RNA结合蛋白Lin28A差异表达可调控牙周膜干细胞的成骨分化  

Differential expression of RNA binding protein Lin28A regulates osteoblastic differentiation of periodontal ligament stem cells

作　　者：何琴[1,2,3] 卜艳 林光磊[1,2] 罗晶 雍敏 黄永清 He Qin;Bu Yan;Lin Guanglei;Luo Jing;Yong Min;Huang Yongqing(Department of Orthodontics,School of Stomatology,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region,Yinchuan 750004,Ningxia Hui Autonomous Region,China;Department of Orthodontics,General Hospital of Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;Department of Oral and Maxillofacial Surgery,School of Stomatology,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China)

机构地区：[1]宁夏医科大学口腔医学院正畸学系,宁夏回族自治区银川市750004 [2]宁夏回族自治区干细胞与再生医学重点实验室,宁夏回族自治区银川市750004 [3]宁夏医科大学总医院口腔正畸科,宁夏回族自治区银川市750004 [4]宁夏医科大学口腔医学院口腔颌面外科学系,宁夏回族自治区银川市750004

出　　处：《中国组织工程研究》2023年第33期5283-5291,共9页Chinese Journal of Tissue Engineering Research

基　　金：宁夏自然科学基金(2019AAC03078),项目负责人:何琴;宁夏医科大学校级特殊人才项目(XT2018019),项目负责人:何琴;宁夏医科大学2020年校级学术技术带头人后备培育对象项目(20012602201),项目负责人:何琴。

摘　　要：背景:Lin28A能影响细胞活动的多个方面,包括胚胎干细胞的自我更新、体细胞的重编程、个体的生长发育、组织代谢及致癌作用等,而它对牙周膜干细胞成骨分化的影响国内外均没有深入研究,其影响机制更未见报道。目的:探索Lin28A对牙周膜干细胞成骨分化能力的影响。方法:经典酶消化法分离、培养人牙周膜干细胞,qRT-PCR检测Lin28A在该类细胞中的分布情况及其在成骨诱导不同时间的差异性表达;构建Lin28A过表达及干扰表达的慢病毒载体转染至牙周膜干细胞,荧光显微镜观察转染效果;qRT-PCR验证慢病毒转染后Lin28A的表达差异,并进一步从正反两个方面检测成骨诱导培养过程中成骨基因碱性磷酸酶、Runt相关转录因子2和骨桥蛋白的表达量以及碱性磷酸酶染色和茜素红染色,Western blot检测Runt相关转录因子2的蛋白表达水平;应用生物信息学技术预测和双荧光素酶结合实验验证与Lin28A有关的let-7家族成员之间的相关关系,并通过成骨诱导培养差异表达Lin28A的牙周膜干细胞,测定细胞成骨分化过程中表达变化最明显的miRNA。结果与结论:(1)Lin28A富集在牙周膜干细胞的细胞核中,且在成骨诱导不同时间后呈表达上升的趋势;(2)与空白对照组比较,在过表达Lin28A的牙周膜干细胞模型中,碱性磷酸酶染色和茜素红染色相较空白对照组明显深染,成骨基因碱性磷酸酶、Runt相关转录因子2及骨桥蛋白的表达量分别增加3.3倍、5.1倍及2.2倍(P<0.01);干扰Lin28A表达的细胞模型中,碱性磷酸酶染色和茜素红染色相较对照组染色减少,成骨基因碱性磷酸酶、Runt相关转录因子2及骨桥蛋白的表达量分别下降19%,61%及10%(P<0.01);(3)成骨相关转录因子Runt相关转录因子2的蛋白表达水平与mRNA的差异趋势一致,过表达组呈上升趋势,干扰组呈下降趋势;(4)生物信息学预测出与Lin28A相关的let-7家族成员有8个,分别�BACKGROUND:The cellular activity of Lin28A can be influenced in several ways,including the self-renewal of embryonic stem cells,somatic cell reprogramming,individual growth and development,tissue metabolism,and carcinogenic consequences.However,neither domestically nor internationally have its effects on periodontal ligament stem cell ability to differentiate into osteoblasts been well explored;the underlying mechanisms are not known.OBJECTIVE:To investigate the impact of Lin28A on periodontal ligament stem cell capacity for osteogenic differentiation.METHODS:By using conventional enzyme digestion,periodontal ligament stem cells were extracted and cultured.The distribution of Lin28A in these cells and the differential expression of Lin28A at different time points of osteogenic induction were detected by qRT-PCR.Lentiviral vectors with Lin28A overexpression and interference expression were constructed and transfected into periodontal ligament stem cells.Fluorescence microscopy was used to observe the transfection effect.qRT-PCR was used to verify the expression difference of Lin28A after lentivirus transfection.The expression levels of osteoblastic genes alkaline phosphatase,Runt-related transcription factor 2 and osteopontin,alkaline phosphatase staining and alizarin red staining results were further detected from positive and negative aspects.The protein expression level of Runt-related transcription factor 2 was detected by western blot assay.Bioinformatics techniques and dual luciferase were used to predict and verify the relationship between Lin28A-related let-7 family members.The periodontal ligament stem cells differentially expressing Lin28A were cultured through osteoblastic induction,and the miRNA with the most obvious expression change during osteoblastic differentiation was determined.RESULTS AND CONCLUSION:(1)Lin28A was enriched in the nucleus of periodontal ligament stem cells,and the expression of Lin28A increased at different time points after osteogenic induction.(2)In the periodontal ligament ste

关 键 词：牙周膜干细胞 成骨分化 Lin28A 慢病毒载体 转染 基因干扰 let-7a 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R781.42