miR-34a对健康及感染状态下MG63细胞成骨分化的影响
发布时间：2019-04-13 14:20
【摘要】：牙周炎是由牙菌斑生物膜引起的牙周组织感染性疾病,以牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)等为代表的牙周致病菌可以分泌多种毒力因子,激发宿主固有免疫应答,引发牙槽骨吸收,进而导致牙周组织破坏。研究显示约47%的美国人患有不同程度的牙周炎,在65岁以上的成年人中,发病率更是高达64%。牙周炎导致的牙槽骨吸收现已成为成人失牙的首要原因,严重影响患者咀嚼、发音,并影响患者生存质量。研究牙周炎所致的牙槽骨吸收机制,抑制炎性牙槽骨吸收并促进骨再生具有重要意义。近期研究发现,微小分子RNA(microRNAs,miRNAs)可能是牙周炎所致牙槽骨吸收的生物学标记及治疗靶点。研究者认为一些miRNAs可以通过调控破骨细胞及成骨细胞功能,影响牙周炎导致的牙槽骨吸收进程。在这些miRNAs中,miR-34a备受关注。miR-34a是一种具有调控功能的内源性非编码RNA,其编码基因位于染色体1p36区域。近年来一些研究发现其可以影响包括炎症反应及骨改建在内的多种病理生理进程。实验证实miR-34a可以抑制LPS诱导的巨噬细胞炎症应答,抑制破骨细胞的形成从而抑制骨质疏松,并且可以促进骨髓间充质干细胞及脂肪干细胞成骨分化。鉴于miR-34a可以促进成骨分化,抑制LPS诱导的炎症应答及破骨细胞功能,我们推测miR-34a可能具有促进骨形成、抑制炎性牙槽骨吸收的作用。因此我们设计本实验检测miR-34a对健康及炎症状态下成骨样细胞成骨分化能力的影响,为进一步探讨miR-34a能否抑制牙周炎所致的牙槽骨吸收奠定实验基础。方法:1选取人成骨样细胞MG63为实验细胞,使用Lipofectamine2000将miR-34a拟态物转染至细胞。2荧光显微镜及实时定量PCR法检测miR-34a转染效率。3使Pg感染MG63细胞,实时定量PCR法检测细胞内miR-34a表达量变化。4实时定量PCR法检测转染miR-34a拟态物后健康或感染状态下MG63细胞中成骨相关因子Runx2、SP7及COLⅠ基因表达变化。结果:Lipofectamine2000可有效将miR-34a拟态物转染至MG63细胞内;miR-34a可以上调健康MG63细胞内Runx2、SP7及COLⅠ基因表达水平;感染状态下MG63细胞内miR-34a表达量降低;miR-34a下调感染状态下MG63细胞内Runx2、SP7及COLⅠ基因表达水平。结论:miR-34a促进健康状态下成骨细胞成骨分化,抑制感染状态下成骨细胞成骨分化。
[Abstract]:Periodontitis is an infectious disease of periodontal tissue caused by dental plaque biofilm. Periodontal pathogenic bacteria, such as Porphyromonas gingivalis (Porphyromonas gingivalis,Pg), can secrete many virulence factors to stimulate host innate immune response. The alveolar bone resorption is induced, which leads to the destruction of periodontal tissue. The study found that about 47 percent of Americans suffer from periodontitis of varying degrees, and that 64 percent of adults over 65 suffer from periodontitis. Alveolar bone absorption caused by periodontitis has become the primary cause of adult tooth loss, which seriously affects the mastication, pronunciation and quality of life of the patients. It is of great significance to study the mechanism of alveolar bone resorption induced by periodontitis, inhibit the inflammatory alveolar bone resorption and promote bone regeneration. Recent studies have found that RNA (microRNAs,miRNAs) may be a biological marker and therapeutic target of alveolar bone resorption induced by periodontitis. The researchers believe that some miRNAs may affect alveolar bone resorption by regulating osteoclast and osteoblast function. Among these miRNAs, miR-34a has attracted much attention. MiR-34a is an endogenous non-coding RNA, with regulatory function, and its coding gene is located in the 1p36 region of chromosome. In recent years, some studies have found that it can affect a variety of pathophysiological processes, including inflammatory reaction and bone remodeling. It was proved that miR-34a could inhibit the inflammatory response of macrophages induced by LPS, inhibit the formation of osteoclasts and inhibit osteoporosis, and promote the osteogenic differentiation of bone marrow mesenchymal stem cells and adipose-derived stem cells. Since miR-34a can promote osteogenic differentiation, inhibit LPS-induced inflammatory response and osteoclast function, we speculate that miR-34a may promote bone formation and inhibit inflammatory alveolar bone resorption. Therefore, we designed this experiment to detect the effect of miR-34a on osteoblast-like osteoblast differentiation in healthy and inflammatory condition, which laid the experimental foundation for further exploring whether miR-34a can inhibit alveolar bone resorption induced by periodontitis. Methods: 1 Human osteoblast-like cells (MG63) were selected as experimental cells, and miR-34a mimicry was transfected into cells by Lipofectamine2000. 