阿仑膦酸钠干预膝关节炎大鼠Wnt通路及Ⅱ型胶原蛋白的实验机制研究(1)
【摘要】 目的：觀察阿仑膦酸钠(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治疗膝关节炎的机制。方法：取4周龄SPF级SD大鼠15只关节软骨细胞原代培养并传至第3代，将第3代软骨细胞平均分为3组：空白对照组(SD CON)软骨细胞用常规DMEM完全培养液培养5 d;IL-1β诱导组(SD IL-1β)软骨细胞用10 ng/mL重组人IL-1β培养2 d后更换为常规DMEM完全培养液培养3 d;
IL-1β+ALN组(SD IL-1β+ALN)软骨细胞用10 ng/mL重组人IL-1β培养2 d后更换为浓度为
1×10-6mol/L的ALN培养3 d。培养后取细胞进行比较观察，检测各组中Collagen Ⅱ、Wnt3a、Wnt5a及Wnt16的变化。结果：Collagen Ⅱ、Wnt3a、Wnt5a和Wnt16在软骨细胞中均有表达，经ALN处理后软骨细胞中Ⅱ型胶原蛋白增加。Wnt3a在空白对照组和IL-1β诱导组表达比较，差异无统计学意义(P>0.05)，在IL-1β+ALN组中表达量明显升高，差异有统计学意义(P<0.05);Wnt5a在IL-1β诱导组中的表达量明显高于空白对照组，差异有统计学意义(P<0.05);ALN处理后，表达量降低，但仍高于空白对照组，差异有统计学意义(P<0.05);Wnt16在IL-1β诱导组中表达量明显低于空白对照组，差异有统计学意义(P<0.05);ALN处理后表达量升高，但仍低于空白对照组，差异有统计学意义(P<0.05)。结论：ALN可能通过Wnt信号通路提高Wnt3a、Wnt16的表达与降低Wnt5a的表达，间接影响RANKL/RANK/OPG信号系统，并影响下游MMP-13、β-catenin及Collagen Ⅱ蛋白的表达来共同调节破骨细胞和成骨细胞的动态平衡，从而保护软骨和软骨下骨。
【关键词】 阿仑膦酸钠; Wnt信号通路; Ⅱ型胶原蛋白; 骨关节炎; 大鼠
Research of Mechanism on Affect of Alendronate Sodium on Wnt Signaling Pathway and Collagen Ⅱ in Knee Osteoarthritis Rats/DENG Bin，WANG Xiyi，XU Dongqing，et al.//Medical Innovation of China，2019，16(21)：0-030
【Abstract】 Objective：To research the effect of Alendronate Sodium(ALN)on chondrocytes induced by IL-1β in vitro in knee osteoarthritis rats and to explore the mechanism of ALN in the treatment of knee osteoarthritis.Method：Fifteen articular chondrocytes of 4-week-old SPF-grade SD rats were cultured and passaged to the third passage.The third generation chondrocytes were divided into three groups：blank control group(SD CON)，chondrocytes were cultured in DMEM complete medium for 5 d.IL-1β induction group(SD IL-1β)，chondrocytes were cultured with 10 ng/mL recombinant human IL-1β for 2 d and replaced with normal DMEM complete culture medium for 3 d.IL-1β+ALN group(SD IL-1β+ALN)，chondrocytes were cultured with 10 ng/mL recombinant human IL-1β for 2 d and replaced with ALN with a concentration of 1×10-6 mol/L for 3 d.The changes of Collagen Ⅱ，Wnt3a，Wnt5a and Wnt16 in each group were detected.Result：The expression of Collagen Ⅱ，Wnt3a，Wnt5a and Wnt16 were found in chondrocytes，the increasing expression of Collagen Ⅱ after ALN treatment.There was no significant difference in the expression of Wnt3a between the blank control group and IL-1β induction group(P>0.05).The expression of Wnt3a in IL-1β+ALN group was significantly higher(P<0.05).The expression of Wnt5a in IL-1βinduction group was significantly higher than that in blank control group(P<0.05)，after ALN treatment it decreased，but it was still higher than that in the blank control group(P<0.05).And the expression level of Wnt16 in IL-1βinduction group was significantly lower than that in the blank control group(P<0.05)，after ALN treatment it increased，but it was still lower than that of the blank control group(P<0.05).Conclusion：By Wnt signaling pathway Alendronate Sodium can increase the expression of Wnt3a，Wnt-16 and decrease that of Wnt5a and indirectly affect the RANKL/RANK/OPG signaling pathway and the downstream factor，for example MMP-13，β-catenin and Collagen Ⅱ proteins，co-regulate the dynamic balance of osteoclasts and osteoblasts，protect cartilage and subchondral bone., http://www.100md.com(邓斌 王锡奕 许冬青 康书鹏 张广波 庄鑫泓 王晓跃 许镇文)
