氧化钇纳米球通过溶酶体—线粒体通路诱导骨髓间充质干细胞凋亡的机制研究

发布时间：2018-06-05 10:28

本文选题：Y_2O_3NSs + 溶解性　；参考：《河北大学》2017年硕士论文

【摘要】：随着纳米技术的发展,稀土纳米材料由于其独特的光、电、磁的性质已广泛的应用于生物医学领域。氧化钇纳米材料被大量用作荧光探针、药物载体、骨支架材料等,文献报道称稀土纳米材料易于在骨中蓄积且不易排出,然而氧化钇纳米材料(Y_2O_3 NPs)对骨代谢的影响还不是很清楚。本文研究了氧化钇纳米球(Y_2O_3 NSs)与骨髓间充质干细胞(BMSCs)之间的相互作用。水热法制备Y_2O_3 NSs,通过SEM、TEM、DLS等手段进行表征。MTT法等等方法检测Y_2O_3 NSs对细胞活力的影响。在细胞水平上,Annexin V/PI双染法,摄取机制研究,定位,组织蛋白酶的释放,Bid和Bax的表达,细胞色素C(Cyt C)的释放,Caspase 3的激活等方法研究了Y_2O_3 NSs对BMSCs溶酶体-线粒体依赖的凋亡通路的影响。在溶酶体模拟液中合成了磷酸根包覆的氧化钇(Y_2O_3@YPO4),采用TEM、Zeta、FTIR等方法进行表征,Y_2O_3 NSs及Y_2O_3@YPO4对组织蛋白酶的释放及Caspase 3表达的比较,探讨了Y_2O_3 NSs在溶酶体内的物种变化及Y_2O_3@YPO4安全性的设计思路。在动物水平上,通过HE染色,末端标记法(TUNEL)、Caspase 3的表达等手段检测了Y_2O_3 NSs对ICR小鼠的体内骨代谢的影响。结果表明,合成了粒径约为160 nm的Y_2O_3 NSs,MTT法表明Y_2O_3 NSs对BMSCs具有浓度、时间依赖的细胞毒性。Y_2O_3NSs通过大胞饮的形式进入细胞,定位于溶酶体,并且在溶酶体内溶解,释放出的Y3+与溶酶体内的磷酸根络合,形成蜘蛛网状结构,破坏了溶酶体的稳态,最终通过Caspase3依赖的方式诱导了细胞凋亡,而Y_2O_3@YPO4减少了组织蛋白酶的释放,降低了Caspase3的表达以及Cyt C的释放,体内实验结果表明Y_2O_3 NSs诱导部分肝细胞坏死,肺泡的破裂,骨小梁变细,减少,并且激活骨组织中Caspase 3的表达,而Y_2O_3.@YPO4对骨组织没有明显的毒性。因此,通过Y_2O_3 NSs在体内外的毒性评价表明Y_2O_3 NSs在溶酶体内的溶解是引起细胞凋亡的关键,Y_2O_3@YPO4为稀土氧化物对生物体内的安全性设计提供了一种新思路。
[Abstract]:With the development of nanotechnology, rare earth nanomaterials have been widely used in biomedical fields due to their unique optical, electrical and magnetic properties. Yttrium oxide nanomaterials are widely used as fluorescent probes, drug carriers, bone scaffolds and so on. However, the effect of Y _ S _ 2O _ 3 NPs on bone metabolism is unclear. The interaction between yttrium oxide nanospheres (Ys) and bone marrow mesenchymal stem cells (BMSCs) has been studied. Ys _ 2O _ 3 NSs were prepared by hydrothermal method. The effects of Ys _ 2O _ 3 NSs on cell viability were detected by means of SEMT-TEML-DLS and other methods, such as MTT method. At the cellular level, Annexin V / Pi double staining, uptake mechanism studies, localization, cathepsin release and expression of bid and Bax, The effects of Y _ 2O _ 3 NSs on lysosome-mitochondrial dependent apoptosis pathway of BMSCs were studied by the activation of Caspase 3 by the release of cytochrome Cyt C, and the activation of Caspase 3. Yttrium phosphate coated yttrium oxide YPO4 was synthesized in lysosomal mimic solution. The expression of cathepsin and expression of Caspase 3 were characterized by means of TEMN ZetaI FTIR and the comparison of the release of cathepsin and the expression of Caspase 3 in Y2O3 / YPO4 and Y2O3 / YPO4, respectively. The species changes in lysosomes of Ys _ 2O _ 3NSs and the safety of Ys _ 2O _ 3NSs in lysosomes were discussed. At the animal level, the effects of Y _ 2O _ 3 NSs on bone metabolism in ICR mice were detected by HE staining and end labeling method. The results showed that Ys _ 2O _ 3 NSs with a diameter of about 160nm were synthesized. The results showed that Ys _ 2O _ 3NSs had concentration and time-dependent cytotoxicity to BMSCs. Ys _ 2O _ 3NSs entered the cells through the form of large cell drink, located in lysosomes, and dissolved in lysosomes. The released Y3 complex with phosphates in lysosomes to form spider reticular structure, destroy the steady-state of lysosome, induce cell apoptosis by Caspase3-dependent manner, and Y2O3YPO4 reduce the release of cathepsin, and Y2O3-dependent YPO4 reduces the release of cathepsin, and Y2O3YPO4 reduces the release of cathepsin. The expression of Caspase3 and the release of Cyt C were decreased. The results of in vivo experiments showed that Y2O3 NSs induced partial hepatocyte necrosis, alveolar rupture, bone trabecula thinning, and activation of Caspase 3 expression in bone tissue. But Ys _ 2 O _ 3. No obvious toxicity to bone tissue. Therefore, the evaluation of the toxicity of Y2O3NSs in vivo and in vitro shows that the dissolution of Y _ 2O _ 3NSs in the lysosome is the key to the apoptosis of the cells. Y2O3OP-YPO4 provides a new idea for the safety design of rare earth oxides to organisms.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R99;TB383.1

【参考文献】