摘要
目的 探讨不同形貌羟基磷灰石（HA）微粒对体外RAW264.7细胞凋亡的影响. 方法 采用水热合成及pH调节的方法获取微棒与微球HA微粒.实验分为3组：空白对照组、微棒HA组和微球HA组.将微棒HA与微球HA分别与RAW264.7细胞共培养24 h后,应用细胞乳酸脱氢酶分泌量检测HA微粒对细胞毒性的影响,茜素红染色检测细胞-材料的相互关系,Hoechst凋亡染色及Western blot分析HA微粒对细胞凋亡的影响.结果 共培养24 h,除微棒HA微粒浓度为200 μg/mL时对RAW264.7细胞活性有轻微影响外,在其他浓度下微棒HA与微球HA均具有良好的生物相容性.微棒HA组茜素红染色强度（20.28 ±2.23）显著高于微球HA组（11.72±0.82）,差异有统计学意义（P＜0.05）.微棒HA组与微球HA组的阳性凋亡细胞荧光强度（0.59±0.08、0.55 ±0.03）高于空白对照组（0.46 ±0.07）,微棒HA组的阳性凋亡细胞荧光强度义高于微球HA组,差异均有统计学意义（P＜0.05）.微棒HA组Bcl-2蛋白表达（0.05 ±0.01）显著低于空白对照组（0.25±0.05）与微球HA组（0.21 ±0.04）,差异均有统计学意义（P＜0.05）.微棒HA组与微球HA组Caspase-3蛋白表达（0.32 ±0.04、0.27 ±0.06）较空白对照组（0.13±0.03）上调,差异均有统计学意义（P＜0.05）. 结论 相对于微球HA微粒,微棒HA微粒与RAW264.7细胞发生了更为密切的接触或内吞,从而诱发了更高程度的细胞损伤,表现为较高的细胞凋亡水平.
Objective To investigate the in vitro effect of hydroxyapatite （HA） microparticles of different shapes on apoptosis of RAW264.7 cells.Methods Microrod and microsphere HA particles were synthesized through hydrothermal treatment with further pH adjustments.The experiments were conducted in 3 groups,including a blank control group,microrod HA-treated and microsphere HA-treated groups.After the HA particles were co-cultured with RAW264.7 cells for 24 h,lactate dehydrogenase （LDH） secretion was detected to evaluate the cytotoxicity of HA particles and alizarin red staining （ARS） was used to investigate the cell-material interaction.Hoechst apoptotic staining and western blot skill were applied to analyze the apoptotic effect on RAW264.7 cells of differently shaped HA particles,respectively.Results The LDH assay validated that the microrod and microsphere HA particles were biocompatible after 24 h co-culture except for the microrod HA particles at 200 μg/mL concentration.The ARS detection showed that treatment with microrod HA particles initiated significantly higher ARS intensity （20.28 ± 2.23） than microsphere HA particles （11.72 ± 0.82） （P 〈 0.05）.The fluorescence intensity analysis of apoptotic staining suggested that,compared with control group （0.46±0.07）,microrod HA group （0.59±0.08） and microsphere HA group （0.55 ± 0.03） triggered significantly higher cell apoptotic levels,and microrod HA group did significantly higher than microsphere HA group （P 〈 0.05）.Expression levels of apoptotic relevant proteins indicated microrod HA group inhibited significantly more anti-apoptotic protein Bcl-2 expression （0.05 ± 0.01） than control group （0.25 ± 0.05） and microsphere HA group （0.21 ± 0.04） （P 〈 0.05）.Moreover,microrod HA group and microsphere HA group significantly increased expression of apoptotic pathway related protein Caspase-3 （0.32±0.04 and 0.27±0.06,respectively） relative to the control group （0.13±0.03） （P 〈0.05）.Conclusion Compared with microsphere HA particles,microrod HA particles may initiate more closer interaction with RAW264.7 cells,resulting in a higher severity of lesion to the cells that is indicated by a higher level of cellular apoptosis.
出处
《中华创伤骨科杂志》
CAS
CSCD
北大核心
2015年第5期428-432,共5页
Chinese Journal of Orthopaedic Trauma
基金
国家重点基础研究发展计划（2012CB619100）
国家自然科学基金（81171724,81371924）
国家重大科学研究计划（“863”计划）（2012AA020504）