2 fluorescent microscope and real-time quantitative PCR were used to detect the transfection efficiency of miR-34a. 3. Pg was used to infect MG63 cells. The expression of miR-34a in MG63 cells was detected by real-time quantitative PCR. (4) the expression of osteogenic factors Runx2,SP7 and COL 鈪，
本文编号：2457648
[Abstract]:Periodontitis is an infectious disease of periodontal tissue caused by dental plaque biofilm. Periodontal pathogenic bacteria, such as Porphyromonas gingivalis (Porphyromonas gingivalis,Pg), can secrete many virulence factors to stimulate host innate immune response. The alveolar bone resorption is induced, which leads to the destruction of periodontal tissue. The study found that about 47 percent of Americans suffer from periodontitis of varying degrees, and that 64 percent of adults over 65 suffer from periodontitis. Alveolar bone absorption caused by periodontitis has become the primary cause of adult tooth loss, which seriously affects the mastication, pronunciation and quality of life of the patients. It is of great significance to study the mechanism of alveolar bone resorption induced by periodontitis, inhibit the inflammatory alveolar bone resorption and promote bone regeneration. Recent studies have found that RNA (microRNAs,miRNAs) may be a biological marker and therapeutic target of alveolar bone resorption induced by periodontitis. The researchers believe that some miRNAs may affect alveolar bone resorption by regulating osteoclast and osteoblast function. Among these miRNAs, miR-34a has attracted much attention. MiR-34a is an endogenous non-coding RNA, with regulatory function, and its coding gene is located in the 1p36 region of chromosome. In recent years, some studies have found that it can affect a variety of pathophysiological processes, including inflammatory reaction and bone remodeling. It was proved that miR-34a could inhibit the inflammatory response of macrophages induced by LPS, inhibit the formation of osteoclasts and inhibit osteoporosis, and promote the osteogenic differentiation of bone marrow mesenchymal stem cells and adipose-derived stem cells. Since miR-34a can promote osteogenic differentiation, inhibit LPS-induced inflammatory response and osteoclast function, we speculate that miR-34a may promote bone formation and inhibit inflammatory alveolar bone resorption. Therefore, we designed this experiment to detect the effect of miR-34a on osteoblast-like osteoblast differentiation in healthy and inflammatory condition, which laid the experimental foundation for further exploring whether miR-34a can inhibit alveolar bone resorption induced by periodontitis. Methods: 1 Human osteoblast-like cells (MG63) were selected as experimental cells, and miR-34a mimicry was transfected into cells by Lipofectamine2000. 2 fluorescent microscope and real-time quantitative PCR were used to detect the transfection efficiency of miR-34a. 3. Pg was used to infect MG63 cells. The expression of miR-34a in MG63 cells was detected by real-time quantitative PCR. (4) the expression of osteogenic factors Runx2,SP7 and COL 鈪，
本文编号：2457648
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