小鼠软骨前体细胞ATDC5中沉默骨膜蛋白基因能延缓软骨退变

Attenuation of cartilage degeneration via silencing Postn gene in mouse chondroprogenitor cell line ATDC5 cells

目的观察骨膜蛋白（Postn）在骨关节炎软骨面的表达，探讨其对软骨退变的作用及作用机制。方法把C57/BL6小鼠随机分为实验组和对照组，实验组小鼠右膝关节采用内侧半月板不稳定（DMM）法建立骨关节炎模型，对照组则只切开皮肤和关节囊，采用番红O-固绿及甲苯胺蓝染色观察关节软骨面的变化及用免疫组织化学检测关节软骨面Postn的表达水平。然后通过体外实验用白细胞介素（IL）-1β诱导ATDC5软骨前体细胞株发生骨关节炎，再采取siRNA oligo干扰Postn基因的表达，继而通过实时定量反转录聚合酶链反应（RT-qPCR）检测OA相关因子的表达水平。结果动物实验中，相对于Sham组，DMM组关节软骨厚度明显变薄（DMM 4周比Sham 4周，P＝0.000；DMM 8周比Sham 8周，P＝0.001）；国际骨关节炎研究学会（OARSI）评分明显升高（DMM 4周比Sham 4周，P＝0.005；DMM 8周比Sham 8周，P＝0.002）；Postn的阳性细胞率明显增多（DMM 4周比Sham 4周，P＝0.000；DMM 8周比Sham 8周，P＝0.004）。体外实验中，相对于NC组，IL-1β＋siScramble组中Postn （1.65±0.23，P＝0.008）、基质金属蛋白酶-13（MMP-13），1.75±0.24，P＝0.005）、含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶5（ADAMTS-5，4.63±1.58，P＝0.017）、血管内皮生长因子（VEGF，6.24±0.90，P＝0.001）、Runt相关转录因子2（Runx2，1.54±0.14，P＝0.003）表达升高，但10型胶原A1（COL10a1，1.12±0.12，P＝0.161）差异无统计学意义。而相对于IL-1β＋siScramble组，IL-1β＋siPostn组Postn （0.51±0.17，P＝0.002）、MMP-13 （0.79±0.09，P＝0.003）、ADAMTS-5 （0.90±0.11，P＝0.015）、VEGF （1.04±0.62，P＝0.001）、Runx2（0.82±0.14，P＝0.003）、COL10a1 （0.77±0.11，P＝0.020）的表达均不同程度的下降。结论Postn在DMM骨关节炎软骨面表达升高。ATDC5软骨前体细胞中沉默Postn基因能延缓软骨退变。

ObjectiveTo investigate the expression of Periostin （Postn） in osteoarthritic cartilage and explore its influence and machanism in cartilage degeneration.MethodsC57/BL6 mice were randomly divided into two groups： experimental group and control group, the right knee joints of experimental gruop were underwent destabilization of the medial meniscus （DMM） surgery, while that of control group were only incised skin and joint capsule. Safranin O-fast green and Toluidine Blue staining were used to examinate the change of articular cartilage, and immunohistochemistry staining was used to detect the expression of Postn in articular cartilage. We induced ATDC5 mouse chondrogenic cells to osteoarthritis with interleukin （IL）-1β, and real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR） was used to detect the levels of osteoarthritic related factors after silencing Postn gene via small interfering RNA （siRNA） oligo.ResultsIn animal experiments, the thickness of articular cartilage was obviously decreased （DMM 4 w vs. Sham 4 w, P=0.000; DMM 8 w vs. Sham 8 w, P=0.001）, International Society for the study of osteoarthritis （OARSI） scoring was obviously increased （DMM 4 w vs. Sham 4 w, P=0.005; DMM 8 w vs. Sham 8 w, P=0.002） and the rates of Postn positive cells were obviously increased in DMM groups compared to Sham groups （DMM 4 w vs. Sham 4 w, P=0.000; DMM 8 w vs. Sham 8 w, P=0.004）. In vivo experiment, Postn （1.65±0.23, P=0.008）, matrix metalloproteinase-13 （MMP-13, 1.75±0.24, P=0.005）, a disintegrin and metalloproteinase with thrombospondin motifs 5 protein （ADAMTS-5, 4.63±1.58, P=0.017）, vascular endothelial growth factor （VEGF, 6.24±0.90, P=0.001） and Runt related transcription factor 2 protein （Runx2, 1.54±0.14, P=0.003） were up-regulated in IL-1β＋ siScramble group compared to NC group, while no statistics difference was observed in collagen type X, alpha 1 （COL10a1, 1.12±0.12, P=0.161）. Postn （0.51±0.17, P=0.002）, MMP-13 （0.79±0.09, P=0.003）, ADAMTS-5 （0.90±0.11, P=0.015）, VEGF （1.04±0.62, P=0.001）, Runx2 （0.82±0.14, P=0.003）, COL10a1 （0.77±0.11, P=0.020） were differently down-regulated in IL-1β＋ siPostn group compared to IL-1β＋ siScramble group.ConclusionPostn was elevated in osteoarthritic articular cartilage in DMM model. Silencing Postn gene in ATDC5 chondrogenic cells could attenuate cartilage degeneration.