二甲双胍对微弧氧化羟基磷灰石涂层金属钛表面的糖尿病大鼠BMSCs细胞增殖和成骨分化的影响及机制研究  

Effects and mechanisms of metformin on proliferation and osteogenic differentiation of BMSCs in diabetes rats exposed to micro arc oxidation of hydroxyapatite coated Titanium

作　　者：谢振杰 卢燕雪 袁晓诗 钟汉铭[1] XIE Zhenjie;LU Yanxue;YUAN Xiaoshi;ZHONG Hanming(Department of Stomatology,The Tenth Affiliated Hospital of Southern Medical University,Dongguan People's Hospital,Dongguan 523059,Guangdong,China)

机构地区：[1]南方医科大学第十附属医院·东莞市人民医院口腔科,广东东莞523059

出　　处：《西部医学》2024年第8期1107-1114,共8页Medical Journal of West China

基　　金：2022年东莞市社会发展科技项目(20221800901622)。

摘　　要：目的探究二甲双胍(Met)对微弧氧化羟基磷灰石(MAO-HA)涂层金属钛表面的2型糖尿病(T2DM)大鼠骨髓间充质干细胞(BMSCs)细胞增殖和成骨分化的影响及作用机制。方法20只SPF级雄性大鼠随机分为正常组(NC组)、T2DM组、低剂量二甲双胍组(Met-Low组)、高剂量二甲双胍组(Met-High组),每组5只。采用高糖高脂饲料喂养4周联合腹腔注射链脲佐菌素(STA,50 mg/kg)建立T2DM大鼠模型。Met-Low、Met-high组大鼠分别进行二甲双胍150 mg·kg^(-1)·d^(-1)、300 mg·kg^(-1)·d^(-1)灌胃,NC组、T2DM组以等量0.9%氯化钠溶液灌胃。灌胃4周后,处死大鼠并分离股骨、胫骨中的BMSCs。采用电化学法(ELC)和微弧氧化法(MAO)制备羟基磷灰石(HA)涂层金属钛材料。将正常大鼠来源的BMSCs种植于ELC-HA、MAO-HA涂层表面,培养48 h后,扫描电镜观察涂层表面形态及BMSCs在其表面的吸附情况。将不同来源的BMSCs种植于MAO-HA涂层表面,并分为NC组、T2DM组、Met-Low组、Met-High组。培养48 h后,CCK-8、EdU染色检测BMSCs增殖能力;碱性磷酸酶(ALP)染色、茜素红染色检测BMSCs成骨分化能力;Western blot检测细胞增殖相关标志物[增殖细胞核抗原(PCNA)、Ki67]、成骨分化相关标志物[碱性磷酸酶(ALP)、骨钙素(OCN)、骨桥蛋白(OPN)]、Wnt/βcatenin通路相关蛋白[Wnt3a、β连环蛋白(βcatenin)、糖原合酶激酶-3β(GSK-3β)]蛋白水平。结果MAO-HA涂层表面具有均匀粗糙的孔隙,其上BMSCs充分伸展,细胞突起粗大,而ELC-HA涂层表面十分光滑,其上BMSCs皱缩,突起较细。与NC组相比,T2DM组BMSCs细胞存活率、EdU阳性细胞率及成骨分化能力明显降低,BMSCs中PCNA、Ki67、ALP、OCN、OPN、Wnt3a、βcatenin蛋白水平明显降低,GSK-3β蛋白水平明显升高(均P<0.05)。与T2DM组相比,Met-Low组和Met-High组BMSCs细胞存活率、EdU阳性细胞率及成骨分化能力明显升高,BMSCs中PCNA、Ki67、ALP、OCN、OPN、Wnt3a、βcatenin蛋白水平明显升高,GSKObjective To explore the effects and mechanisms of metformin(Met)on proliferation and osteogenic differentiation of bone marrow stem cells(BMSCs)in type 2 diabetes mellitus(T2DM)rats exposed to micro arc oxidation of hydroxyapatite(MAO-HA)coated Titanium.Methods High sugar and high-fat diet(fed for 4 weeks)combined with intraperitoneal injection of streptozotocin(STZ)(50 mg/kg)was used to establish T2DM rat model.Rats were randomly divided into Normal group,T2DM group,low-dose metformin group and high-dose metformin group.Metformin group rats were treated with metformin(150 mg/kg/d,300 mg/kg/d)by gavage.After 4 weeks of lavage,rats were euthanized and BMSCs were separated from the femur and tibia.Electrochemical method(ELC)and micro arc oxidation method(MAO)were used to prepare hydroxyapatite(HA)coated titanium metal materials.BMSCs from normal rat were implanted on the surface of ELC-HA and MAO-HA coatings.After 48 hours of cultivation,scanning electron microscopy was used to observe the surface morphology of the coating and the adsorption of BMSCs on its surface.BMSCs from different sources were implanted on the surface of MAO-HA coating and divided into NC group,T2DM group,Met-Low group and Met-High group.After 48 hours of cultivation,CCK-8 and EdU staining were used to detect the proliferation ability of BMSCs;Alkaline phosphatase(ALP)staining and Alizarin red staining were used to detect the osteogenic differentiation ability of BMSCs;Western blot was used to detect proliferation related markers[proliferating cell nucleus antigen(PCNA),Ki67],osteogenic differentiation related markers[ALP,osteocalcin(OCN),osteopontin(OPN)],Wnt/βcatenin pathway related proteins[Wnt3a,βCatenin,glycogen synthase Kinase-3β(GSK-3β)]protein levels.Results The surface of MAO-HA coating had uniformly rough pores,on which BMSCs were fully extended and cell protrusions were coarse,while the surface of the ELC-HA coating was very smooth,with BMSCs wrinkled and protrusions finer on it.Compared with NC group,the survival rate,EdU posi

关 键 词：二甲双胍 微弧氧化羟基磷灰石涂层金属钛 糖尿病 骨髓间充质干细胞 成骨分化 Wnt/βcatenin通路 

分 类 号：R587.1[医药卫生—内分泌] R392.2[医药卫生—内科学]