摘要
背景:近年来的研究显示,纳米羟基磷灰石在抗肿瘤领域展现出潜在价值,它能够选择性杀伤肿瘤细胞且对正常细胞没有明显毒性,但纳米羟基磷灰石抗肿瘤的机制尚不完全清楚。目的:探索纳米羟基磷灰石在体外诱导肿瘤细胞发生免疫原性细胞死亡的能力。方法:采用化学沉淀法合成纳米羟基磷灰石,通过X射线衍射、傅里叶变换红外光谱和透射电镜表征纳米羟基磷灰石的物相、官能团及形貌尺寸,利用马尔文粒度仪测量纳米羟基磷灰石在PBS(pH=7.4)中的表面电荷。将不同质量浓度的纳米羟基磷灰石悬液、阿霉素溶液分别与L929小鼠成纤维细胞(或黑色素瘤细胞B16)共培养,CCK-8法检测细胞活力。将B16细胞分4组处理,分别加入PBS、100μg/mL纳米羟基磷灰石悬液、500μg/mL纳米羟基磷灰石悬液、1μg/mL阿霉素溶液,检测钙网蛋白暴露情况以及细胞上清中高迁移率族蛋白B1、三磷酸腺苷水平。结果与结论:①X射线衍射、傅里叶变换红外光谱检测显示纳米羟基磷灰石物相纯度高、结晶度良好,透射电镜下可见纳米羟基磷灰石呈均匀的棒状结构,长度70-90 nm,宽度10-20 nm,形貌规则,纳米羟基磷灰石的电位为(-12.83±2.04)mV。②CCK-8检测结果显示,100,500,2000μg/mL的纳米羟基磷灰石悬液均未显著影响L929成纤维细胞活力,0.1,1μg/mL阿霉素溶液显著降低了L929成纤维细胞活力;0.1,1μg/mL阿霉素溶液与100,500,2000μg/mL的纳米羟基磷灰石悬液均可降低B16细胞活力,并且纳米羟基磷灰石悬液对B16细胞活力的影响弱于阿霉素。③免疫荧光染色结果显示,500μg/mL纳米羟基磷灰石组钙网蛋白表达高于PBS组、阿霉素组(P<0.05);4组间高迁移率族蛋白B1水平比较差异无显著性意义(P>0.05);阿霉素组三磷酸腺苷水平高于PBS组(P<0.05)。④结果表明,纳米羟基磷灰石可能是一种潜在的安全有效的免疫原性细胞死亡诱导剂。
BACKGROUND:Recent studies have shown that nanohydroxyapatite has shown potential value in the field of anti-tumor.It can selectively kill tumor cells and has no obvious toxicity to normal cells,but the anti-tumor mechanism of nanohydroxyapatite is not yet fully understood.OBJECTIVE:To explore the ability of nanohydroxyapatite to induce immunogenic cell death in tumor cells in vitro.METHODS:Nanohydroxyapatite was synthesized by chemical precipitation method.The phase,functional group,and morphology of nanohydroxyapatite were characterized by X-ray diffraction,Fourier transform infrared spectroscopy,and transmission electron microscopy.The surface charge of nanohydroxyapatite in PBS(pH=7.4)was measured by Malvern particle size analyzer.Nanohydroxyapatite suspensions with different mass concentrations and doxorubicin solutions were co-cultured with L929 mouse fibroblasts(or melanoma cells B16).Cell viability was detected by CCK-8 assay.B16 cells were divided into four groups and treated with PBS,100μg/mL nanohydroxyapatite suspension,500μg/mL nanohydroxyapatite suspension,and 1μg/mL doxorubicin solution,respectively.The exposure of calreticulin and the levels of high-mobility group protein B1 and adenosine triphosphate in the cell supernatant were detected.RESULTS AND CONCLUSION:(1)X-ray diffraction and Fourier transform infrared spectroscopy showed that nanohydroxyapatite had high phase purity and good crystallinity.Under transmission electron microscopy,nanohydroxyapatite showed a uniform rod-like structure with a length of 70-90 nm,a width of 10-20 nm,and a regular morphology.The potential of nanohydroxyapatite was(-12.83±2.04)mV.(2)CCK-8 assay results showed that 100,500,and 2000μg/mL nanohydroxyapatite suspensions did not significantly affect the viability of L929 fibroblasts,while 0.1 and 1μg/mL doxorubicin solutions significantly reduced the viability of L929 fibroblasts.0.1 and 1μg/mL doxorubicin solutions and 100,500,and 2000μg/mL nanohydroxyapatite suspensions could reduce the viability of B16 cells.The effect of nanohydroxyapatite suspension on B16 cell viability was weaker than that of doxorubicin.(3)Immunofluorescence staining results showed that the expression of calreticulin in the 500μg/mL nanohydroxyapatite group was higher than that in the PBS group and doxorubicin group(P<0.05).There was no significant difference in the level of high mobility group protein B1 among the four groups(P>0.05).The level of adenosine triphosphate in the doxorubicin group was higher than that in the PBS group(P<0.05).(4)The results show that nanohydroxyapatite may be a potential safe and effective immunogenic cell death inducer.
作者
李恕
赵正宜
曾琴
朱向东
Li Shu;Zhao Zhengyi;Zeng Qin;Zhu Xiangdong(National Engineering Research Center for Biomaterials,Chengdu 610064,Sichuan Province,China;College of Biomedical Engineering,Chengdu 610064,Sichuan Province,China;West China School of Pharmacy,Sichuan University,Chengdu 610064,Sichuan Province,China;NMPA Key Laboratory for Quality Research and Control of Tissue Regenerative Biomaterials&Institute of Regulatory Science for Medical Devices&NMPA Research Base of Regulatory Science for Medical Devices,Sichuan University,Chengdu 610041,Sichuan Province,China)
出处
《中国组织工程研究》
北大核心
2026年第20期5143-5151,共9页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金项目(32171333),项目负责人:曾琴。
