【摘要】：牙周炎是由牙菌斑生物膜引起的牙周組織感染性疾病,以牙齦卟啉單胞菌(Porphyromonas gingivalis,Pg)等為代表的牙周致病菌可以分泌多種毒力因子,激發(fā)宿主固有免疫應(yīng)答,引發(fā)牙槽骨吸收,進而導(dǎo)致牙周組織破壞。研究顯示約47%的美國人患有不同程度的牙周炎,在65歲以上的成年人中,發(fā)病率更是高達64%。牙周炎導(dǎo)致的牙槽骨吸收現(xiàn)已成為成人失牙的首要原因,嚴重影響患者咀嚼、發(fā)音,并影響患者生存質(zhì)量。研究牙周炎所致的牙槽骨吸收機制,抑制炎性牙槽骨吸收并促進骨再生具有重要意義。近期研究發(fā)現(xiàn),微小分子RNA(microRNAs,miRNAs)可能是牙周炎所致牙槽骨吸收的生物學(xué)標記及治療靶點。研究者認為一些miRNAs可以通過調(diào)控破骨細胞及成骨細胞功能,影響牙周炎導(dǎo)致的牙槽骨吸收進程。在這些miRNAs中,miR-34a備受關(guān)注。miR-34a是一種具有調(diào)控功能的內(nèi)源性非編碼RNA,其編碼基因位于染色體1p36區(qū)域。近年來一些研究發(fā)現(xiàn)其可以影響包括炎癥反應(yīng)及骨改建在內(nèi)的多種病理生理進程。實驗證實miR-34a可以抑制LPS誘導(dǎo)的巨噬細胞炎癥應(yīng)答,抑制破骨細胞的形成從而抑制骨質(zhì)疏松,并且可以促進骨髓間充質(zhì)干細胞及脂肪干細胞成骨分化。鑒于miR-34a可以促進成骨分化,抑制LPS誘導(dǎo)的炎癥應(yīng)答及破骨細胞功能,我們推測miR-34a可能具有促進骨形成、抑制炎性牙槽骨吸收的作用。因此我們設(shè)計本實驗檢測miR-34a對健康及炎癥狀態(tài)下成骨樣細胞成骨分化能力的影響,為進一步探討miR-34a能否抑制牙周炎所致的牙槽骨吸收奠定實驗基礎(chǔ)。方法:1選取人成骨樣細胞MG63為實驗細胞,使用Lipofectamine2000將miR-34a擬態(tài)物轉(zhuǎn)染至細胞。2熒光顯微鏡及實時定量PCR法檢測miR-34a轉(zhuǎn)染效率。3使Pg感染MG63細胞,實時定量PCR法檢測細胞內(nèi)miR-34a表達量變化。4實時定量PCR法檢測轉(zhuǎn)染miR-34a擬態(tài)物后健康或感染狀態(tài)下MG63細胞中成骨相關(guān)因子Runx2、SP7及COLⅠ基因表達變化。結(jié)果:Lipofectamine2000可有效將miR-34a擬態(tài)物轉(zhuǎn)染至MG63細胞內(nèi);miR-34a可以上調(diào)健康MG63細胞內(nèi)Runx2、SP7及COLⅠ基因表達水平;感染狀態(tài)下MG63細胞內(nèi)miR-34a表達量降低;miR-34a下調(diào)感染狀態(tài)下MG63細胞內(nèi)Runx2、SP7及COLⅠ基因表達水平。結(jié)論:miR-34a促進健康狀態(tài)下成骨細胞成骨分化,抑制感染狀態(tài)下成骨細胞成骨分化。
[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，

本文編號：2457648