短刺法对兔膝骨关节炎软骨细胞Sox9、VEGF和ColX表达的影响

Effect of eletroacupuncture with close-to-bone needling treatment on expression of Sox9,VEGF and ColX in impaired cartilage of rabbits with knee osteoarthritis

目的观察短刺法对兔膝骨关节炎(KOA)关节软骨细胞Y染色体的性别决定区域(Sox9)、血管内皮细胞生长因子(VEGF)和Ⅹ型胶原(ColⅩ)表达的影响,探讨短刺治疗KOA的作用机制。方法 40只新西兰兔利用随机对照表随机分为正常组(N组)10只、造模组30只,对造模组行Hulth手术法复制膝骨关节炎模型,X线评估造模情况,造模成功后随机分为对照组(M组)、短刺组(CN组)和普通针刺组(NTN组),术后6周开始治疗,M组不予任何处理,CN组和NTN组取左膝关节单侧足三里、内外膝眼、梁丘和阴陵泉穴,留针20 min,1次/d,一疗程5 d,共4个疗程,分别在治疗前和治疗结束后随机选取各组新西兰兔观察其行为学改变;实验结束后观察软骨大体形态及组织学评分;Westem-Blot法检测Sox9和ColⅩ蛋白的表达;免疫组化检测VEGF和ColⅩ蛋白的表达;RT-PCR检测Sox9和VEGFm RNA的表达。结果 M、CN、NTN组在治疗前和治疗结束后兔行为学及软骨大体观察发生明显改变;HE染色显示CN组和NTN组软骨组织损伤减轻,有修复趋势;N组、M组、CN组和NTN组软骨Mankin评分差异均有统计学意义(P<0.01);CN组和NTN组较M组Sox9蛋白和m RNA表达升高、VEFG蛋白和m RNA表达降低、ColⅩ蛋白表达降低(P<0.01);CN组和NTN组差异有统计学意义(P<0.01)。结论短刺法能有效治疗KOA,其作用机制可能是提高软骨细胞Sox9的含量,降低VEGF和ColⅩ的表达,抑制肥大细胞分化,从而维持软骨细胞的正常表型,使软骨得到修复。

Objective To investigate the effect of eletroacupuncture with close-to-bone needling treatment on expression of Sox9,vascular endothelial growth factor（VEGF） and typeX collagen（ColX） in impaired cartilage of rabbits with knee osteoarthritis（KOA） and explore its possible mechanisms.Methods Forty New Zealand rabbits were randomized equally into normal control group,KOA model group,eletroacupuncture with close-to-bone needling group（CN group）,and normal thrust needing group（NTN group）.In the latter 3 groups,KOA was induced by Hulth-Telhag treatment and evaluated with X-ray examination,and 6 weeks after the modeling,eletroacupuncture for 20 min was administered in CN and NTN groups at the acupoints ＂Zusanli＂,＂Waixiyan＂,＂Neixiyan＂,＂Liangqiu＂ and ＂Yinlingquan＂ in the left knee joints once daily for 5 days as a treatment cycle.After 5 treatment cycles,the rabbits were examined for behavioral changes,cartilage morphology,and Mankin scores; The protein and m RNA expressions of S0x9,VEGF,and Col X were examined using Westen blotting,immunohistochemistry,and RT-PCR as appropriate.Results The rabbits in the model,CN and NTN groups showed significant changes in behaviors and cartilage histomorphology after the modeling and after the treatments.HE staining showed that cartilage injury was repaired and tended to recovery in CN and NTN groups.The cartilage pathologies was severer in the model group than in the normal control,CN and NTN groups（P〈0.01）; Sox9 protein increased and VEGF m RNA level decreased in CN and NTN groups after treatment as compared with those in the model group（P〈0.01）.Conclusion Eletroacupuncture with close-to-bone needling can effectively improve KOA in rabbits probably by enhancing Sox9 and reducing VEGF and Col X expressions in the cartilage to inhibit hypertrophic differentiation of the chondrocytes,maintain chondrogenic phenotype and repair cartilage cells.