敲除牙龈卟啉单胞菌菌毛次要组分FimCDE的载体构建

目的：构建重组质粒pPHU281-C-Spec-E,用于敲除菌毛蛋白次要组分基因fimCDE。构建fimCDE缺陷的牙龈卟啉单胞菌,为进一步研究FimCDE在感染中的作用奠定基础。方法：厌氧罐培养牙龈卟啉单胞菌菌株 ATCC 33277,提取基因组DNA后,PCR扩增获得含有人工设计的酶切位点的fimC基因的上游片段及fimE的下游片段,将其克隆到自杀质粒pPHU281内,命名为pPHU281-C-E;再将抗大观霉素的抗性基因插入到C及E片段之间,最终获得重组质粒pPHU281-C-Spec-E。结果：酶切及DNA序列分析验证重组质粒pPHU281-C-Spec-E构建成功。结论：成功构建了含有牙龈卟啉单胞菌菌毛蛋白次要组分基因fimC上游及fimE下游片段的重组质粒pPHU281-C-Spec-E,可用于构建菌毛蛋白次要组分FimCDE缺陷的的突变体。     

人骨形态发生蛋白-2基因克隆及真核表达载体的构建

目的克隆人骨形态发生蛋白- 2（hBMP2）基因片段,构建pcDNA3.1- hBMP2真核表达质粒。方法采用逆转录聚合酶链式反应（RT- PCR）技术,从人骨肉瘤中扩增出人骨形态发生蛋白- 2基因片段,通过DNA重组技术将该基因片段重组于pcDNA3.1真核表达载体上,构建pcDNA3.1- hBMP2重组质粒,通过用PCR扩增、酶切电泳分析及DNA测序的方法对重组DNA进行鉴定。结果经PCR扩增、酶切电泳分析和DNA测序证实,本实验构建的重组质粒目的基因片段为人BMP2- cDNA。结论本实验成功克隆了hBMP2基因并构建成其真核表达质粒。     

Design and preparation of cloned DNA fragment from pac gene of Streptococcus mutans

目的 :为克隆构建DNA防龋疫苗制备出表达片段。方法 :酶切电泳鉴定含pac基因的重组质粒 ;用计算机辅助选定pac基因中重要区域并设计引物 ,对其进行PCR扩增 ,酶切电泳鉴定扩增产物。结果 :pPC4 1重组质粒包含pac基因 ;自pac基因中选定的核苷酸序列为模板所扩增的DNA片段为预期目的片段。结论 :对含pac基因重要抗原决定簇区域的成功扩增为构建基因重组防龋疫苗完成了重要的准备工作。     

变形链球菌F-ATP酶基因重组质粒的构建和初步分析

目的：构建用于转化变形链球菌，含有F-ATP酶基因的重组质粒。方法：采用PCR方法，以变形链球菌基因组DNA为模板，扩增F-ATP酶β亚基5′末端序列，将克隆片段与载体pVA891酶切后连接，形成重组质粒，并对变形链球菌的转化作了初步分析。结果：构建的重组质粒经PCR鉴定、酶切鉴定和DNA序列测定，显示插入的目的片段序列正确。结论：变形链球菌目的片段与穿梭载体重组后能有效克隆，为通过同源重组特异突变变形链球菌染色体基因奠定基础。     

真核表达质粒pBABE-hygro-PLAP-1的构建及鉴定

目的:构建并鉴定含有plap-1基因和已知FLAG序列的真核细胞表达载体pBABE-hygro-PLAP-1.方法:采用PCR扩增PLAP-1的目的片段,然后连接到已知FLAG序列的载体p3×FLAG-CMV-10上:再次通过PCR扩增出PLAP-1的目的片段及已知序列的FLAG,最后将其插入真核细胞表达载体pBABE-hygro的多克隆位点上.对该重组体进行酶切鉴定和测序验证.结果:通过对重组质粒进行酶切鉴定以及DNA序列测定分析.证明真核表达载体pBABE-hygro-PLAP-1构建成功.结论:成功构建php-1基因的真核表达载体,为进一步研究plap-1基因的功能奠定了实验基础.     

