【摘要】：慢性牙周炎是指發(fā)生在牙周支持組織上的炎癥性疾病。牙菌斑生物膜為其發(fā)病的始動因子，隨著致病菌數(shù)量的增加，細菌及其毒性產(chǎn)物可直接侵入并破壞牙周組織，誘發(fā)初期的炎癥反應，然而，目前更多的資料表明，宿主對病原菌不恰當?shù)拿庖叻磻窃斐裳乐芙M織破壞的主要原因。牙齦卟啉單胞菌(Porphyromonas gingivalis, P.gingivalis)作為重要的牙周致病菌之一，，其多種毒力因子可通過直接作用或間接引發(fā)宿主免疫炎癥反應造成牙周組織的破壞。 脫氧寡核苷酸(oligodeoxynucleotide, ODN)是一類由數(shù)十個核苷酸單體連接而成的短鏈DNA分子的總稱。ODN具有免疫調(diào)節(jié)功能，可抑制由病原體感染所引起的過度免疫反應，從而維持機體的免疫平衡狀態(tài)。ODN易于合成并可進行修飾，具有結(jié)構(gòu)、性質(zhì)穩(wěn)定，高效低毒，無需構(gòu)建載體便可自行進入細胞發(fā)揮作用等生物學特性。研究已證實ODN是一個安全、可靠的制劑，已廣泛用于臨床研究并展現(xiàn)出良好的應用前景。 慢性牙周炎的總體治療目標不僅要控制細菌感染、消除炎癥，而且要抑制由細菌及其代謝產(chǎn)物所引發(fā)的宿主免疫炎癥反應產(chǎn)生的牙周組織破壞，從而促進牙周組織不同程度的修復和再生，恢復牙周組織的生理外形和功能。其中成骨細胞的增殖在牙槽骨的再生修復中發(fā)揮著重要作用。由于ODN具有種屬特異性和個體差異性，因此本研究對不同序列的ODN進行篩選，篩選出對P.gingivalis侵入成骨細胞增殖活性具有影響作用的ODN，為應用ODN調(diào)控牙周組織免疫炎癥反應，促進牙周組織的修復再生奠定實驗基礎。 方法：實驗所選用的ODN均由吉林大學基礎醫(yī)學院分子生物學教研室設計、大連TaKaRa公司合成。選擇P.gingivalis模式株ATCC33277作為實驗菌株,常規(guī)進行厭氧培養(yǎng)。選擇人成骨樣細胞系MG63細胞作為實驗細胞，并建立P.gingivalis內(nèi)化MG63細胞的模型，采用MTT法檢測ODN對P.gingivalis感染MG63細胞2h、4h、8h和12h增殖活性的影響，篩選出具有影響作用的ODN，進一步采用MTT比色法檢測其對P.gingivalis內(nèi)化MG63細胞2h、4h、24h和48h增殖活性的影響。 結(jié)果：與PBS對照組相比，ODN BW001、FC003、FC004、SAT05f和MT01對P.gingivalis感染MG63細胞2h、4h、8h和12h均具有促增殖作用，結(jié)果具有統(tǒng)計學意義（P0.05，P0.01）；與PBS對照組相比，ODN FC003對P.gingivalis內(nèi)化的MG63細胞有促增殖作用，結(jié)果具有統(tǒng)計學意義（P0.05）。 結(jié)論：ODN BW001、FC003、FC004、SAT05f和MT01能促進P.gingivalis感染的MG63細胞增殖；ODN FC003能促進P.gingivalis內(nèi)化的MG63細胞增殖。
[Abstract]:Chronic periodontitis is an inflammatory disease that occurs in periodontal supporting tissue. Dental plaque biofilm is the initiator of the disease. With the increase of the number of pathogenic bacteria, bacteria and its toxic products can directly invade and destroy periodontal tissue and induce the initial inflammatory reaction. However, more data show that, The host's improper immune response to pathogenic bacteria is the main cause of periodontal tissue damage. Porphyromonas gingivalis (Porphyromonas gingivalis, P.gingivalis) as one of the most important periodontal pathogens, its virulence factors can cause periodontal tissue damage by direct or indirect host immune inflammation. Deoxyoligodeoxynucleotide (oligodeoxynucleotide, ODN) is a class of short-chain DNA molecules linked by dozens of nucleotide monomers. ODN has immunomodulatory function and can inhibit the excessive immune response caused by pathogen infection. In order to maintain the immune balance of organism. ODN is easy to synthesize and can be modified, has the biological characteristics of structure, stability, high efficiency and low toxicity, no need to construct the vector to enter the cell to play a role in the biological characteristics. It has been proved that ODN is a safe and reliable preparation, which has been widely used in clinical research and has shown a good application prospect. The overall goal of treatment for chronic periodontitis is not only to control bacterial infection and eliminate inflammation, but also to suppress periodontal tissue damage caused by host immune inflammation caused by bacteria and their metabolites. It can promote the restoration and regeneration of periodontal tissue, and restore the physiological shape and function of periodontal tissue. The proliferation of osteoblasts plays an important role in alveolar bone regeneration and repair. Because of the species-specific and individual differences of ODN, different sequences of ODN were screened in this study, and ODN, which had an effect on the proliferation of P.gingivalis invading osteoblasts, was selected to regulate the periodontal tissue immune inflammation by ODN. To promote the restoration and regeneration of periodontal tissue lay the experimental foundation. Methods: the selected ODN was designed by the Department of Molecular Biology, College of basic Medicine, Jilin University, and synthesized by Dalian TaKaRa Company. The P.gingivalis model strain ATCC33277 was selected as the experimental strain for anaerobic culture. Human osteoblast-like cell line MG63 cells were selected as experimental cells and P.gingivalis internalized MG63 cells were established. The effects of ODN on the proliferation of MG63 cells infected with P.gingivalis for 2 h, 4 h and 12 h were detected by MTT assay. The effect of ODN, on the proliferation of P.gingivalis internalized MG63 cells was further determined by MTT colorimetric assay for 24 h and 48 h respectively. Results: compared with PBS control group, ODN BW001,FC003,FC004,SAT05f and MT01 could promote the proliferation of MG63 cells infected with P.gingivalis for 2 h, 4 h and 12 h, the results were statistically significant (P0.05 + P0.01), and compared with PBS control group, ODN FC003 could promote the proliferation of MG63 cells internalized by P.gingivalis. The results were statistically significant (P0.05). Conclusion BW001,FC003,FC004,SAT05f and MT01 can promote the proliferation of MG63 cells infected with P.gingivalis. ODN FC003 can promote the proliferation of MG63 cells internalized by P.gingivalis.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R781.4

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