| 郭 硕,刘文文,魏星辉,王 宁,李小康,郭 征.PCL/β-TCP调控巨噬细胞极化对成血管效应的影响[J].,2020,(11):2012-2018 |
| PCL/β-TCP调控巨噬细胞极化对成血管效应的影响 |
| Effect of PCL/β-TCP on Angiogenesis Through Regulating Macrophage Polarization |
| 投稿时间：2020-02-23 修订日期：2020-03-18 |
| DOI：10.13241/j.cnki.pmb.2020.11.003 |
| 中文关键词: 骨修复支架 炎症反应 成血管效应 生物陶瓷 巨噬细胞极化 |
| 英文关键词: Bone repair scaffold Inflammation response Vascularization effect Bioceramics Macrophage polarization |
| 基金项目:国家重点研究发展计划项目（2017YFC1104902）；国家自然科学基金面上项目（51771227） |
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| 中文摘要: |
| 摘要 目的：探究生物可降解材料聚己内酯/β-磷酸三钙（PCL/β-TCP）通过调控巨噬细胞极化对骨组织内血管生成的作用，为其临床应用提供依据。方法：采用3D打印技术制备试样并加以表征。体内实验采用模型为SD大鼠股骨远端植入模型。双侧植入PCL/β-TCP支架后采取免疫荧光染色观察支架内部成血管标记物CD31的表达差异，并采用血管灌注方法进行血管造影，评价支架内部血管体积。采用免疫荧光染色检测炎症标记物iNOS，抑炎标记物Arg-1的表达情况。体外实验采用细胞共培养的方式检测PCL/β-TCP对巨噬细胞极化的调控作用以及免疫介导的血管形成改变。实验分为两组，空白组（Control）及PCL/β-TCP（PT5）组。将巨噬细胞系Raw264.7接种于材料表面并对其极化水平及分泌改变进行检测。通过免疫荧光染色检测M1巨噬细胞标记物iNOS、M2巨噬细胞标记物Arg-1的表达情况。通过RT-qPCR检测CCR-7，CD206，血管内皮生长因子（VEGF），血小板源性生长因子（PDGF-BB），肿瘤坏死因子α（TNF-α），白细胞介素-10（IL-10）的转录情况。酶联免疫吸附试验（ELISA）检测VEGF、PDGF-BB、IL-10、TNF-α的分泌情况。应用Transwell迁移实验检测PCL/β-TCP刺激下巨噬细胞分泌作用对人脐静脉内皮细胞（HUVECs）迁移能力的影响，应用免疫荧光染色检测PCL/β-TCP刺激下巨噬细胞分泌作用对HUVECs表面血管形成指标CD31表达情况的影响。结果：在体内实验中，支架周围组织CD31表达升高（P<0.001），血管灌注结果提示支架内部血管体积显著增加（P<0.001），同时炎症标记物iNOS表达下调（P<0.001），抗炎标记物Arg-1升高（P<0.001）。在体外实验中，与Control相比，PT5组巨噬细胞中炎症标记物iNOS合成无明显差异，抑炎标记物Arg-1合成增加；炎症标记物CCR-7及TNF-α转录水平下调（P<0.01，P<0.01），抗炎标记物CD206及IL-10转录上调（P<0.001，P<0.001）；VEGF转录水平下调（P<0.01），PDGF-BB转录上调（P<0.01）；VEGF分泌水平下降（P<0.001），PDGF-BB分泌增加（P<0.01），IL-10分泌水平提高（P<0.001），TNF-α分泌水平下降（P<0.05）。在巨噬细胞分泌作用下，HUVECs的迁移能力提高（P<0.001），CD31表达上调（P<0.001）。结论：骨修复材料PCL/β-TCP可通过调控巨噬细胞向M2方向极化进而促进血管形成，可作为骨修复材料的候选材料之一。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the angiogenesis changes caused by biodegradable material polycaprolactone/β-tricalcium phosphate (PCL/β-TCP) through the regulation of macrophage polarization, and to provide a basis for its clinical application. Methods: The samples were prepared by 3D printing technology and characterized. In vivo experiments, SD rats were implanted in distal femur. Immunofluorescence staining was performed to detect the expression of angiogenesis marker CD31 and angiography was used to evaluate the vascular volume in the stent. Pro-inflammatory marker iNOS and anti-inflammatory marker Arg-1 in the scaffold were detected by immunofluorescence staining after implantation. PCL/β-TCP and its effect on angiogenesis through the regulation of macrophage polarization were detected by cell co-culture in vitro. The experiment was divided into two groups, the blank group (Control) and the PCL/β-TCP (PT5) group. Macrophage cell line Raw264.7 was inoculated on the surface of the material and the polarization and secretion changes were detected. The expression of the M1 macrophage marker iNOS and M2 macrophage marker Arg-1 were detected by immunofluorescence staining. The transcription of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB), M1 macrophage marker CCR-7, and M2 macrophage marker CD206 were detected by RT-qPCR. The secretion of VEGF, TNF-α, PDGF-BB and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA). Transwell migration experiment was used to detect the effect of macrophage secretion on HUVECs migration ability of human umbilical vein endothelial cells stimulated by PCL/β-TCP. Results: In vivo experiments, the volume and expression of CD31 were enhanced (P<0.001, P<0.001). Expression of inflammatory marker iNOS decreased (P<0.001), while the anti-inflammatory marker Arg-1 increased (P<0.001). In vitro study, compared with Control, there was no significant difference in synthesis of inflammatory marker iNOS (P>0.05), and the synthesis of anti-inflammatory marker Arg-1 increased in PT5 (P<0.001). Transcription level of inflammatory marker CCR-7 and TNF-α was decreased (P<0.01, P<0.01) in PT5, while the transcription of anti-inflammatory markers CD206 and IL-10 were up-regulated (P<0.001, P<0.001). Transcription of VEGF was down-regulated (P<0.01) in PT5, and PDGF-BB transcription level was up-regulated (P<0.001). Treated with PT5, the secretion level of VEGF decreased (P<0.01), while the secretion level of PDGF-BB was upregulated (P<0.01) as well as IL-10(P<0.001). Compared with Control, the secretion of TNF-α was impaired in PT5(P<0.05). Under the action of macrophage secretion, HUVECs migration ability and expression of CD31 were enhanced (P<0.001). Conclusion: Bone repair material PCL/β-TCP can promote angiogenesis by regulating the polarization of macrophages towards M2, and can be one of the candidate materials for bone repair materials. |
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