口腔基础医学进展重点全覆盖.doc

口腔基础医学进展重点niche: niche is the function unit of an organism in a particular habitathabitat: habitat is the site where micro-organisms growecological succession: the phenomenon that species or bacteria population shift with the change of environment overtime.climax community: the complex community can be gradually established and continues its homeostasis. A fully developed community is said to be a climax community.normal flora: It is one of the non-specific immunological factors for human. There are 3 barriers: the first is physical barrier; the second is associated with chemical secretions like saliva or tears; lastly the biological barrier includes the indigenous bacteria. The presence of indigenous bacteria inhibits the colonization of other pathogens.oral indigenous flora: buccal epithelial ecosystem; dorsum of the tongue ecosystem; supragingival plaque ecosystem; subgingival plaque ecosystem.plaque: it is the soft nonmineralized bacterial deposit, which adhere to the teeth and dental prostheses. Plaque is a network of bacteria that interconnected by bacteria and salivary polymers.The formation of dental plaque biofilm:1. Pellicle formation: The compositions of saliva can attach to the surface of tooth that lead to the formation of an acellular proteinaceous pellicle. The selective adherence is thought to be another important role of pellicle. The selective manner depends on the components of the bacteria surface and the receptors in the pellicle. 2. Microbial colonization: The initial colonizers in pellicle are called pioneer bacteria. As the pioneer bacteria grow rapidly and in a perpendicular orientation to the surface of the tooth, the environment of dental plaque has changed to allow colonization of other strains of bacteria.3. Mature dental plaque: As the dental plaque becomes thicker, lower oxygen concentration and reducing of redox potential are responsible for the growth of anaerobes. The structure of mature dental plaque can be divided into 3 layers: the inner layer is the pellicle; the intermediate layer is composed of variable bacteria; the outer layer is boned more loosely by gram-positive and gram-negative bacteria and intermingled with bacillus. Kochs postulates: 1. There must be enough numbers of pathogenic microbes. 2. A high level of antibody against the micro-organism can be detected. 3. Pathogenic microorganism should carry with related virulent factors. 4. The disease caused by microorganism can be reproduced in animal model. 5. Removing the microorganism can lead to the improvement of clinical symptoms. Quorum sensing: It is a system of stimulus and response correlated to population density. Many species of bacteria use quorum sensing to coordinate the gene expression. Bacteria achieve quorum sensing through releasing, sensing and accepting signal molecules from the surrounding environment. These signal molecules are called autoinducers. Bacteria can distinguish the flora density through the autoinducers and then control the expression of genes to regulate the number of bacteria within the colony. The quorum sensing is different with the different composition of bacteria.multistep process of oral cancer:The process of oral cancer can be separated into several stages: 1) normal condition but suffering tobacco, carcinogens or chronic infections; 2) 3p, 9p deletion and telomerase activation; 3) promoter methylation and 17p deletion; 4) P53 Notch1 mutation; 5) cancer. Multiple genetic and epigenetic alterations accumulate in oral epithelial cells, leading to malignant transformation and progression. Identifying and characterizing these alterations can accelerate the biomarker development to assess cancer risk and classify tumors. It can also provide molecules target for personalized prevention and treatment.microsatellite: Microsatellite are repeated short sequences of DNA, and have high variability on size from one individual to another individual. microsatellite instability: If there are defects in mismatch repair machinery, the length of microsatellite may be altered, either increase or decrease the numbers of the repeats, a feature called microsatellite instability, which may cause functional