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目的 探讨骨髓间充质干细胞(BMSC)来源的外泌体(exosome)对钛(Ti)颗粒诱导的破骨细胞形成的影响。方法 全骨髓法分离培养BMSC,流式细胞术检测其表面分子CD105、CD44及CD45,通过成骨和成脂诱导培养并鉴定其分化潜能；收集BMSC的培养上清，提取外泌体并通过电镜观察外泌体的形态，流式细胞术检测外泌体表面分子CD63和CD81;将RAW264.7细胞培养后分为对照组、Ti颗粒(0.1 mg/L)刺激组、Ti颗粒(0.1 mg/L)+exosome(1 mg/L)联合刺激组；同时10周龄的雌性BALB/c小鼠随机分为3组，生理盐水对照组、Ti颗粒(10 mg/L)组、Ti颗粒(10 mg/L)+exosome(20 mg/L)联合刺激组，建立小鼠气囊骨片模型，药物干预两周后取出骨片；对各细胞和小鼠骨片组，用Real-time PCR检测肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6 (IL-6)以及核因子-κB受体活化因子(RANK)、基质金属蛋白酶9(MMP-9)和活化T细胞核因子1 (NFATc1)的mRNA表达水平，抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞，Western blot检测RANK、MMP-9和NFATc1的蛋白表达水平，TRAP染色观察破骨细胞。结果 BMSC表面分子CD44呈阴性，CD45和CD105呈阳性，具有分化为成骨细胞和成脂细胞的能力；外泌体呈囊泡状，大小为175 nm,表面分子CD63和CD81呈阳性；RAW264.7细胞经Ti颗粒(0.1 mg/L)刺激可见多个细胞融合，多核巨噬细胞数量较对照组和Ti颗粒(0.1 mg/L)+exosome(1 mg/L)联合刺激组增多；Ti颗粒(0.1 mg/L)刺激组TNF-α、IL-1β、IL-6以及RANK、MMP-9和NFATc1的表达较对照组和Ti颗粒(0.1 mg/L)+exosome(1 mg/L)联合刺激组增多，RNAK、MMP-9和NFATc1表达升高(P<0.05);Ti颗粒(10 mg/L)诱导的溶骨现象相对于对照组和Ti颗粒(10 mg/L)+exosome(20 mg/L)联合刺激组更加明显，且TNF-α、IL-1β、IL-6以及RANK、MMP-9和NFATc1表达增多，RNAK、MMP-9和NFATc1表达也增强(P<0.05)。结论 BMSC来源的外泌体抑制破骨细胞的形成。
Abstract：Objective To explore the effect of bone marrow mesenchymal stem cells(BMSC)-derived exosome on the development of osteoclasts, bone tissue destruction, and osteolysis induced by Ti particles. Methods BMSC were isolated from bone marrow, and the expressions of surface molecules(CD105, CD44, and CD45) were detected by flow cytometry. Differentiation potential was identified by osteogenic and lipogenic induction culture. The culture supernatant of the BMSC was collected for extracting exosome by ultracentrifugation. The morphology of the exosomes was observed by electron microscope, and the surface molecular CD63 and CD81 of the exosomes was detected by flow cytometry. The RAW264.7 cells were divided into three groups, including the normal group, Ti particle(0.1 mg/L) stimulation group, and Ti particle(0.1 mg/L) combined with exosomes(1 mg/L) stimulation group. Some cells were collected to detect of the expression of tumour necrosis factor-α(TNF-ɑ), interleukin-1β(IL-1β), interleukin-6(IL-6), receptor activator of nuclear factor-κB(RANK), matrix metalloproteinase 9(MMP-9), and nuclear factor of activated T cells(NFATc1) by real-time PCR, and other cells were collected for tartrate resistant acid phosphatase(TRAP) staining. The protein expression levels of RANK, MMP-9, and NFATc1 were detected by Western blot. The 10-week-old female BALB/c mice were randomly divided into three groups, including the normal saline control group, Ti particles(10 mg/L) group, and Ti particles(10 mg/L) combined with exosome(20 mg/L) stimulation group. The mouse model of airbag bone slices was established. Two weeks later, bone tissues were collected for TRAP staining, detecting the expression of by Real time-PCR and western blot detection. Results The surface molecular CD44 was negatively expressed in BMSCs, and the CD45 and CD105 were positive expressed in BMSCs. Alizarin red staining and oil red O staining showed that the cells had the ability to differentiate into osteoblasts and adipocytes. The exosome was vesicular, as observed by electron microscope. Through Nanosight analysis, the size of the exocrine was about 175 nm. The surface molecular CD63 and CD81 of the exosome was positive. There were several multinucleated macrophages in RAW264.7 cells stimulated by Ti particle(0.1 mg/L), while the number of multinucleated macrophages stimulated by Ti particle(0.1 mg/L) combined with exosomes(1 mg/L) decreased, compared with the Ti particle(0.1 mg/L) stimulation group. The cytoplasm of multinucleated macrophages was purplish red and the nucleus was blue after TRAP staining. The results of real-time PCR showed that the expression of TNF-α, IL-1β, IL-6, RANK, MMP-9, and NFATc1 in the combined stimulation group were increased, and the TNF-α, IL-1β, IL-6, RANK, MMP-9, and NFATc1 expression in the combined stimulation group were decreased. Western blot analysis showed that RANK, MMP-9, and NFATc1 were significantly expressed in the Ti particle(0.1 mg/L) stimulated group. The TRAP staining showed that Ti particles(10 mg/L) induced osteolysis was obvious compared with the combined stimulation group. The real-time PCR showed that the TNF-α, IL-1β, IL-6, and RANK expressions in the combined stimulation group were increased, and the expressions of TNF-α, IL-1β, IL-6, and RANK in the combined stimulation group were decreased, and Western blot analysis showed that RNAK was significantly expressed in the Ti particle(10 mg/L) stimulated group(P<0.05). Conclusion BMSC-derived exosomes inhibit osteoclast migration.
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基本信息:
DOI：10.19367/j.cnki.2096-8388.2026.01.003
中图分类号:R329.2
引用信息:
[1]李宏,王淑敏,李玉秀,等.骨髓间充质干细胞来源的外泌体对破骨细胞形成的影响[J].贵州医科大学学报,2026,51(01):27-36.DOI:10.19367/j.cnki.2096-8388.2026.01.003.
基金信息:
国家自然科学基金项目(81860154)
2026-01-20
2026-01-20