加载miRNA-21壳聚糖基纳米粒对牙周膜干细胞成骨分化的影响  

Effect of miRNA-21-loaded Chitosan-based Nanoparticles on Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

作　　者：吴广升 王亚男 惠光艳 梅涛[3] 王忠山 WU Guang-sheng;WANG Ya-nan;HUI Guang-yan;MEI Tao;WANG Zhong-shan(Department of Stomatology,Qingdao Special Servicemen Recuperation Center of PLA Navy,Qingdao,Shandong 266071,China;Department of Oral and Maxillofacial Surgery,Qingdao Stomatological Hospital,Qingdao,Shandong 266001,China;Graduate School,Ocean University of China,Qingdao,Shandong 266100,China;State Key Laboratory of Military Stomatology&National Clinical Research Center for Oral Diseases&Shaanxi Key Laboratory of Stomatology,Department of Prosthodontics,School of Stomatology,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]海军青岛特勤疗养中心口腔科,山东青岛266071 [2]青岛市口腔医院口腔颌面外科,山东青岛266001 [3]中国海洋大学研究生院,山东青岛266100 [4]军事口腔医学国家重点实验室/口腔疾病国家临床医学研究中心/陕西省口腔医学重点实验室/空军军医大学口腔医院修复科,西安710032

出　　处：《解放军医药杂志》2021年第1期1-6,共6页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

基　　金：国家自然科学基金青年项目(81700930)。

摘　　要：目的观察壳聚糖/三聚磷酸钠/透明质酸/miR-21纳米粒(CTH/miR-21 NPs)对牙周膜干细胞(PDLSCs)成骨分化的作用。方法采用离子交联法制备CTH/miR-21 NPs,与0.2%的明胶溶液混合后涂覆细胞培养板底部,制备miR-21功能化的反向转染培养板。通过扫描电镜观察纳米粒的分布及交联效果。分析CTH/miR-21 NPs的粒径、电位及形态。原代培养PDLSCs,并对其成骨、成脂分化潜能及细胞表面标记物进行鉴定。将PDLSCs接种于含CTH/miR-21 NPs涂层的培养板,通过荧光显微镜、流式细胞仪观察CTH/miR-21 NPs反向转染PDLSCs的效率,并通过RT-PCR观察其成骨相关基因的表达情况。结果 CTH/miR-21 NPs粒径由147.3 nm增加至288.5 nm,Zeta电位由44.5 mV降至21.7 mV,CTH/miR-21 NPs被明胶交联于培养板底部,呈球形。PDLSCs可以成功地被CTH/miR-21 NPs反向转染,且300、450 pmol/孔组RUNT相关转录因子2(RUNX2)、骨桥素(OPN)、Ⅰ型胶原酶(COLⅠ)及骨钙素(OCN)表达水平高于0 pmol/孔组(P<0.05,P<0.01)。结论 CTH/miR-21 NPs可以高效反向转染PDLSCs并促进成骨相关基因的表达,可以为基因转染技术应用于牙周组织再生提供新的思路。Objective To investigated the effect of chitosan(CS)/tripolyphosphate(TPP)/Hyaluronic acid(HA)/miRNA-21(miR-21)nanoparticles(CTH/miR-21 NPs)on osteogenic differentiation of periodontal ligament stem cells(PDLSCs).Methods The CTH/miR-21 NPs were prepared by the ionotropic gelation technique,and then mixed with 0.2%gel solution to fabricate CTH/miR-21 NPs pre-coated culture plates for gene reverse transfection.The distribution of nanoparticles and cross-linking effect were observed under scanning electron microscope.For the characterization of CTH/miR-21 NPs,the particle size,zeta potential and surface morphology were sequentially investigated.PDLSCs were primary cultured and characterized by their osteogenic/adipogenic differentiation potential and cell surface markers.PDLSCs were seeded on CTH/miR-21 NPs pre-coated culture plates and the reverse transfection was assessed by fluorescence microscopy and flow cytometry.The osteogenic expression level of PDLSCs were evaluated by real-time quantitative PCR(RT-PCR).Results The size of CTH/miR-21 NPs was inreased from 147.3 nm to 288.5 nm,and the potential was decreased from 44.5 mV to 21.7 mV.CTH/miR-21 NPs was cross-linked by gelatin on culture plates presenting with a spherical shape.The PDLSCs could be successfully reverse transfected by CTH/miR-21 NPs and the expression of RUNT-related transcription factor-2(RUNX2),genes osteopontin(OPN),osteocalcin(OCN)and collagen typeⅠ(COLⅠ)in the 300 and 450 pmol/hole groups were significantly higher than those in the 0 pmol/hole group(P<0.05,P<0.01).Conclusion The CTH/miR-21 NPs can efficiently reverse transfect PDLSCs and enhance osteogenic expression level,which might promisingly provide a new avenue for application of gene transfection technique in periodontal tissue regeneration.

关 键 词：微小RNA 壳聚糖 牙周膜干细胞 RUNX2 骨桥素 Ⅰ型胶原酶 骨钙素 

分 类 号：R780.2[医药卫生—口腔医学]