1.北京大学第三医院骨科,北京 100191;2.骨与关节精准医学工程研究中心,北京 100191; 3.脊柱疾病研究北京市重点实验室,北京 100191
R-33
1.Peking University Third Hospital, Department of Orthopaedics, Beijing 100191, China. 2. Engineering Research Center of Bone and Joint Precision Medicine, Beijing 100191. 3. Beijing Key Laboratory of Spinal Disease Research, Beijing 100191
目的 探究雌激素缺乏对终板软骨细胞退变的影响。 方法 选取 6 月龄大鼠 40 只,分为两组:双侧卵巢切除实验组(OVX)和假手术对照组(SHAM)。 术后 9 周取材,分别进行软骨终板组织的提取和原代终板软骨细胞的培养。免疫组化实验对比 SHAM 组和 OVX 组的软骨终板组织 II 型胶原(COL-II)的表达情况。 倒置相差显微镜和甲苯胺蓝细胞染色观察细胞形态来鉴定终板软骨细胞。 CCK-8 法对比两组终板软骨细胞的活力情况。罗丹明标记的鬼笔环肽荧光染色观察 OVX 后终板软骨细胞 F-actin 的改变情况。 细胞免疫荧光检测终板软骨细胞在雌激素缺乏后 COL-II 的改变情况。 RT-qPCR 法对比两组的 SOX9、ACAN、ADAMTS-5、MMP13 和 COL-X 的表达有无差异。 结果 与对照组相比较,OVX 组的软骨终板的 COL-II 蛋白表达减低。终板软骨细胞的形态大多呈多角形和梭形,铺路石样排列。 OVX 的终板软骨细胞活力降低,细胞骨架更加紊乱,应力纤维增多,迁移能力下降, COL-II 表达降低。与对照组相比,OVX 组的终板软骨细胞 SOX9 和 ACAN 表达降低,MMP13、ADAMTS-5 和 COL-X 表达升高。 结论 雌激素缺乏会使终板软骨细胞发生退变。 OVX 后的终板软骨细胞活力下降、迁移能力降低、软骨合成减低伴随细胞外基质的降解和细胞肥大分化增加。
Objective To investigate the effect of estrogen deficiency on the degeneration of spinal endplate chondrocytes. Methods Forty 6-month-old rats were divided into ovariectomy (OVX) and sham operation groups. All of the rats were sacrificed at week 9 after operation. The cartilage endplate tissue was extracted, and endplate chondrocytes were cultured. The expression of COL-II in the cartilage endplate tissue was evaluated by immunohistochemistry. The cells were stained by toluidine blue to identify endplate chondrocytes. The viability of endplate chondrocytes was detected by CCK-8. Fluorescence staining of rhodamine-labeled phalloidin was used to observe the changes of F-actin after OVX. Cellular immunofluorescence was performed to detect the changes of COL-II of endplate chondrocytes. The expression levels of SOX9, ACAN, ADAMTS-5, MMP13 and COL-X were compared between the two groups by RT-qPCR. Results After OVX, the expression of COL-II protein in the cartilage endplates was decreased. Most of the chondrocytes in the endplates were polygonal and fusiform, arranged like paving stones. Endplate chondrocytes of OVX demonstrated more disordered cytoskeleton, decreased viability, increased stress fibers, decreased migration ability, and lower COL-II expression. For the OVX group, the expression of SOX9 and ACAN in the endplate chondrocytes was decreased, while that of ADAMTS-5, MMP13 and COL-X was increased. Conclusions Estrogen deficiency can cause degeneration of endplate chondrocytes.
孙 尚,邹 达,赵振达,蒋 嫒,杨子欢,陈龙艇,李危石,宋纯理,冷慧杰.基于大鼠 OVX 模型研究雌激素缺乏对终板软骨细胞退变的影响[J].中国比较医学杂志,2022,32(5):16~22.
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