[1]胡 琛,胡萧旬,覃 媛,等.磷酸钙骨水泥/印第安刺猬蛋白/聚乳酸羟基共聚物微球复合物制备及体外释放性能初步研究[J].陕西医学杂志,2023,52(3):261-266.[doi:DOI:10.3969/j.issn.1000-7377.2023.03.004]
　HU Chen,HU Xiaoxun,QIN Yuan,et al.Preliminary study on preparation of CPC/Ihh/PLGA microsphere composite and its performance in vitro[J].,2023,52(3):261-266.[doi:DOI:10.3969/j.issn.1000-7377.2023.03.004]
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磷酸钙骨水泥/印第安刺猬蛋白/聚乳酸羟基共聚物微球复合物制备及体外释放性能初步研究
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《陕西医学杂志》[ISSN:1000-7377/CN:61-1281/TN]

卷:
52
期数:
2023年3期
页码:
261-266
栏目:
基础研究
出版日期:
2023-03-05

文章信息/Info

Title:
Preliminary study on preparation of CPC/Ihh/PLGA microsphere composite and its performance in vitro
作者:
胡 琛1; 2; 胡萧旬1; 覃 媛2; 黄宏莉1; 廖红兵1; 2
(1.广西医科大学附属口腔医学院 广西口腔颌面修复与重建研究重点实验室,广西 南宁 530021; 2.广西医科大学附属口腔医院口腔修复科,广西 南宁 530021)
Author(s):
HU Chen; HU Xiaoxun; QIN Yuan; HUANG Hongli; LIAO Hongbing
(Guangxi Key Laboratory of Oral and Maxillofacial Restoration and Reconstruction,School of Stomatology,Guangxi Medical University,Nanning 530021,China)
关键词:
印第安刺猬蛋白;  磷酸钙骨水泥;  骨髓来源单核巨噬细胞;  聚乳酸羟基共聚物;  体外释放性能;  小鼠
Keywords:
Indian hedgehog;  Calcium phosphate cement;  BMDMs;  PLGA;  Release properties in vitro;  Mouse
分类号:
R 96
DOI:
DOI:10.3969/j.issn.1000-7377.2023.03.004
文献标志码:
A
摘要:
目的:观察不同浓度印第安刺猬蛋白(Ihh)对小鼠骨髓来源单核巨噬细胞(BMDMs)增殖分化的影响,筛选最适浓度用于后续实验,并初步探索磷酸钙骨水泥/印第安刺猬蛋白/聚乳酸羟基共聚物(CPC/Ihh/PLGA)微球复合物的制备工艺及体外释放情况。方法:选择6～8周龄SPF级C57BL/6J雄性小鼠10只,处死后分离骨髓,纯化BMDMs并进行培养。根据培养基不同分为空白对照组、100 ng/ml Ihh组、200 ng/ml Ihh组、300 ng/ml Ihh组、400 ng/ml Ihh组和500 ng/ml Ihh组。利用CCK-8法检测不同浓度Ihh对小鼠BMDMs增殖速率的影响。采用抗酒石酸酸性磷酸酶(TRAP)染色法评估不同浓度Ihh促小鼠BMDMs破骨向分化能力。采用荧光定量聚合酶链反应(RT-PCR)检测不同浓度Ihh对小鼠BMDMs破骨向分化相关基因mRNA表达的影响。应用复乳溶剂挥发法制备Ihh/PLGA微球。利用扫描电子显微镜观察微球形态并计算其粒径大小。通过酶联免疫吸附试验(ELISA)检测微球中Ihh包封率。最后制备CPC/Ihh/PLGA微球复合物并对其体外释放特性进行研究。结果:与空白对照组比较,不同浓度Ihh组在细胞接种后第1、3、5天时对小鼠BMDMs均有促进增殖的作用(均P<0.05),但不同浓度组比较差异无统计学意义(均P>0.05)。随着Ihh浓度的升高,破骨细胞生成数目、破骨向分化相关基因[抗酒石酸酸性磷酸酶5(Acp5)、组织蛋白酶K(Ctsk)和核因子κB受体活化因子(RANK)]mRNA表达表现为先升高再降低的趋势,且300 ng/ml Ihh组破骨细胞生成数目及破骨向分化相关基因mRNA相对表达量高于其他浓度组(均P<0.05)。制备负载最适Ihh浓度的PLGA微球后,该微球粒径为(54.02±8.63)μm,微球中Ihh包封率为65.41%。制备CPC/Ihh/PLGA微球复合物后,其体外累积释放量为78.04%。结论:Ihh能够促进小鼠BMDMs增殖及破骨向分化,最适浓度为300 ng/ml,将其负载于PLGA后成功制备CPC/Ihh/PLGA微球复合物,且该复合物在体外具有缓释作用。
Abstract:
Objective:To investigate the effects of Indian hedgehog(Ihh)on the proliferation and differentiation of mouse bone marrow-derived mononuclear macrophage cells(BMDMs),and to select the optimal concentration for subsequent experiments,and to preliminarily explore the preparation technology and in vitro release of CPC/Indian hedgehog(Ihh)/PLGA microsphere composite.Methods:A total of 10 aged 6 to 8 weeks male SPF C57/BL 6J mice were selected,bone marrow was isolated after sacrifice,BMDMs were purified and cultured.According to the different medium,BMDMs were divided into blank control group,100 ng/ml Ihh group,200 ng/ml Ihh group,300 ng/ml Ihh group,400 ng/ml Ihh group and 500 ng/ml Ihh group.The effect of Ihh concentration on the cell proliferation rate was analyzed by CCK-8 assay.The ability of different concentrations of Ihh to induce osteoclastogenesis of BMDMs was assessed by TRAP staining.The effect of different concentrations of Ihh on mRNA expression of osteoclastogenesis-related genes in BMDMs was measured by RT-PCR.Ihh/PLGA microspheres were prepared by W/O/W method.The morphology of the microspheres was observed by SEM,and the particle size was calculated.The encapsulation efficiency of Ihh protein in the microspheres was detected by ELISA.Finally,CPC/Ihh/PLGA microspheres composite was prepared and there in vitro release characteristics were studied.Results:Compared with blank control group,different concentrations of Ihh were found to promote proliferation of BMDMs on days 1,3,and 5 after cell inoculation(all P<0.05),but there was no statistically significant difference between the different concentrations(all P>0.05).With the increase of Ihh concentration,osteoclastogenesis and osteoclastogenesis-related genes(Acp5,Ctsk and RANK)mRNA expression showed a tendency to first increase and then decrease,and the relative expression of osteoclastogenesis and osteoclastogenesis-related gene mRNAs were higher in the 300 ng/ml Ihh group than those in the other concentration groups(all P<0.05).PLGA microspheres loaded with optimal Ihh concentration were prepared,and the particle size of the microspheres were(54.02±8.63)μm,and the encapsulation rate of Ihh in the microspheres were 65.41%.CPC/Ihh/PLGA microsphere composite was prepared with a cumulative release of 78.04% in vitro.Conclusion:Ihh was able to promote proliferation and osteoclastic differentiation of BMDMs in mice,and the optimum concentration was 300 ng/ml.CPC/Ihh/PLGA microspheres were successfully prepared,and the composite has sustained-release effect in vitro.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金资助项目(81860201)
更新日期/Last Update: 2023-03-06