IL-1β+ALN组(SD IL-1β+ALN)软骨细胞用10 ng/mL重组人IL-1β培养2 d后更换为浓度为
1×10-6mol/L的ALN培养3 d。培养后取细胞进行比较观察，检测各组中Collagen Ⅱ、Wnt3a、Wnt5a及Wnt16的变化。结果：Collagen Ⅱ、Wnt3a、Wnt5a和Wnt16在软骨细胞中均有表达，经ALN处理后软骨细胞中Ⅱ型胶原蛋白增加。Wnt3a在空白对照组和IL-1β诱导组表达比较，差异无统计学意义(P>0.05)，在IL-1β+ALN组中表达量明显升高，差异有统计学意义(P<0.05);Wnt5a在IL-1β诱导组中的表达量明显高于空白对照组，差异有统计学意义(P<0.05);ALN处理后，表达量降低，但仍高于空白对照组，差异有统计学意义(P<0.05);Wnt16在IL-1β诱导组中表达量明显低于空白对照组，差异有统计学意义(P<0.05);ALN处理后表达量升高，但仍低于空白对照组，差异有统计学意义(P<0.05)。结论：ALN可能通过Wnt信号通路提高Wnt3a、Wnt16的表达与降低Wnt5a的表达，间接影响RANKL/RANK/OPG信号系统，并影响下游MMP-13、β-catenin及Collagen Ⅱ蛋白的表达来共同调节破骨细胞和成骨细胞的动态平衡，从而保护软骨和软骨下骨。
【关键词】 阿仑膦酸钠; Wnt信号通路; Ⅱ型胶原蛋白; 骨关节炎; 大鼠
Research of Mechanism on Affect of Alendronate Sodium on Wnt Signaling Pathway and Collagen Ⅱ in Knee Osteoarthritis Rats/DENG Bin，WANG Xiyi，XU Dongqing，et al.//Medical Innovation of China，2019，16(21)：0-030
【Abstract】 Objective：To research the effect of Alendronate Sodium(ALN)on chondrocytes induced by IL-1β in vitro in knee osteoarthritis rats and to explore the mechanism of ALN in the treatment of knee osteoarthritis.Method：Fifteen articular chondrocytes of 4-week-old SPF-grade SD rats were cultured and passaged to the third passage.The third generation chondrocytes were divided into three groups：blank control group(SD CON)，chondrocytes were cultured in DMEM complete medium for 5 d.IL-1β induction group(SD IL-1β)，chondrocytes were cultured with 10 ng/mL recombinant human IL-1β for 2 d and replaced with normal DMEM complete culture medium for 3 d.IL-1β+ALN group(SD IL-1β+ALN)，chondrocytes were cultured with 10 ng/mL recombinant human IL-1β for 2 d and replaced with ALN with a concentration of 1×10-6 mol/L for 3 d.The changes of Collagen Ⅱ，Wnt3a，Wnt5a and Wnt16 in each group were detected.Result：The expression of Collagen Ⅱ，Wnt3a，Wnt5a and Wnt16 were found in chondrocytes，the increasing expression of Collagen Ⅱ after ALN treatment.There was no significant difference in the expression of Wnt3a between the blank control group and IL-1β induction group(P>0.05).The expression of Wnt3a in IL-1β+ALN group was significantly higher(P<0.05).The expression of Wnt5a in IL-1βinduction group was significantly higher than that in blank control group(P<0.05)，after ALN treatment it decreased，but it was still higher than that in the blank control group(P<0.05).And the expression level of Wnt16 in IL-1βinduction group was significantly lower than that in the blank control group(P<0.05)，after ALN treatment it increased，but it was still lower than that of the blank control group(P<0.05).Conclusion：By Wnt signaling pathway Alendronate Sodium can increase the expression of Wnt3a，Wnt-16 and decrease that of Wnt5a and indirectly affect the RANKL/RANK/OPG signaling pathway and the downstream factor，for example MMP-13，β-catenin and Collagen Ⅱ proteins，co-regulate the dynamic balance of osteoclasts and osteoblasts，protect cartilage and subchondral bone., http://www.100md.com(邓斌 王锡奕 许冬青 康书鹏 张广波 庄鑫泓 王晓跃 许镇文)