VPS26基因通过Wnt/β-联蛋白通路促进高脂血症大鼠种植体骨结合的机制研究  

Mechanism of VPS26 gene promoting implant osseointegration through Wnt/β-catenin pathway in hyperlipidemia rats

作　　者：慕宇青 袁孟绮 袁欣 朱丽娜 郭美画 蓝菁 Mu Yuqing;Yuan Mengqi;Yuan Xin;Zhu Lina;Guo Meihua;Lan Jing(Department of Prosthodontics,School and Hospital of Stomatology,Cheeloo College of Medicine,Shandong University&Shandong Key Laboratory of Oral Tissue Regeneration&Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration,Jinan 250012,China)

机构地区：[1]山东大学齐鲁医学院口腔医学院·口腔医院修复科、山东省口腔组织再生重点实验室、山东省口腔生物材料与组织再生工程实验室,济南250012

出　　处：《中华口腔医学杂志》2023年第4期345-353,共9页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82170999);山东省重点研发计划(2021SFGC0502)。

摘　　要：目的研究VPS26在高脂环境中对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSC)成骨、成脂分化作用的机制,探讨VPS26对高脂大鼠种植体骨结合和裸鼠异位成骨的影响。方法普通成骨诱导液(成骨组)和高脂成骨诱导液(高脂组)培养BMSC,高脂组促进或抑制VPS26的表达,检测成骨和成脂相关基因的表达。细胞诱导第7、14天后行碱性磷酸酶(alkaline phosphatase,ALP)和油红O染色;成骨组细胞采用免疫荧光染色和免疫共沉淀方法检测VPS26与β-联蛋白(β-catenin)的结合,双荧光素酶报告实验检测萤火虫荧光值/海肾荧光值(以下简称TOP/FOP)比值。取18只雄性12周龄高脂血症Wista大鼠(体质量为160~200 g),于其双侧股骨干骺端植入种植体,分为3组,每组6只,分别注射VPS26过表达慢病毒(LV-VPS26组)、阴性对照慢病毒(LV-nc组)和生理盐水(空白对照组),种植体及周围骨组织行显微CT分析和HE、油红O染色评估种植体骨结合和股骨脂滴形成。20只雌性6周龄裸鼠(体质量为30~40 g)均分为5组,每组4只,于背部皮下分别植入不转染和转染了LV-VPS26、LV-nc、shVPS26、shscr慢病毒的成骨组BMSC,取样观察异位成骨。结果过表达VPS26后高脂组BMSC中ALP的mRNA表达水平(1.56±0.09)显著高于阴性对照(1.01±0.03)(t=10.09,P<0.001),过氧化物酶增殖物活化受体-γ(peroxisome proliferator-activated receptor-γ,PPAR-γ)和脂肪酸结合蛋白4(fatty acid-binding protein4,FABP4)的mRNA表达水平则显著低于阴性对照(t=6.44,P<0.001;t=10.01,P<0.001)。蛋白质印迹法结果显示过表达VPS26后高脂组BMSC中ALP、Runt相关转录因子2的蛋白表达较阴性对照增强,PPAR-γ、FABP4则减弱,高脂组骨髓间充质干细胞ALP活性在过表达VPS26后更强,脂滴的形成较阴性对照更弱,数量更少。免疫荧光染色、免疫共沉淀和双荧光素酶报告实验结果显示VPS26与β-catenin存在共定位和相互作用,且TOP/FOP比值显著上升43Objective To investigate the mechanism of VPS26 effect on osteogenesis and adipogenesis differentiation of rat bone marrow mesenchymal stem cells(BMSC)in high fat environment,and to explore the effect of VPS26 on implants osseointegration of high fat rats and ectopic osteogenesis in nude mice.Methods BMSC were cultured under normal osteogenic induction(osteogenic group)and high-fat osteogenic induction(high-fat group).High-fat group was transfected with VPS26 enhancer and inhibitor,and the expression levels of osteogenesis related genes and adipogenesis related genes were examined.Osteogenesis and adipogenesis of BMSC were detected by alkaline phosphatase(ALP)staining and oil red O staining after 7 and 14 days of induction.In osteogenic group,the binding of VPS26 toβ-catenin was detected by immunofluorescence staining and immunoprecipitation,and dual luciferase reporter assay(TOP Flash)was used to analyze the TOP/FOP ratio.Eighteen male 12-week hyperlipidemic Wista rats(160-200 g)were implanted with implants,and six in each group were injected with VPS26 overexpression lentivirus(LV-VPS26 group),negative control lentivirus(LV-nc group)and saline(blank control group).Micro-CT analysis,HE and oil red O staining were used to evaluate the osseointegration of the implants and lipid droplets formation of the femur samples.Twenty female 6-week nude mice(30-40 g)were divided into five groups and subcutaneously implanted with osteogenic BMSC non-transfected and transfected LV-VPS26,LV-nc,shVPS26,and shscr lentivirus on the back.Samples were used to observe ectopic osteogenesis.Results The mRNA expression levels of ALP in the high-fat group BMSC after overexpression of VPS26(1.56±0.09)were significantly higher than those of the negative control(1.01±0.03)(t=10.09,P<0.001),while those of peroxisome proliferator-activated receptor-γ(PPAR-γ)(t=6.44,P<0.001)and fatty acid-binding protein4(FABP4)(t=10.01,P<0.001)were lower than those of the negative control.Western blotting results showed that compared with the negative co

关 键 词：牙种植体 骨结合 高脂血症 VPS26 Wnt/β-联蛋白通路 

分 类 号：R589.2[医药卫生—内分泌]