软骨细胞外泌体在大鼠颞下颌关节骨关节炎软骨异常钙化中的作用研究

Study on the role of chondrocyte-exosomes in abnormal calcification of cartilage in temporomandibular joint osteoarthritis in rats

目的探索软骨细胞外泌体在大鼠颞下颌关节(temporomandibular joint,TMJ)骨关节炎(osteoarthritis,OA)退变软骨异常钙化中的作用。方法选取48只6周龄雌性SD大鼠,根据实验时间点(2、4、8周)随机分为三大组,每大组再随机分为对照组与单侧前牙反(unilateral anterior crossbite,UAC)实验组,每组8只大鼠。对UAC实验组大鼠施加UAC刺激诱导TMJ OA,于2、4、8周后分别处死各组大鼠,对TMJ髁突软骨进行CD63、基质羧基谷氨酸蛋白(matrix gla protein,MGP)的免疫组化染色和mRNA含量检测,同时透射电镜观察软骨中钙化痕迹及外泌体结构。提取大鼠TMJ髁突软骨细胞体外培养并施加流体剪切力刺激,茜素红染色检测钙化结节形成情况,对软骨细胞和外泌体蛋白进行Western Blot检测。结果 UAC实验组大鼠TMJ髁突软骨内CD63分子高表达,同时在钙化痕迹周围存在大量外泌体样结构,而MGP含量则在4周和8周时显著减少。流体剪切力可显著促进TMJ髁突软骨细胞钙化结节形成,同时外泌体数目增多,但外泌体中MGP蛋白含量显著降低。对正常培养的软骨细胞施加外泌体后可显著促进钙化结节的生成。结论大鼠TMJ OA软骨中外泌体数目增加,同时外泌体内MGP含量降低,两方面共同促进软骨异常钙化的发生。

Objective To explore the role of chondrocyte-exosomes in abnormal calcification of degenerative cartilage in temporomandibular joint osteoarthritis(TMJ OA)in rats. Methods Totally forty-eight 6-weeks-old female SD rats were randomly divided into 3 groups according to the experimental time point(2,4 and 8 weeks). Each group was then randomly divided into control subgroup and unilateral anterior crossbite(UAC)subgroup,with 8 rats in each subgroup.TMJ OA in SD rats of UAC subgroup were induced by UAC,and the animals were sacrificed after 2,4 and 8 weeks,respectively. CD63 and matrix gla protein(MGP)expressed in condylar cartilage of TMJ were detected by immunohistochemical staining and mRNA assay. Meanwhile,trace of calcification and exosome structure in the cartilage were observed by transmission electron microscopy. TMJ condylar chondrocytes was extracted from rats for in vitro culture,and was stimulated by fluid flow shear force. Alizarin red staining was used to detect the formation of calcified nodules,and Western blot was used to detect the protein in chondrocytes and exosomes. Results CD63 was highly expressed in the condylar cartilage of the TMJ in the UAC subgroups,and there were a large number of exosomlike structures around the calcification,while MGP expression was significantly reduced at 4 and 8 weeks. Fluid flow shear force could significantly promote the formation of calcified nodules in cultured TMJ condylar chondrocytes and increase the number of exosomes,but the content of MGP protein in exosomes was significantly reduced. The addition of exosomes to normal cultured chondrocytes could significantly promote the formation of calcified nodules. Conclusion The increased number of exosomes in TMJ OA cartilage and the decreased content of MGP in exosomes promote the occurrence of abnormal calcification in cartilage of TMJ OA in rats.