ODN对牙龈卟啉单胞菌侵入成骨细胞增殖活性的影响

发布时间：2018-09-11 14:11

【摘要】：慢性牙周炎是指发生在牙周支持组织上的炎症性疾病。牙菌斑生物膜为其发病的始动因子，随着致病菌数量的增加，细菌及其毒性产物可直接侵入并破坏牙周组织，诱发初期的炎症反应，然而，目前更多的资料表明，宿主对病原菌不恰当的免疫反应是造成牙周组织破坏的主要原因。牙龈卟啉单胞菌(Porphyromonas gingivalis, P.gingivalis)作为重要的牙周致病菌之一，，其多种毒力因子可通过直接作用或间接引发宿主免疫炎症反应造成牙周组织的破坏。脱氧寡核苷酸(oligodeoxynucleotide, ODN)是一类由数十个核苷酸单体连接而成的短链DNA分子的总称。ODN具有免疫调节功能，可抑制由病原体感染所引起的过度免疫反应，从而维持机体的免疫平衡状态。ODN易于合成并可进行修饰，具有结构、性质稳定，高效低毒，无需构建载体便可自行进入细胞发挥作用等生物学特性。研究已证实ODN是一个安全、可靠的制剂，已广泛用于临床研究并展现出良好的应用前景。慢性牙周炎的总体治疗目标不仅要控制细菌感染、消除炎症，而且要抑制由细菌及其代谢产物所引发的宿主免疫炎症反应产生的牙周组织破坏，从而促进牙周组织不同程度的修复和再生，恢复牙周组织的生理外形和功能。其中成骨细胞的增殖在牙槽骨的再生修复中发挥着重要作用。由于ODN具有种属特异性和个体差异性，因此本研究对不同序列的ODN进行筛选，筛选出对P.gingivalis侵入成骨细胞增殖活性具有影响作用的ODN，为应用ODN调控牙周组织免疫炎症反应，促进牙周组织的修复再生奠定实验基础。方法：实验所选用的ODN均由吉林大学基础医学院分子生物学教研室设计、大连TaKaRa公司合成。选择P.gingivalis模式株ATCC33277作为实验菌株,常规进行厌氧培养。选择人成骨样细胞系MG63细胞作为实验细胞，并建立P.gingivalis内化MG63细胞的模型，采用MTT法检测ODN对P.gingivalis感染MG63细胞2h、4h、8h和12h增殖活性的影响，筛选出具有影响作用的ODN，进一步采用MTT比色法检测其对P.gingivalis内化MG63细胞2h、4h、24h和48h增殖活性的影响。结果：与PBS对照组相比，ODN BW001、FC003、FC004、SAT05f和MT01对P.gingivalis感染MG63细胞2h、4h、8h和12h均具有促增殖作用，结果具有统计学意义（P0.05，P0.01）；与PBS对照组相比，ODN FC003对P.gingivalis内化的MG63细胞有促增殖作用，结果具有统计学意义（P0.05）。结论：ODN BW001、FC003、FC004、SAT05f和MT01能促进P.gingivalis感染的MG63细胞增殖；ODN FC003能促进P.gingivalis内化的MG63细胞增殖。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4

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