PRF负载的Genistein促进肥胖小鼠骨缺损修复的实验研究

doi:10.19439/j.sjos.2024.01.003

摘要/Abstract

摘要： 目的: 明确染料木素(genistein, GEN）对成骨分化的作用并探讨由富血小板纤维蛋白（platelet- rich fibrin,PRF）负载的GEN对肥胖小鼠骨缺损修复过程的影响。方法: 体外实验中,采用CCK8测定7天内不同浓度GEN（0、0.1、1、10、50 μmol/L）对小鼠胚胎成骨细胞前体细胞（mouse embryo osteoblast precursor cells, MC3T3-E1）增殖的影响;利用碱性磷酸酶（ALP）染色及ALP活性定量检测,明确细胞中ALP活性的变化。采用实时荧光定量PCR (qRT-PCR)及蛋白免疫印迹法检测成骨分化过程中ALP、骨桥蛋白（osteopontin,OPN）和骨钙素（osteocalcin, OCN）的RNA及蛋白表达水平,用茜素红染色明确GEN对MC3T3-E1矿化程度的影响。利用扫描电镜观察PRF载药前、后超微结构变化,验证PRF载药的可行性。体内实验中,采用高脂饮食饲喂法建立肥胖C57小鼠模型。制作直径2.8 mm的颅骨缺损模型,将制备好的GEN/PRF复合物置入骨缺损区。利用Micro-CT扫描及H-E染色评估GEN对肥胖小鼠颅骨缺损修复的影响。采用GraphPad Prism 5.0软件包对数据进行统计学分析。结果: CCK8结果显示,7天内,0.1、1 μmol/L GEN促进细胞增殖（P<0.05）;10 μmol/L GEN对细胞增殖过程无明显影响; 50 μmol/L GEN从第2天开始,显著抑制细胞生长,具有细胞毒性（P<0.05）。在MC3T3-E1成骨分化过程中,0.1 μmol/L GEN在一定程度上增加ALP、OPN和OCN蛋白表达（P<0.05）。1、10 μmol/L GEN显著增强ALP活性（P<0.05）,上调ALP、OPN和OCN的RNA及蛋白表达水平（P<0.05）,促进MC3T3-E1中钙结节形成（P<0.05）,2个浓度促进细胞成骨分化的效果相似。扫描电镜发现,PRF内部呈现三维网状结构,为加载药物分子进行局部缓释提供了空间。体内实验中,高脂饮食组小鼠体重大于正常饮食组体重的27.7%（P<0.05）,并出现葡萄糖耐量异常（P<0.05）。Micro-CT显示,与正常饮食组相比,肥胖小鼠股骨中骨小梁数目减少（P<0.05）,骨小梁间距增宽（P<0.05）,骨密度降低（P<0.05）。此外,PRF加载的GEN（0.1、1.0 μmol/L）可增加肥胖小鼠颅骨的骨体积分数（P<0.05）。H-E染色结果显示,GEN/PRF复合物可促进骨缺损愈合。结论: GEN显著促进MC3T3-E1成骨分化, 加载PRF后可有效加快肥胖小鼠颅骨缺损愈合。

关键词: 染料木素, 肥胖, 骨缺损, 小鼠

Abstract: PURPOSE: To clarify the effect of genistein(GEN) on osteogenic differentiation and explore the effect of GEN loaded by platelet-rich fibrin (PRF) on the repair process of bone defects in obese mice. METHODS: In in vitro experiments, the effect of GEN(0, 0.1, 1, 10, 50 μmol/L) on the proliferation of mouse embryonic osteoblast precursor cells (MC3T3-E1) was determined by CCK 8. Alkaline phosphatase(ALP) staining and quantitative detection of ALP activity were performed to determine the changes of ALP activity in cells; RNA and protein expression levels of ALP, osteopontin (OPN) and osteocalcin (OCN) were detected by quantitative real-time PCR(qRT-PCR) and Western blot. Alizarin red staining was used to define the effect of GEN on mineralization of MC3T3-E1. To verify the feasibility of the PRF drug loading, the ultrastructure of PRF was subsequently observed under SEM. In in vivo experiments, obese C57 mouse models were established by high-fat diet feeding. On this basis, skull defect models with a diameter of 2.8 mm were established, and the prepared GEN/PRF complexes were placed into the bone defect area. The effects of GEN on skull defect repair in obese mice were evaluated by Micro-CT scanning and hematoxylin-eosin(H-E) staining. Statistical analysis was performed with GraphPad Prism 5.0 software package. RESULTS: CCK 8 results showed that 0.1, 1 μmol/L GEN promoted cell proliferation within 7 days(P<0.05); 10 μmol/L GEN had no significant effect on the process of cell proliferation. From the second day, 50 μmol/L GEN significantly inhibited cell growth and showed cytotoxicity(P<0.05). These two concentrations had similar effects in promoting cellular osteogenic differentiation. SEM results showed that PRF presented a 3-dimensional network structure, providing space for loading drug molecules. In in vivo experiments, the body weight of mice in the high-fat diet (HFD) group was 27.7% greater than that in the normal diet group(P<0.05) and had abnormal glucose tolerance (P<0.05). Micro-CT showed that compared with the normal diet group, the number of bone trabeculae in the femur of obese mice was decreased(P<0.05), the distance between bone trabeculae was widened(P<0.05), and the bone density was decreased (P<0.05). In addition, GEN (0.1, 1.0 μmol/L) loaded by PRF increased bone volume fraction in the skull of obese mice (P<0.05). H-E results showed that GEN/PRF promoted the healing of the bone defects. CONCLUSIONS: GEN promotes osteogenic differentiation of MC3T3-E1, and it can effectively accelerate the healing of cranial bone defects after loading with PRF in obese mice.

Key words: Genistein, Obesity, Bone defect, Mice

中图分类号:

R782

张雪冰, 李琦. PRF负载的Genistein促进肥胖小鼠骨缺损修复的实验研究[J]. 上海口腔医学, 2024, 33(1): 13-21.

ZHANG Xue-bing, LI Qi. Genistein loaded by PRF improved bone healing in obese mice[J]. Shanghai Journal of Stomatology, 2024, 33(1): 13-21.

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