miR-27a调控比格犬上颌窦黏膜干细胞成骨分化的研究  

Role of miR-27a in the osteogenic differentiation of beagle maxillary sinus membrane stem cells

作　　者：张静 朱双喜[2] 荣琼 彭伟[2] 李祥[2] 陈松龄[2] ZHANG Jing;ZHU Shuangxi;RONG Qiong;PENG Wei;LI Xiang;CHEN Songling(Department of Stomatology,Clifford Hospital,Guangzhou University of Chinese Medicine,Guangzhou 511495,China;Department of Stomatology,The First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China;Department of Stomatology,The First People＇ s Hospital of Yunnan Province,The Affiliated Hospital of Kunming University of Science and Technology,Kunming 650032,China.)

机构地区：[1]广州中医药大学祈福医院口腔科,广东广州511495 [2]中山大学附属第一医院口腔科,广东广州510080 [3]云南省第一人民医院口腔科、昆明理工大学附属医院口腔科,云南昆明650032

出　　处：《口腔疾病防治》2018年第8期484-490,共7页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金项目(81371111);广东省医学科学技术研究基金项目(A2018419);云南省科技厅-昆明医科大学应用基础研究联合专项项目(2017FE468-168)

摘　　要：目的检测miR-27a在犬上颌窦黏膜干细胞(maxillary sinus membrane stem cells,MSMSCs)成骨分化过程中的表达情况,探讨其在犬上颌窦黏膜干细胞成骨分化中的作用。方法体外培养犬上颌窦黏膜干细胞,经成骨诱导培养后,RT-PCR检测miR-27a的表达。犬上颌窦黏膜干细胞转染pre-miR-27a和anti-miR-27a后,RT-PCR检测Runt相关转录因子2(runt-related transcription factor 2,Runx2)和骨桥蛋白(osteopontin,OPN)m RNA的表达,Western blot检测Runx2和OPN蛋白的表达。转染pre-miR-27a的细胞成骨诱导培养后与BioOss复合,建立裸鼠皮下异位成骨模型,观察miR-27a体内抑制成骨情况。结果犬上颌窦黏膜干细胞经成骨诱导培养后,miR-27a表达量降低。miR-27a 1 d(tD=3.795,P=0.023),3 d(tD=4.493,P=0.011),7 d(tD=11.591,P<0.001),14 d(tD=12.542,P<0.001),21 d(tD=5.621,P=0.008)的表达量均高于0 d组。转染pre-miR-27a的犬上颌窦黏膜干细胞经成骨诱导培养后,与阴性对照组相比,成骨标志物Runx2 m RNA(t=4.923,P=0.007)及蛋白(t=4.425,P=0.008)和OPN m RNA(t=5.253,P=0.006)及蛋白(t=5.132,P=0.006)表达量明显降低。相反,转染anti-miR-27a的犬上颌窦黏膜干细胞经成骨诱导培养后,Runx2(t=3.925,P=0.013)和OPN(t=3.712,P=0.019)m RNA表达量比阴性对照组明显升高。单纯培养细胞与Bio-Oss复合后,在裸鼠皮下有成骨表现;但转染pre-miR-27a的犬上颌窦黏膜干细胞与Bio-Oss复合后,犬上颌窦黏膜干细胞在裸鼠皮下异位新骨形成面积减少(t=7.219,P=0.002)。结论 miR-27a负向调控上颌窦黏膜干细胞成骨分化。Objective To detect the expression level of miR-27a during the osteogenic difl＇erentiation of beagle maxillary sinus membrane stem cells （MSMSCs） and explore the role of miR-27a in the osteogenic differentiation of MSMSCs. Methods Beagle MSMSCs ,were cultured in vitro. The expression level of miR-27a was detected via RT-PCR after an osteogenic inductive culture was prepared. The mRNA expression levels of Runx2 and OPN were examined via RT-PCR, and the protein expression levels of Runx2 and OPN were examined via Western blot after the cells were transfected with pre-miR-27a or anti-miR-27a. Finally, osteoprogenitor cells transfected with pre-miR-27a were composited with Bio-Oss particles and subcutaneously implanted into nude mice to form ectopic bone formation models, and then the inhibition of bone formation from miR-27a was observed in vivo. Results The expression level of miR- 27a in the beagle MSMSCs decreased after osteogenic inductive culturing. The relative miR-27a levels were significant- ly decreased at day 1 （t = 3.795, P = 0.023）, day 3 （t = 4.493, P = 0.011）, day 7 （t = 11.591, P 〈 0.001）, day 14 （t = 12.542, P 〈 0.001）, and day 21 （t = 5.621, P = 0.008） compared with day 0. In addition, the expression levels of Runx2 mRNA （t = 4.923, P = 0.007） and protein （t = 4.425, P = 0.008） were reduced after the cells were transfected with pre- miR-27a. The expression levels OPN mRNA （t= 5.253, P= 0.006） and protein （t= 5.132, P= 0.006） were also reduced. In contrast, the mRNA expression levels of Runx2 （t = 3.925, P = 0.013） and OPN （t = 3.712, P = 0.019） were in- creased after the cells were transfected with anti-miR-27a, and bone formation was observed after the subcutaneous implantation of beagle MSMSCs composited with Bio-Oss in nude mice. Nevertheless, ectopic bone formation was inhibit- ed by pre-miR-27a-transfected beagle MSMSCs composited with Bio-Oss （t = 7.219, P = 0.0020）. Conclusion MiR- 27a negatively regulates the osteogenic differentiati

关 键 词：miR-27a 上颌窦黏膜干细胞 成骨分化 Runt相关转录因子2 骨桥蛋白 

分 类 号：R782.13[医药卫生—口腔医学]