MiR-146a-5p促进APTG-CM诱导的PDLSCs向成骨细胞分化的功能研究

发布时间：2018-05-20 18:07

本文选题：牙周膜干细胞 + 根端牙胚细胞　；参考：《福建医科大学》2015年硕士论文

【摘要】：正畸治疗过程中,常常发生患者口腔卫生情况不佳导致的牙槽骨吸收,牙槽骨作为牙周组织,借助牙周膜纤维与牙齿紧密相连,在维持牙齿的稳定和行使正常生理功能上发挥着重要作用。在临床上发现,由于牙周疾病或者正畸力量过大,往往导致牙槽骨吸收。人牙周韧带中存在有牙周膜干细胞(periodontal ligament stem cells,PDLSCs),其具有自我增殖和多向分化潜能,可作为组织工程最直接可靠的种子细胞。研究表明,h PDLSCs在不同的培养环境和细胞因子的作用下,可不断增殖分化成为成骨细胞、成牙骨质细胞和成纤维细胞;其可修复牙周缺损,使牙周组织再生。Micro RNA在间充质干细胞向成骨细胞分化过程中起着重要作用。h PDLSCs属于间充质干细胞,具有多向分化潜能,然而在h PDLSCs向成骨细胞方向分化过程中,hsa-mi R-146a是否起到了重要的调控作用尚未得知。目的为了研究在特定体外微环境中人牙周膜干细胞向成骨细胞方向分化过程中mi R46a-5p是否发挥调控作用,从而为牙槽骨的再生及发育机制提供基础性研究。实验方法本实验首先构建慢病毒特异性上/下调mi R146a-5p,进而通过由根端牙胚细胞(Apical Tooth germ cells,APTGC)构成的体外微环境诱导h PDLSCs向成骨细胞方向分化,对诱导前后的h PDLSCs进行碱性磷酸酶(Alkaline Phosphatase,ALP)活性检测以及成骨相关基因:(ALP、BSP及OCN)检测。1.h PDLSCs的体外培养和筛选;2.APTG诱导培养基的制备;3.RT-QPCR验证mi R146a-5p表达差异;4.慢病毒转染h PDLSCs;5.ALP活性检测;6.Real-time PCR检测特异性上/下调mi R146a-5p后成骨相关基因:(ALP、BSP及OCN)的变化。实验结果1.通过免疫磁珠法筛选获得h PDLSCs。2.RT-QPCR检测验证mi R146a-5p在h PDLSCs向成骨细胞方向分化过程中表达增高。3.慢病毒特异性转染效率检测显示LV-has-mi R146a-5p及LV-has-mi R146a-5pinhibitor导入人牙周膜干细胞后其mi R146a-5p特异性表达为上调及下调。4.ALP活性检测显示,LV-has-mi R146a-5p上调组细胞ALP活性较对照组有显著升高,LV-has-mi R146a-5pinhibitor组细胞ALP活性较对照组有显著降低。5.RT-PCR检测显示LV-has-mi R146a-5p上调组细胞成骨细胞相关基因(ALP、BSP及OCN)的转录水平表达量增加,LV-has-mi R146a-5pinhibitor组细胞成骨细胞相关基因(ALP、BSP及OCN)的转录水平表达量降低。结论mi R-146a-5p促进人牙周膜干细胞向成骨细胞分化。
[Abstract]:During orthodontic treatment, alveolar bone resorption often occurs due to poor dental hygiene. Alveolar bone, as a periodontal tissue, is closely connected with teeth by periodontal ligament fibers. It plays an important role in maintaining dental stability and exercising normal physiological function. Clinically, alveolar bone resorption is often due to periodontal disease or excessive orthodontic force. There are periodontal ligament stem cells (PDLSCs) in human periodontal ligament, which have the potential of self-proliferation and multi-differentiation, and can be used as the most direct and reliable seed cells in tissue engineering. The results showed that PDLSCs could proliferate and differentiate into osteoblasts, cementoblasts and fibroblasts under different culture conditions and cytokines, and could repair periodontal defects. Making periodontal tissue regenerate. Micro RNA play an important role in the differentiation of mesenchymal stem cells into osteoblasts. H PDLSCs belongs to mesenchymal stem cells and has the potential of multiple differentiation. However, whether hsa-mi R-146a plays an important regulatory role in the differentiation of h PDLSCs into osteoblasts is not known. Objective to investigate whether mi R46a-5p plays a regulatory role in the differentiation of human periodontal ligament stem cells into osteoblasts in a specific microenvironment in vitro, so as to provide basic research for the regeneration and development of alveolar bone. Methods Lentivirus specific up-/ down-regulated mi R146a-5p was constructed firstly, and then h PDLSCs was induced to differentiate into osteoblast by microenvironment composed of apical Tooth germ cells (Apical Tooth germ cells). The alkaline phosphatase (ALP) activity of h PDLSCs before and after induction and the osteoblast-associated genes (BSP and OC) were detected by alkaline phosphatase and osteoblasts. 1. The culture and screening of 2. APTG induced culture medium were carried out. 3. RT-QPCR was used to verify the difference of mi R146a-5p expression. The activity of ALP in lentivirus transfected hPDLSCs5. Real-time PCR was used to detect the changes of osteoblast-associated genes (BSP and OCNs) after specific up-/ down-regulation of mi R146a-5p. Experimental results 1. H PDLSCs.2.RT-QPCR detection was performed by immunomagnetic bead assay to verify the increased expression of mi R146a-5p during the differentiation of h PDLSCs into osteoblasts. Detection of lentivirus-specific transfection efficiency showed that the specific expression of mi R146a-5p was up-regulated and down-regulated after the introduction of LV-has-mi R146a-5p and LV-has-mi R146a-5pinhibitor into human periodontal ligament stem cells. 4. Detection of ALP activity showed that the ALP activity of LV-has-mi R146a-5p upregulated group was significantly higher than that of control group. The activity of ALP in R146a-5pinhibitor group was significantly lower than that in control group. 5. RT-PCR assay showed that LV-has-mi R146a-5p up-regulated the expression of osteoblast-associated genes (ALPG-BSP and OCN-C) in LV-has-mi R146a-5pinhibitor group, and increased the expression level of ALPH-BSP and OCN-related genes in LV-has-mi R146a-5pinhibitor group. Conclusion mi R-146a-5p can promote the differentiation of human periodontal ligament stem cells into osteoblasts.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R783.5

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