disease if these sequences locate at particular regions.EZH2: Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of Polycomb repression complex 2 (PRC2), a highly conserved histone methyltransferase. EZH2 is involved in methylation and silencing of a subset of genes involved in cell differentiation, suggesting that EZH2 may play an important role in cell differentiation and maintenance of adult stem cell populations.cell proliferation: An increase in the number of cells as a result of cell growth and cell division.cell apoptosis: Programed cell death, is a type of cell death in which a series of molecule steps lead to cell death. This is the bodys normal way of getting rid of the unneeded cells or abnormal cells.signal pathways in oral cancer: 1. Proliferative signal: RAS, PI3K 2. Apoptosis signal: extrinsic pathway: TNF, Fas/FasL; intrinsic pathway: Bcl-2; final pathway: Caspase 3. Negative feedback mechanism of proliferation: RAS, PTEN, mTOR 4. Regulation of apoptosis signaling: p53, NFB, PI3K 5.Signaling transduction of differentiation and dedifferentiation: Hedgehog/GIL, Wnt/catenin, Notch, TGFNFB signal pathway: NFB is a nuclear transcription factor that regulates expression of a large number of genes involved in the regulation of apoptosis, viral replication, tumorigenesis, inflammation and many autoimmune diseases. NFB is sequestered in the cytoplasm, bound with the inhibitor proteins of the IB family. NFB signal pathway can be separated into 3 parts: 1) The various stimuli that activate NFB cause the phosphorylation of IB, which is followed by its degradation; 2) This results in exposure of nuclear localization signals on NFB subunits and the subsequent translocation of the molecule to nucleus; 3) NFB binds with the consensus sequence of various genes and thus activates their transcription.Signal transduction of cell differentiation and dedifferentiation and their role in tumor development: 1. Hedgehog/GLI signal pathway: Epithelial-mesenchymal transitions (EMT) is characterized by the loss of epithelial markers and the gain of mesenchymal makers. This signal pathway controls cancer cell differentiation, cell proliferation, tumor migration and invasion, EMT, tumor angiogenesis and CSC maintenance. 2. Wnt/catenin signal pathway: 1) Wnt 3 mutations were associated with cleft lip with or without cleft palate; 2) mutations in the Wnt10A gene are associated with ectodermal dysplasia; 3) calcifying odontogenic cyst (COC) is associated with gain-of-function mutation of catenin; 4) Wnt pathway are overexpressed in adenoid cystic carcinoma (ACC); 5) Expression analyses of HNSCC and HNSCC-derived cell lines have found that most tumor and cell lines presented overexpress Wnt signal; 6) In leukoplakia, a precancerous lesion, Wnt3 and catenin are detected in individuals with lesions, but not in normal oral epithelium.3. Notch signal pathway: recent work in this pathway shows EMT promotion and enhancement of CSC, and interestingly, implicates Notch signaling in several cancers displaying resistance to conventional chemotherapy.4. TGF signal pathway: TGFpromotes EMT in late stages of tumorigenesis by a combination of Smad-dependent and Smad-independent effects on cell junction complexes. The majority of oral squamous cancer cells retain a full or partial response to the anti-proliferation effects of TGF.checkpoints: Checkpoints are regulatory pathways that ensure that the biochemically independent processes of the cell cycle are coupled.分子分型的定义和意义：疾病的分子分型（molecular classification）是通过综合的分子分析技术为疾病分类提供更多的信息，从而使疾病分类的基础从宏观形态学转向以分子特征为基础的新的分类体系（molecular characteristics-based classification）。目前，可以在DNA、RNA和蛋白质水平上进行疾病分子分型研究。根据基因表达谱（mRNA）水平差异进行分型是目前分子分型研究的主要方面。意义：在传统肿瘤分型、分级和分期的基础上，开展以肿瘤特异性分子靶标检测为核心的分子分类诊断（遗传学变化、表观学变化）是精确、客观预测肿瘤预后、指导治疗方案的制定和疗效监测的前提和基础，对于肿瘤个体化治疗的实施具有重要意义。1.肿瘤的早期预测2.转移复发的风险进行判断3.预测放化疗的敏感性，耐药性甚至不良反应4.最终判断病人的预后Dynamics between cancer and immune system: Cancer immunoediting process is composed of 3 phases termed “elimination”, “equilibrium” and “escape”: 1) In the elimination phase, innate and adaptive immune systems are able to destroy cancer cells; this has yet to be demonstrated definitively. 