软骨终板干细胞源性外泌体对椎间盘退变的影响△

Effect of exosomes derived from cartilage endplate stem cells on intervertebral disc degeneration

摘要:

［目的］ 探讨大鼠软骨终板干细胞 （cartilage endplate stem cells, CESC） 来源的外泌体对髓核细胞 （nucleus pulposus cells, NPC） 以及椎间盘退变 （intervertebral disc degeneration, IVDD） 的影响。［方法］ 从大鼠椎间盘中提取并培养 CESC 和 NPC。利用超速离心方法提取并纯化正常 CESC 分泌的外泌体 （Nor-Exos） 和高糖诱导衰老 CESC 分泌的外泌体 （Eld-Exos）， 分别与 NPC 共培养。Tunel 染色和流式细胞检测三组的细胞凋亡数，Western blot 检测 NPC 中 Bax，Bcl-2，Cyclin D1 和 Cyclin D3 蛋白表达水平。9 只 SD 大鼠随机分三组，分别为空白对照组、Nor-Exos 组和 Eld-Exos 组，通过针刺法建立 IVDD 模型并给予相应处理，术后 5 周行 MRI 检查，按 Pfirrmann 评级标准评分。［结果］体外实验，细胞凋亡由低至高依次为空白对照组 < Nor-Exos 组 < Eld-Exos 组，总体组间差异和两两比较差异均有统计学意义（P<0.05）。Bax 的蛋白表达由低至高依次为空白对照组 < Nor-Exos < Eld-Exos 组，而 Bcl-2、Cyclin D1 和 Cyclin D3 蛋白相表达低至高依次为 Eld-Exos 组 < Nor-Exos 组 < 空白对照组，总体组间差异和两两比较差异均有统计学意义 （P<0.05）。体内实验，Pfirrmann 分级评分由低至高依次为，空白对照组 < Nor-Exos 组 < Eld-Exos 组，总体组间差异和两两比较差异均有统计学意义（P<0.05）。［结论］ Eld-Exos 显著促进 NPC 的凋亡， 增加椎间盘组织中凋亡蛋白水平，加快 IVDD 的进展。

Abstract:

[Objective] To explore the effect of exosomes derived from rat cartilage endplate stem cells (CESC) on nucleus pulposus cells (NPC) and intervertebral disc degeneration (IVDD) . [Methods] The CESC and NPC were isolated and cultured from rat intervertebral disc. The Nor-Exos from normal CESC and Eld-Exos from high glucose induced aging CESC were extracted and purified by super centrifu- gation, and co-cultured with NPC, respectively. The Tunel staining and flow cytometry were used to detect apoptotic cells, while western blot was used to detect the protein expression levels of Bax, Bcl-2, Cyclin D1 and Cyclin D3 in NPC. In addition, 9 SD rats were randomly divided into three groups, namely blank control group, Nor-Exos group and Eld-Exos group. IVDD model was established by acupuncture method and corresponding treatment was given. MRI scan was performed 5 weeks after operation, and scores were marked according to Pfir- rmann criteria. [Results] In vitro, the cell apoptosis was ranked down-up in order of: blank control group ＜ Nor-Exos group ＜ Eld-Exos group, with statistically significant differences in term of total groups and pairwise comparison (P＜0.05) . The protein expression of Bax was down-up of blank control group ＜ Nor-Exos group ＜ Eld-Exos group, whereas the expression levels of Bcl-2, Cyclin D1 and Cyclin D3 in ascending order were of Eld- Exos group ＜ Nor- Exos group ＜ blank control group, with statistically significant differences among total groups and pairwise comparison (P＜0.05) . In vivo, Pfirrmann score was marked from low to high as blank control group ＜ Nor- Exos group ＜ Eld-Exos group, with statistically significant differences (P＜0.05) . [Conclusion] The Eld-Exos does considerably promote apopto- sis of NPC, increase the presentation of apoptotic protein in intervertebral disc tissue, and accelerate the progression of IVDD.