甲苯胺蓝染色观察肥大带宽度、Ⅱ型胶原(collagenⅡ,ColⅡ)、Ⅹ型胶原(collagenⅩ,ColⅩ)免疫组化染色观察软骨细胞分化及TUNEL染色观察软骨细胞凋亡变化。结果与Blank组比较,LPS处理后跖骨和胫骨TL-rate及CZ-rate明显降低(跖骨TL-rate P=0.025;跖骨CZ-rate P=0.019;胫骨TL-rate P=0.010;胫骨CZ-rate P=0.024)。臧红固绿、甲苯胺蓝染色及形态计量显示LPS组胫骨肥大带较Blank组明显变短(P<0.001)。与Blank组比较,LPS组胫骨的软骨分化相关蛋白ColⅡ、ColⅩ表达降低,软骨细胞凋亡明显增加。结论 LPS通过抑制软骨增生、分化及促进肥大带软骨细胞凋亡来抑制骨骼发育及矿化。
Objective To investigate the effects of lipopolysaccharides (LPS) stimulation on bone development and mineralization in vitro and its mechanism. Methods The metatarsal and tibial bone of E18.5 d mice were isolated and divided into control group (Blank group) and LPS group (10 μg / mL LPS). The images of TL-rate, CZ-rate and the proportion of the total length of the mineralized zone on the 7th day (CZ %). Cultured for 7 days, fixed, paraffin-embedded sections, Zang Hong solid green and toluidine blue staining observed hypertrophy zone width, collagen Ⅱ (ColⅡ), collagen Ⅹ, Col Ⅹ immunohistochemical staining for chondrocyte differentiation TUNEL staining was used to observe chondrocyte apoptosis. Results Compared with Blank group, the TL-rate and CZ-rate of metatarsal and tibia decreased significantly after LPS treatment (metatarsal TL-rate P = 0.025; metatarsal CZ-rate P = 0.019; tibia TL-rate P = P = 0.024). Zanghong solid green, toluidine blue staining and morphometry showed that LPS group tibial hypertrophy compared with the blank group was significantly shorter (P <0.001). Compared with Blank group, the expressions of ColⅡand ColⅩ in cartilage differentiation of LPS group decreased and chondrocyte apoptosis increased obviously. Conclusion LPS suppresses bone development and mineralization by inhibiting the proliferation and differentiation of cartilage and promoting the apoptosis of hypertrophic chondrocytes.