掺锂羟基磷灰石纳米线促进骨质疏松斑马鱼骨形成的研究

Study on promoting bone formation in osteoporotic zebrafish by lithium-doped hydroxyapatite nanowires

目的:观察掺锂羟基磷灰石纳米线对地塞米松诱导的斑马鱼骨质疏松模型的骨代谢调控作用。方法:采用水热法合成纯羟基磷灰石纳米线(nHA)和掺杂10%锂离子的羟基磷灰石纳米线(Li-nHA),对其进行表征。选取正常条件下培养3天(3dpf)的斑马鱼幼鱼,加入nHA和Li-nHA浸提液共同培养至7dpf,设阴性对照组(含0.1%DMSO),利用转基因品系Tg(ola.sp7:nlsGFP)斑马鱼筛选出促成骨效果明显的最佳浓度。通过地塞米松诱导,构建斑马鱼骨质疏松模型,加入nHA和Li-nHA浸提液培养,对野生型斑马鱼进行茜素红染色,通过显微荧光分析转基因斑马鱼的成骨分化。采用实时定量PCR检测成骨标志基因,如锌指转录因子(SP7)、碱性磷酸酶(ALP)、骨保护蛋白(OPG)、Runt相关转录因子2(Runx2)和骨钙素(OCN)等的表达。采用GraphPad Prism 9.3软件对数据进行统计学分析。结果:nHA、Li-nHA均能促进骨质疏松斑马鱼的骨形成,上调ALP、OCN、Runx2、SP7和OPG表达水平。与nHA相比,Li-nHA能显著增加斑马鱼骨矿化特异性染色面积及累积吸光度,其成骨标志基因表达较nHA显著增加。结论:在纳米羟基磷灰石中掺杂锂离子可以增强骨诱导性能,与nHA相比,Li-nHA能有效促进骨质疏松斑马鱼的骨形成。

PURPOSE:To observe the regulatory effect of lithium-doped hydroxyapatite nanowires on bone metabolism in osteoporotic zebrafish induced by dexamethasone.METHODS:Pure hydroxyapatite nanowires(nHA)and hydroxyapatite nanowires doped with 10%lithium ions(Li-nHA)were prepared by using hydrothermal method,and then material characterization was performed.The juvenile zebrafish cultured for 3 days(3dpf)were selected and co-cultured with nHA and Li-nHA extracts up to 7dpf.A negative(0.1%DMSO)control group was set up and transgenic zebrafish Tg(ola.sp7:nlsGFP)was used to select the best concentration for promoting bone formation.The osteoporotic zebrafish were induced by dexamethasone and incubated with nHA and Li-nHA extracts.The wild-type zebrafish was stained with alizarin red and the osteogenic differentiation was observed in transgenic zebrafish.Real-time quantitative PCR was adopted to detect osteogenic maker genes,such as zinc finger transcription factor(SP7),alkaline phosphatase(ALP),osteoprotegerin(OPG),Runt related transcription factor 2(Runx2)and osteocalcin(OCN).Statistical analysis was performed with GraphPad Prism 9.3 software.RESULTS:nHA and Li-nHA promoted bone formation and up-regulated expression levels of ALP,OCN,Runx2,SP7 and OPG of osteoporotic zebrafish.Compared with nHA,Li-nHA significantly increased the mineralization specific staining area and cumulative optical density of zebrafish bone,and the expression of osteogenic maker genes was also significantly increased.CONCLUSIONS:Doping lithium ions in nano hydroxyapatite can enhance its osteoinductive properties,and Li-nHA can effectively improve bone formation of osteoporotic zebrafish.