PRF负载的Genistein促进肥胖小鼠骨缺损修复的实验研究  

Genistein loaded by PRF improved bone healing in obese mice

作　　者：张雪冰 李琦[2] ZHANG Xue-bing;LI Qi(Stomatology College of Inner Mongolia Medical University,Huhehaote 010030;Department of Stomatology,Affiliated Hospital of Inner Mongolia Medical University.Huhehaote 010030,Inner Mongolia Autonomous Region,China)

机构地区：[1]内蒙古医科大学口腔医学院,内蒙古呼和浩特010030 [2]内蒙古医科大学附属医院口腔科,内蒙古呼和浩特010030

出　　处：《上海口腔医学》2024年第1期13-21,共9页Shanghai Journal of Stomatology

基　　金：内蒙古自然科学基金(2020MS08036)。

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、50μmol/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、1μmol/L GEN促进细胞增殖(P<0.05);10μmol/L GEN对细胞增殖过程无明显影响;50μmol/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,0.1μmol/L GEN在一定程度上增加ALP、OPN和OCN蛋白表达(P<0.05)。1、10μmol/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�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(0,0.1,1,10,50μmol/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 0.1,1μmol/L GEN promoted cell proliferation within 7 days(P<0.05);10μmol/L GEN had no significant effect on the process of cell proliferation.From the second day,50μmol/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 trabecul

关 键 词：染料木素 肥胖 骨缺损 小鼠 

分 类 号：R782[医药卫生—口腔医学]