纳米羟基磷灰石颗粒能够影响大鼠腹腔单核巨噬细胞的凋亡吗？  

Whether nano-hydroxyapatite particles can influence apoptosis of mononuclear macrophage in rat abdominal cavity?

作　　者：丁婷婷[1] 孙皎[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院,上海生物材料研究测试中心,上海市口腔医学重点实验室,上海市200023

出　　处：《中国组织工程研究与临床康复》2009年第51期10093-10096,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金项目(30470479);上海市科委项目(08DZ2291600,0752nm026);上海市重点学科建设项目(S30206)~~

摘　　要：背景:由于尺寸效应,纳米颗粒可以结合细胞内大分子从而引起细胞坏死或凋亡。针对纳米颗粒的生物安全性,目前缺乏对颗粒致细胞凋亡的机制分析。目的:从细胞及分子水平,探讨纳米羟基磷灰石颗粒对大鼠腹腔单核巨噬细胞凋亡的影响。设计、时间及地点:材料-细胞学体外观察,于2007-01/2008-12在上海生物材料研究测试中心完成。材料:2级SD雄性大鼠购自上海西普尔-必凯实验动物有限公司,纳米颗粒由中科院硅酸盐所提供。方法:无菌条件下吸取大鼠腹腔液,贴壁法体外分离培养单核巨噬细胞,调整细胞浓度为2×109L-1。在300W/40kHz超声条件下制备含20,100,200mg/L纳米羟基磷灰石颗粒的悬浮液,分别诱导单核巨噬细胞24h,并设立正常对照组。主要观察指标:应用透射电镜观察细胞超微结构表征;AnnexinV-EGFP/PI双染检测细胞凋亡率;WesternBlot法检测凋亡调控相关基因P53的表达变化。结果:正常对照组单核巨噬细胞伪足完整、核膜正常、核质均匀;20,100,200mg/L纳米羟基磷灰石诱导24h后,单核巨噬细胞具有典型的凋亡形态学特征。与正常对照组比较,20,100mg/L纳米羟基磷灰石诱导后细胞凋亡率均显著升高(P<0.01),且100mg/L诱导组升高幅度明显大于20mg/L组(P<0.05)。正常对照组单核巨噬细胞中几乎不表达P53蛋白;经不同质量浓度的纳米羟基磷灰石诱导后,单核巨噬细胞P53蛋白的表达均明显增高,且蛋白的表达与纳米颗粒的质量浓度呈正相关。结论:纳米羟基磷灰石颗粒能够通过蛋白磷酸化上调P53蛋白的表达,从而启动下游相关基因,最终导致细胞凋亡。BACKGROUND： Because of their size effect, nanometer particles （NPs） can combine molecular within cells, which can result in cell necrosis or apoptosis. But there are no systematic mechanisms of apoptosis induced by NPs about biological safety of NPs. OBJECTIVE： To investigate the effect of nano-hydroxyapatite particles on mononuclear macrophage in rat abdominal cavity at celluar and molecular level. DESIGN, TIME AND SETTING： A materials-cytology observation was performed at Shanghai Biomaterials Research ＆ Testing Center from January 2001 to December 2008. MATERIALS： SD rats of clean grade were provided by SINe-BRITISH SIPPR/BK LAB. ANIMAL Co., Ltd.; NPs were provided by Shanghai Institute of Ceramics. METHODS： Peritoneal fluid was extracted under a sterility environment to in vitro separate and culture mononuclear macrophage using adherence method. The concentration of cell was adjusted at 2×109/L. At 300 W/40 kHz ultrasound, teal suspension containing 20, 100 and 200 mg/L nano-hydroxyapatite particles was prepared to induce mononuclear macrophage for 24 hours, respectively. A normal control group was established. MAIN OUTCOME MEASURES： Ultrastructural phenotype was detected using transmission electron microscope; apoptotic rate was measured using AnnexinV-EGFP/PI staining; variation of apoptosis-related P53 gene expression was detected using Western Blot. RESULTS： Pseudopodia of mononuclear macrophage in the normal control group were intact, nuclear membrane was normal, and nucleoplasm was uniformed. After inducing by 20, 100, 200 mg/L nano-hydroxyapatite particles for 24 hours, apoptotic morphological characteristics were typical in mononuclear macrophage. Compared with normal control group, apoptotic rate was significantly increased following the induction of 20 and 100 mg/L nano-hydroxyapatite （P 〈 0.01）, and the increasing in the 100 mg/L nano-hydroxyapatite particle group was greater than 20 mg/L nano-hydroxyapatite particle group （P 〈 0.05）. P53 protein was not observed in

关 键 词：纳米羟基磷灰石 颗粒 凋亡 P53 

分 类 号：R318[医药卫生—生物医学工程]