2) In the equilibrium phase, the host immune system and any tumor cell variant that have survived the elimination phase enter into a dynamic equilibrium, wherein lymphocytes, perforin and IFN exert potent and relentless selection pressure on the tumor cells that is enough to contain, but not fully extinguish. 3) In the escape phase, tumor cell variants selected in the equilibrium phase now can grow in an immunologically intact environment. This hosts immune defenses most likely occurs when genetic and epigenetic changes in the tumor cell confer resistance to immune detection and/or detection, allowing the tumors to expand and become clinically detectable.Role of checkpoint inhibitor in cancer immunotherapy:Immune checkpoint can control the balance between co-stimulatory signals and co-inhibitory signals. These two kinds of signals have presented important roles in maintaining self-tolerance and regulating T cell response. There are two kinds of main checkpoints, CTLA4 and PD-1, which are negative regulatory factors for the stimulation of T cells. CTLA4 and PD-1 could induce the down-regulation of immune system by inhibiting the activity of T cells. The overexpression of these molecular would inhibit the immune response to cancer cells. Checkpoint inhibitors could block this pathway to make cancer cells attacked. This is the mechanism of immune checkpoint therapy.similarities and differences between miRNA and siRNA:1. miRNAs are single-stranded RNA, while siRNAs are double-stranded RNA.2. miRNAs are not perfectly complimentary to their targets.3. miRNAs regulate their targets by 2 means: mRNA degradation and inbition of translation initiation, while siRNA focus on gene silence and target cleavage.4. miRNAs are encoded by the host genome, whereas siRNAs in most cases originate from outer source. What is non-coding RNA and its category: non-coding RNA (ncRNA) is functional RNA molecule which is not translated into a protein. According to different functions, it can be divided into 2 categories: 1) Regulatory ncRNAs: miRNA, lncRNA and piRNA. This kind of ncRNAs are associated with transcription, translation, regulation of protein function and regulation of RNA and protein distribution. 2) House keeping ncRNA: tRNA, rRNA, snRNA, snoRNA and telomerase RNA. This kind of RNA can directly or indirectly participate in the editing of coding protein.免疫印迹（WB）的原理及适用范围：原理：应用蛋白质特异的单克隆抗体或多克隆抗体检测特异性的目的蛋白，通过对未知蛋白进行SDS聚丙烯酰胺凝胶电泳（SDS-PAGE），在电场作用下，根据相应的分子量分离未知蛋白，再转膜至固相支持物，而后用特异性的抗体对目的蛋白进行检测的一种实验技术。适用范围及条件：1.检测细胞或组织内的目的蛋白的表达；2.检测细菌、真菌、血清等目的蛋白的表达；3.非原位检测，不能对目的蛋白进行细胞定位；4.必须有目的蛋白的特异性抗体才能进行；5.属于半定量检测，需以内参蛋白，如-肌动蛋白，作为对照WB结果出现高背景及杂带，如何优化试验：原因及优化：1.对于多克隆抗体有可能出现，选用特异性更强的单克隆抗体，避免交叉反应；2.对于发光试剂盒过于敏感，选用敏感度稍低的试剂盒；3.对于封闭不够，根据室温，适当延长封闭时间，同时防止过度洗脱；4.对于抗体稀释比例过高及抗体过浓，根据样品特性选择合理的抗体浓度；5.对于曝光时间过长，降低曝光时间；6.对于干膜现象的出现，要保证实验过程中充分的反应液WB结果未见到条带，原因：实验操作：1.样品制备是否有问题，上样量是否足够；2.电泳是否得当，电转是否完全；试剂问题：1.电转液配制是否有问题；2.抗体是否适合用于做免疫印记，稀释比例是否得当；3.二抗种属的选择是否混淆；4.发光试剂盒敏感度是否比较低或者已经过期；目的蛋白特性：1.目的蛋白在组织中表达的丰度是否可以检测；2.分子量过大或过小的蛋白是否优化了条件；3.目的蛋白是否是特殊蛋白，如膜蛋白及极少数不易溶解的蛋白Fluorescent substance：荧光物质，某些物质在特定波长范围内的光线照射下，可发出波长比照射光长的光线荧光。受激发后能产生荧光的物质称荧光物质或荧光素。Excitation：激发光，能特异性地激发某种荧光素的一定波长范围内的光线。Emission：发射光，在激发光的作用下，荧光物质可以特征性的发射另一波长的光，此为发射光。Fluorescence probe：荧光探针，能产生荧光的特异性生物染料、标有荧光素的特异性蛋白结合物。Autofluorescence：自发荧光，组织或细胞中的某些成分受激发时可发出荧光。Bleach：漂白，荧光物质受激发时，其发射荧光的能力逐渐下降，并最终丧失。激光共聚焦显微镜的优势：结构方面：1.激光做光源，光色纯，波长固定，成像效果好，分辨率高，图象清晰，荧光检测信噪比高；2.可实现分层扫描；3.可实现连续扫描，可动态记录变化；4.可多根激光管同时扫描，多色荧光同时成像；5.扫描速度快，对样品损伤小功能方面：形态观察-高清晰成像；半定量-荧光强度分析，荧光探针表达量测定；定位研究-分层扫描；多种荧光标记同时检测；荧光标记物绝对定量分析；扩展应用-如细胞切割、细胞筛选等。效果方面：1.更灵敏：共聚焦显微镜采用了光电倍增技术，可将很微弱的荧光信号放大。只要有信号就能被检测到。2.定位更精确：不仅可以定位到细胞水平，还可以定位到亚细胞水平、和分子水平。这也是荧光标记的抗体被称为分子探针的原因。定量测试：1.静态的定量测量：对于不同组的样品，可以单纯比较一种成分，也可以同时比较两种甚至三种成分。也可以在同一张切片上比较不同成分含量。2.动态的定量测量：共聚焦显微镜还能对活细胞内的特定成分进行动态变化测量，并给出动态变化曲线；还可同时测量几种成分，并给出含量比值。快速性：由于利用光源光束点扫描，检测过程快，时间短，计算机精确控制激发光强度，光漂白和荧光淬灭作用很小。同时，数据图像可及时输出或长期储存。共聚焦显微镜与普通光学显微镜的主要区别：1.普通显微镜简单、快捷，所看到的图像是样本的全部结构。2.共聚焦显微镜看到的只是样本荧光的信号，而且仅仅是某一层面的结构。即普通显微镜是各层平面叠加的效果，共聚焦是一种断层扫描的效果3.普通光学显微镜，成像部分发生重叠，从而造成图像对比度的降低。而共聚焦显微镜却可以获得对比度非常高的图像，也提高了显微镜的分辨能力。4.共聚焦显微镜高度方向测定以及焦点深度扩张的原理，使其图像景深很长富有立体感。5.共聚焦显微镜可实现实时观测。 6.共聚焦显微镜可实现高精度微小尺寸，并测定机能。 7.共聚焦显微镜可获得多维图像，如立体三维的扫描成像。Flow Cytometry，FCM：流式细胞术，是一种利用流式细胞仪对生物细胞的理化特性和生物学特性进行多参数定量分析，和对特定细胞群体分选的技术。流式细胞仪：集激光技术、