磷酸三钙磨损颗粒诱导小鼠假体周围骨细胞损伤的作用

doi:10.12047/j.cjap.5608.2018.021

中国应用生理学杂志 ›› 2018, Vol. 34 ›› Issue (1): 83-87.doi: 10.12047/j.cjap.5608.2018.021

磷酸三钙磨损颗粒诱导小鼠假体周围骨细胞损伤的作用

严嘉琦^1,2, 张云¹, 刘方舒¹, 蔡听听¹, 童康强¹, 朱灿¹, 胡露琦¹, 吕淑敏¹

1. 绍兴文理学院医学院基础医学部, 浙江绍兴 312000;
2. 宁波市杭州湾医院儿科, 浙江宁波 315336

收稿日期:2017-05-31 修回日期:2017-10-27 出版日期:2018-01-28 发布日期:2018-06-19
通讯作者: 张云,Tel:0575-88345638;E-mail:zhangyunbme@126.com E-mail:zhangyunbme@126.com
基金资助:
浙江省自然科学基金资助项目（LY13H060003，LY17H060007）；浙江省重大科技专项重点社会发展项目（2014C03017）

TCP wear particles causes injury of periprosthetic osteocytes in the mouse calvaria

YAN Jia-qi^1,2, ZHANG Yun¹, LIU Fang-shu¹, CAI Ting-ting¹, TONG Kang-qiang¹, ZHU Can¹, HU Lu-qi¹, LV Shu-min¹

1. Department of Basic Medicine, College of Medicine, Shaoxing University, Shaoxing 312000;
2. Department of Pediatrics, Hangzhou Bay Hospital of Ningbo, Ningbo 315336, China

Received:2017-05-31 Revised:2017-10-27 Online:2018-01-28 Published:2018-06-19
Supported by:
浙江省自然科学基金资助项目（LY13H060003，LY17H060007）；浙江省重大科技专项重点社会发展项目（2014C03017）

摘要/Abstract

摘要： 目的：研究磷酸三钙（TCP）磨损颗粒是否能诱导小鼠颅骨假体周围骨细胞损伤，并探讨其作用机制。方法：36只雄性ICR小鼠随机分为3组（n=12）：假手术组（Sham）、模型（TCP）组和3-甲基腺嘌呤（3-MA）组，采用TCP磨损颗粒30 mg置于小鼠颅骨中缝骨膜外缝合皮肤构建小鼠颅骨溶解模型。3-MA处理组小鼠于术后第2天颅顶皮下注射自噬的特异性抑制剂3-MA （1.0 mg/kg），2 d 1次，2周后处死动物取血清和颅骨。Micro-CT分析各组小鼠颅骨骨密度（BMD）、骨体积分数（BVF）和骨吸收孔（porosity）数；HE染色和流式细胞术检测各组假体周围骨细胞活性及凋亡情况；ELISA法检测各组血清中骨细胞特征蛋白牙本质基质蛋白（DMP-1）和骨硬化蛋白（SOST）水平；Western blot法检测各组假体周围骨细胞中DMP-1、SOST和自噬关键分子Beclin-1及微管相关蛋白1轻链3（LC-3）等蛋白的表达。结果：与Sham组比较，TCP组假体周围骨细胞活性明显降低，骨细胞死亡及凋亡显著增加（P<0.05），血清SOST水平及其蛋白表达明显增加，而血清DMP-1水平及其蛋白表达显著减少（P<0.05），Beclin-1表达增加，LC-3I向LC-3Ⅱ转换明显增加；与TCP组比较，3-MA组假体周围骨细胞损伤明显加重，骨细胞凋亡显著增加（P<0.05）。结论：TCP磨损颗粒可通过激活凋亡和自噬而诱导假体周围骨细胞损伤，促进假体周围骨溶解和关节无菌性松动。

关键词: TCP磨损颗粒, 小鼠, 骨细胞, 凋亡, 自噬

Abstract: Objective: To study whether tricalcium phosphate(TCP) wear particles cause injuries of periprosthetic osteocytes in the mouse calvaria, and to explain its molecular mechanism.Methods: Thirty six-week(ICR)male mice were randomly divided into sham group, model (TCP) group and 3-methyladenine (3-MA) group. A murine calvarial model of osteolysis was established by 30 mg of TCP wear particles implantation over the periosteum around the middle suture of calvaria in mice. On the second postoperative day, the autophagy specific inhibitor 3-MA (1.0 mg/kg) was subcutaneously injected to the calvaria in the 3-MA-treated mice every other day. After 2 weeks, blood and the calvaria were obtained. Micro-CT was used to detect bone mineral density(BMD), bone volume fraction (BVF) and porosity number. HE staining and flow cytometry were performed to analyze the viability and apoptosis of periprosthetic osteocytes. The serum levels of dentin matrix protein 1(DMP-1) and sclerostin (SOST) were determined by ELISA. The proteins expressions of DMP-1, SOST, Beclin-1 and microtuble-associated protein 1 light chain 3 (LC-3) were detected by Western blot in the calvaria osteocytes.Results: Compared with the sham group, the mice in the TCP group showed that a significant decrease in the viability of periprosthetic osteocytes, but obvious increases in number of osteocytes death and osteocytes apoptosis (P<0.05), and in serum level and protein expression of SOST; significant decreases in serum level and protein expression of DMP-1 (P<0.05), and remarkable up-regulation of autophagy-related factors beclin-1 and the conversion of LC3-Ⅱ from LC3-I in the calvaria osteocytes. Compared with TCP group, the mice in the 3-MA group showed that injuries of calvaria osteocytes were obviously aggravated, and osteocytes apoptosis was significantly increased (P<0.05).Conclusion: TCP wear particles can cause injuries of periprosthetic osteocytes via activation of apoptosis and autophagy, which promotes osteolysis around the prosthesis osteolysis and joint aseptic loosening.

Key words: TCP wear particles, mouse, osteocytes, apoptosis, autophagy

严嘉琦, 张云, 刘方舒, 蔡听听, 童康强, 朱灿, 胡露琦, 吕淑敏. 磷酸三钙磨损颗粒诱导小鼠假体周围骨细胞损伤的作用[J]. 中国应用生理学杂志, 2018, 34(1): 83-87.

YAN Jia-qi, ZHANG Yun, LIU Fang-shu, CAI Ting-ting, TONG Kang-qiang, ZHU Can, HU Lu-qi, LV Shu-min. TCP wear particles causes injury of periprosthetic osteocytes in the mouse calvaria[J]. CJAP, 2018, 34(1): 83-87.

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磷酸三钙磨损颗粒诱导小鼠假体周围骨细胞损伤的作用

TCP wear particles causes injury of periprosthetic osteocytes in the mouse calvaria

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