载槲皮素明胶微球对MC3T3-E1增殖和分化的影响

doi:10.13591/j.cnki.kqyx.2024.07.003

摘要/Abstract

摘要：

目的研究负载槲皮素的明胶三维多孔(G-quercetin)微球作为骨组织材料的可行性。方法利用乳化法制备负载槲皮素的多孔明胶微球,扫描电镜观察微球形态,通过免疫荧光染色、活死细胞染色和CCK-8、碱性磷酸酶(ALP)染色及茜素红染色检测微球的细胞毒性及其对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)黏附、增殖及分化的影响,RT-PCR检测成骨相关基因Runx-2、ALP、OPN、OCN表达。结果扫描电镜结果显示制备的载槲皮素明胶三维微孔材料具有多孔结构。细胞黏附检测显示细胞能够在微球表面铺展良好。与对照组相比,活死细胞染色、CCK-8结果显示该微球无明显细胞毒性(P>0.05);与G-quercetin微球共培养的MC3T3-E1 ALP表达和体外矿化增加;PCR结果显示Runx-2、ALP、OCN、OPN表达明显增高(P<0.05)。结论载槲皮素明胶微球具有良好的生物相容性,能够促进MC3T3-E1体外成骨分化,有望作为新型骨组织工程生物材料应用于临床。

关键词: 明胶, 槲皮素, 微球, 成骨分化, 骨组织工程, 小鼠胚胎成骨细胞前体细胞

Abstract:

Objective This study prepared gelatin three-dimensional porous microspheres and investigated the feasibility of gelatin three-dimensional porous microspheres loaded with quercetin (G-quercetin) as bone tissue scaffold material. Methods Porous gelatin microspheres were prepared by emulsification and loaded with quercetin by lyophilization. Scanning electron microscopy was used to observe morphology of the microspheres. The cytotoxicity of G-quercetin microspheres and their effects on the adhesion, proliferation and differentiation of mouse embryonic osteoblast precursor cells (MC3T3-E1) were detected by immunofluorescence staining, live/dead cell staining and CCK-8 assay, alkaline phosphatase (ALP) staining and alizarin red staining. RT-PCR was used to detect the transcriptional levels of osteoblast-related cytokines such as Runx-2, ALP, OPN and OCN. Results The scanning electron microscopy results showed that the prepared three-dimensional microporous material loaded with quercetin gelatin had a porous structure. Cell adhesion showed that the cells could spread well on the surface of the microspheres. Compared with the control group, the results of live/dead cell staining and CCK-8 detection showed that the microspheres had no significant cytotoxicity (P>0.05). Compared with the control group, G-quercetin microspheres showed an increase in ALP expression and mineralization in vitro. PCR results also showed a significant increase in Runx-2, ALP, OCN, OPN (P<0.05). Conclusion The G-quercetin porous microspheres prepared in this experiment have good biocompatibility and can promote the osteogenic differentiation of MC3T3-E1 in vitro. It is expected to be used as a new scaffold material for bone tissue engineering.

Key words: gelatin, quercetin, microspheres, osteogenic differentiation, bone tissue engineering, mouse embryonic osteoblast precursor cells (MC3T3-E1)

中图分类号:

R318.08

董伟杰, 苏庭舒, 忻贤贞. 载槲皮素明胶微球对MC3T3-E1增殖和分化的影响[J]. 口腔医学, 2024, 44(7): 494-499.

DONG Weijie, SU Tingshu, XIN Xianzhen. Effects of quercetin loaded gelatin microspheres on proliferation and differentiation of MC3T3-E1[J]. Stomatology, 2024, 44(7): 494-499.

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基因名称	引物序列(5'→3')
Runx-2	F:ATCCAGCCACCTTCACTTACACC R:GGGACCATTGGGAACTGATAGG
ALP	F:TATGTCTGGAACCGCACTGAAC R:CACTAGCAAGAAGAAGCCTTTGG
OPN	F:GACGGCCGAGGTGATAGCTT R:CATGGCTGGTCTTCCCGTTGC
OCN	F:GCCCTGACTGCATTCTGCCTCT R:TCACCACCTTACTGCCCTCCTG
GAPDH	F:GGCACAGTCAAGGCTGAGAATG R:ATGGTGGTGAAGACGCCAGTA