解剖学报

解剖学报 ›› 2018, Vol. 49 ›› Issue (5): 605-610.doi: 10.16098/j.issn.0529-1356.2018.05.007

• 细胞和分子生物学 • 上一篇    下一篇

 c-Jun氨基末端激酶介导的细胞凋亡和自噬参与调控磷酸三钙磨损颗粒诱导的小鼠颅骨假体周围骨细胞死亡

陈洋 宁乐 杜嘉莉 周春燕 李韩鑫 邹佳军 傅时杰 张云*   

  1. 绍兴文理学院医学院基础医学部生理学教研室，浙江 绍兴 312000
  • 收稿日期:2017-12-08 修回日期:2018-02-22 出版日期:2018-10-06 发布日期:2018-10-06
  • 通讯作者: 张云 E-mail:zhangyunbme@126.com
  • 基金资助:
    浙江省自然科学基金

 c-Jun N-terminal kinase mediated apoptosis and autophagy is involved in osteocyte death induced by tricalciumphosphate wear particles in the mouse calvaria prosthesis

CHEN Yang NING Le DU Jia-li ZHOU Chun-yan LI Han-xin ZOU Jia-jun FU Shi-jie ZHANG Yun*   

  1. Department of Physiology, School of Basic Medicine, College of Medicine, Shaoxing University, Zhejiang Shaoxing 312000, China
  • Received:2017-12-08 Revised:2018-02-22 Online:2018-10-06 Published:2018-10-06
  • Contact: ZHANG Yun E-mail:zhangyunbme@126.com

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摘要：

目的 探讨c-Jun氨基末端激酶（JNK）介导的细胞凋亡和自噬是否参与调控磷酸三钙（TCP）磨损颗粒诱导假体周围骨细胞死亡。 方法 取雄性ICR小鼠36只，随机分为3组：正常对照组（control，n=12）、TCP磨损颗粒组（模型组，n=12）和SP6000125组（n=12）。采用TCP磨损颗粒30 mg置于颅骨顶后缝合皮肤构建小鼠颅骨溶解模型，SP6000125组小鼠于术后第2天颅顶注射JNK通路特异性抑制剂SP600125（1.0 mg/kg），每3日1次。持续干预2周后处死小鼠取颅骨。Calcein-AM探针标记和HE染色观察各组假体周围骨细胞形态和活性变化；通过酶消化法获取假体周围骨细胞，应用流式细胞术检测假体周围骨细胞凋亡情况；Western blotting法检测假体周围骨细胞中Bcl-2、Bax、磷酸化JNK（p-JNK）、Beclin-1和微管相关蛋白1轻链3（LC-3）等蛋白的表达变化。 结果 与control组比较，TCP组假体周围骨细胞活性明显降低，空骨陷窝比例显著增加，骨细胞凋亡明显（P<0.05），JNK通路被活化，p-JNK蛋白表达明显上调（P<0.05）；自噬相关蛋白Beclin-1和LC-3表达显著上调，且LC-3I向LC-3II转换明显增加（P<0.05）；与TCP组比较，SP600125组假体周围骨细胞凋亡显著减少、自噬被明显抑制（P<0.05）。 结论 JNK介导的细胞凋亡和自噬参与调控TCP磨损颗粒诱导的假体周围骨细胞死亡，促进假体周围骨溶解。

关键词: 磷酸三钙磨损颗粒, 骨溶解, 骨细胞, c-Jun氨基末端激酶信号通路, 假体, 免疫印迹法, 小鼠

Abstract:

Objective To investigate whether c-Jun N-terminal kinase(JNK)-mediated apoptosis and autophagy is involved in osteocytes death induced by tricalciumphosphate(TCP) wear particles in the mouse calvaria. Methods Thirty-six 6-week ICR male mice were randomly divided into three groups: normal control groups (n=12), TCP wear particles groups (n=12) and SP600125-treated groups (n=12). A murine calvarial model of osteolysis was established by 30 mg of TCP wear particles implantation onto the surface of bilateral parietal bones following removal of the periosteum. On the second postoperative day, the SP600125-treated mice were locally injected with the specific inhibitor of JNK pathway (SP600125, 1.0 mg/kg) on the the calvaria under the periosteum each time every three days. After 2 weeks of continuous treatment, the animals were sacrificed and the calvaria were obtained. Calcein-AM labelling and HE staining were used to observe the morphology and viability of calvaria osteocytes; flow cytometry was performed to detected apoptosis of calvaria osteocyte obtained by enzyme digestion; Western blotting was used to examine protein expression of Bcl-2, Bax, p-JNK, Beclin-1 and LC-3 in calvaria osteocytes, respectively. Results Compared with the control group, the viability of calvaria osteocytes was significantly reduced, leading to the obvious increases in the percentage of empty lacunae and osteocyte apoptosis in the TCP group (P<0.05); protein expressions of p-JNK, Beclin-1 and LC-3, and the conversion of LC3-Ⅱ from LC3-Ⅰ were both increased remarkably (P<0.05). Compared with the TCP group, the percentage of apoptosis in calvaria osteocytes decreased, and expression of autophagyrelated protein was obviously inhibited in the SP600125 group (P<0.05). Conclusion JNK-mediated apoptosis and autophagy is involved in osteocytes death induced by TCP wear particle in the calvaria.

Key words: Tricalciumphosphate wear particles, Osteolysis, Osteocyte, c-Jun N-terminal kinase pathway, Prosthesis, Werstern blotting, Mouse