摘要
目的探讨在炎症因子白细胞介素(IL)-1β刺激下,骨形态发生蛋白(BMP)-7对小鼠关节软骨细胞合成表型及分解表型的作用,为BMP-7用于骨关节炎(OA)治疗提供科学证据。方法将原代培养的小鼠关节软骨细胞分为6组,对照组包括未加处理的空白组、IL-1β(5 ng/ml)组,实验组包括3个浓度梯度(10、50、200 ng/ml)BMP-7分别与IL-1β(5 ng/ml)共作用组、单独BMP-7(200 ng/ml)组,作用时间为24 h。用逆转录-定量聚合酶链反应(RT-qPCR)检测Ⅱ型胶原(ColⅡ)、聚集蛋白聚糖(aggrecan)、Y染色体性别决定结构域转录因子(Sox)9等合成基因及基质金属蛋白酶(MMP)-3、MMP-13、含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS)-5等分解基因表达变化。再用酶联免疫吸附试验(ELISA)验证MMP-3、MMP-13蛋白表达水平。结果在IL-1β作用下,软骨细胞特异合成基因显著下调并表达分解表型。添加BMP-7后,受抑制的合成基因得到一定程度恢复,MMP-3、MMP-13、ADAMTS-5等蛋白酶表达量明显减少,其中200 ng/ml BMP-7的促合成、抑分解作用最佳。单独BMP-7作用于软骨细胞会上调aggrecan表达,但不影响ColⅡ和Sox9表达。结论 BMP-7对OA的治疗作用与BMP-7调控炎症环境中软骨细胞合成表型和分解表型的能力相关。
Objective To investigate the effects of bone morphogenetic protein (BMP) 7 on anabolic and catabolic phenotypes of murine chondrocytes treated with interleukin (IL) 1β and supply scientific data for osteoarthritis (OA) treatment using BMP7. Methods Primary cultured murine chondrocytes were divided into six groups: nontreatment group and IL-1β (5 ng/ml) group as control groups, groups of different concentrations of BMP-7 (10, 50, 200 ng/ml) mixed with IL-1β (5 ng/ml) and BMP-7 (200 ng/ml) only group as experiment groups. After 24 h treatment, reverse transcription-quantitative polymerase chain reaction (RT qPCR) were conducted to determine the expression levels of anabolic factors, including type Ⅱ collagen (Col Ⅱ), aggrecan and SRYrelated high mobility group-box (Sox) 9, and catabolic factors, including matrix metalloproteinase (MMP)-3, MMP-13 and a disimegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5. The protein levels of MMP-3 and MMP-13 in cell culture medium were measured with enzymedinked immunosorbent assay (ELISA) kits. Results Under IL-1β stimulation, the phenotype of chondrocytes shifted from anabolic to catabolic. With BMP-7 added, the depressed anabolic factors were recovered and catabolic factors were suppressed in a concentration-dependent manner. BMP-7 at a dose of 200 ng/ml demonstrated the best effects of pro-anabolic and anti-catabolic against IL-li3. The BMP-7 only group showed increased expression of aggrecan, but not Col Ⅱ or Soxg. Conclusion The therapeutic effects of BMP-7 for OA are mediated by its pranabolic and anffcatabolic effects on chondrocytes under proinflammatory environment.
出处
《国际骨科学杂志》
2014年第5期333-336,共4页
International Journal of Orthopaedics
基金
上海市科委自然科学基金(14ZR1431500)
上海市科委科技创新计划重点项目(12401903700)
上海市长宁区卫生系统青年科研项目(20134Q03001)