脂肪干细胞来源外泌体通过miR-21a-5p/PTEN途径促进牙周膜干细胞成骨分化  

Adipose stem cell derived exosomes promote osteogenic differentiation of periodontal ligament stem cells through mir-21a-5p/PTEN pathway

作　　者：徐秋 邱新毓[2] 仇珺[3] 高洁[1] 张浩[1] XU Qiu;QIU Xin-yu;QIU Jun;GAO Jie;ZHANG Hao(State Key Laboratory of Military Stomatology National Clinical Research Center for Oral Diseases,Xi'an 710032,China)

机构地区：[1]军事口腔医学国家重点实验室、第四军医大学口腔医学院口腔正畸学教研室,西安710032 [2]军事口腔医学国家重点实验室、第四军医大学口腔预防医学教研室,西安710032 [3]军事口腔医学国家重点实验室、第四军医大学军事口腔医学国家重点实验室,西安710032

出　　处：《北京口腔医学》2023年第3期153-158,共6页Beijing Journal of Stomatology

基　　金：国家自然科学(32101096);陕西省自然科学基金(2020JM-321)。

摘　　要：目的 研究脂肪干细胞(ADSCs)来源外泌体通过miR-21a-5p/第10号染色体缺失性磷酸酶-张力蛋白同源物基因(PTEN)途径促进牙周膜干细胞(PDLSCs)成骨分化的效应。方法 培养ADSCs、分离外泌体并用磷酸盐缓冲液溶解备用。培养PDLSCs、进行成骨诱导分化并分组,外泌体组加入15μg/ml外泌体、对照组加入等体积磷酸盐缓冲液,NC组加入等体积磷酸盐缓冲液并转染NC序列,NC+外泌体组加入15μg/ml外泌体并转染NC序列,miR-21-5p敲低+外泌体组加入15μg/ml外泌体并转染miR-21-5p抑制物;诱导后21 d时进行茜素红染色并检测A540水平,诱导后7 d时检测miR-21a-5p、PTEN、碱性磷酸酶(ALP)、骨钙素(OCN)、Ⅰ型胶原(CoL-Ⅰ)的表达水平。培养PDLSCs并分为转染NC序列的NC组、转染miR-21a-5p模拟物的miR-21a-5p组,采用双荧光素酶报告基因验证miR-21a-5p靶向PTEN。结果 外泌体组PDLSCs的A540水平及miR-21a-5p、ALP、OCN、CoL-Ⅰ的表达水平均高于对照组,PTEN的表达水平低于对照组(P<0.05);miR-21a-5p组PDLSCs中PTEN的表达水平及野生型双荧光素酶报告基因的荧光值均低于NC组(P<0.05);miR-21-5p敲低+外泌体组PDLSCs的A540水平及miR-21a-5p、ALP、OCN、CoL-Ⅰ的表达水平均低于NC+外泌体组,PTEN的表达水平高于NC+外泌体组(P<0.05)。结论 ADSCs来源外泌体通过调控miR-21a-5p/PTEN途径促进PDLSCs成骨分化。Objective To study the effect of exosomes derived from adipose derived stem cells(ADSCs)on promoting osteogenic differentiation of periodontal ligament stem cells(PDLSCs)through miR-21a-5p/chromosome 10 deletion phosphatase tensin homologue gene(PTEN).Methods ADSCs were cultured,exosomes were isolated and dissolved in phosphate buffer.PDLSCs were cultured,osteogenic differentiation was induced and divided into groups.Exosome group was intervened with 15μg/ml exosomes,control group with equal volume phosphate buffer,NC group with equal volume phosphate buffer and transfected with NC sequence,NC+exosomes group with 15μg/ml and transfected with NC sequence.miR-21-5p knockdown+exosomes group was intervened with 15μg/ml exosomes and transfected with miR-21-5p inhibitor.Alizarin red staining was performed 21 days after induction and the level of A540 was detected.The expression levels of miR-21a-5p,PTEN,alkaline phosphatase(ALP),osteocalcin(OCN)and type I collagen(CoL-I)were detected 7 days after induction.PDLSCs were cultured and divided into NC group transfected with NC sequence and miR-21a-5p group transfected with miR-21a-5p mimic.Then double luciferase reporter gene was used to verify that miR-21a-5p targeted PTEN.Results The level of A540 and the expression levels of miR-21a-5p,ALP,OCN and CoL-I in PDLSCs in exosome group were higher than those in control group,and the expression level of PTEN was lower than that in control group(P<0.05).The expression level of PTEN and the fluorescence value of wild-type PTEN double luciferase reporter gene in PDLSCs in miR-21a-5p group were lower than those in NC group(P<0.05).The level of A540 and the expression levels of miR-21a-5p,ALP,OCN and CoL-I in PDLSCs in miR-21-5p knockdown+exosome group were lower than those in NC+exosome group,and the expression level of PTEN was higher than that in NC+exosome group(P<0.05).Conclusions ADSCs derived exosomes promote osteogenic differentiation of PDLSCs by regulating miR-21a-5p/PTEN pathway.

关 键 词：脂肪干细胞 牙周膜干细胞 外泌体 成骨分化 miR-21a-5p 

分 类 号：R782[医药卫生—口腔医学]