基因重组鼠伤寒沙门氏菌防龋疫苗的研究Ⅱ.携带变形链球菌pac基因表达质粒的构建与初步鉴定

目的：构建携带变形链球菌表面蛋白pac基因A区片段的原核高效表达系统。方法：对含变形链球菌表面蛋白pca基因的pPC41质粒进行PCR扩增,获得了pac基因A区片段,将扩增获得pac基因A区片段与表达质粒pET17-b定向克隆,重组质粒用B HI,EcoRV双酶切后电泳鉴定。结果：获得携带pac基因片段的重组持粒,结论：利用基因工程技术获得的重组质粒为后继工作准备了实验基础。     

牙龈卟啉单胞菌血凝素2基因缺陷型突变株的构建

目的 构建牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)血凝素2(hemagglutinin-adhesin 2,HA-2)基因缺陷型突变株,为研究HA-2基因功能奠定基础.方法 PCR扩增PgHA-2基因两翼片段HA_u、HA_1,将抗性基因ermF-ermAM连接到两者之间构建打靶载体HA-ermF-ermAM.将HA-ermF-ermAM电转化PgATCC33277,基因同源重组整合进入PgATCC33277染色体,用选择性培养基筛选获得PgATCC33277HA-2基因缺陷型突变株.进行PgATCC33277HA-2基因缺陷型突变株与其野生株凝血功能试验,比较二者的凝血功能差异.结果 PCR、酶切和测序鉴定结果表明,打靶载体HA-ermF-ermAM成功构建.经PCR鉴定,打靶载体HA-ermF-ermAM通过同源重组整合进入PgATCC33277染色体,成功获得PgATCC33277HA-2基因缺陷型突变株.凝血实验结果表明,PsATCC33277HA-2基因缺陷型突变株与野生株相比凝血能力明显减弱.结论 成功获得PgATCC33277HA-2基因缺陷型突变株,为进一步研究HA-2的生物学性质奠定了基础.     

含增强型绿色荧光蛋白rhBMP-2基因真核表达载体的构建

目的:构建含增强型绿色荧光蛋白的重组人骨形成蛋白-2(rhBMP-2)基因真核表达质粒。方法:采用PCR方法扩增rhBMP-2基因,并在两端添加合适的酶切位点;利用定向克隆技术将rhBMP-2基因和增强型绿色荧光蛋白(EGFP)基因分别插入到载体pIRES的上下游多克隆位点中,两者通过IRES连接,以防止融合表达;重组的pIRES-rhBMP2-EGFP在大肠杆菌DH5α内扩增后,通过酶切电泳鉴定和DNA测序证明质粒构建成功。结果:通过对重组质粒pIRES-rhBMP2-EGFP进行酶切鉴定以及DNA序列测定分析,证明真核表达重组质粒pIRES-rhBMP2-EGFP构建成功,开放阅读框架正确。结论:利用PCR、T/A克隆等分子生物学技术,可成功地将编码rhBMP-2的DNA片段和EGFP片段克隆到载体pIRES中,构建出重组子pIRES-rhBMP2-EGFP。     

变形链球菌gcp基因突变菌株的构建及鉴定

目的:构建变形链球菌gcp基因突变菌株,以便研究变形链球菌gcp基因功能。方法:厌氧培养变形链球菌UA159,以前期研究中pMD19T-gcp为模板,PCR扩增gcp基因内部序列,连接自杀载体pVA8912,酶切鉴定;将鉴定正确的质粒转化变形链球菌UA159,挑取阳性克隆,提取基因组DNA,用PCR结合酶切鉴定。结果:发现PCR产物及插入片段大小与预期值相符,表明成功构建了重组质粒pVA8912/gcp;经PCR鉴定:gcp基因失活株基因组中gcp基因内部成功插入了重组载体片段。结论:成功构建了变形链球菌gcp基因失活株,为该基因功能的研究奠定了基础。     

牙龈卟啉单胞菌FimA蛋白和IL-15真核共表达质粒的构建与鉴定

目的 构建含编码牙龈卟啉单胞菌FimA蛋白和IL-15基因的真核共表达质粒,并检测其在哺乳动物细胞中的表达。方法 应用基因重组技术,构建真核表达载体pIRES-fimA和真核共表达载体pIRES-fimA:IL15,通过酶切、PCR及DNA序列测定鉴定获得的质粒,用Lipofectamine 2000介导转染CHO细胞,用Western blot检测重组质粒在哺乳动物中的表达,酶联免疫吸附试验检测培养上清中的蛋白表达。结果 PCR扩增获得的fimA和IL-15目的基因与预计相同,定向插入真核表达质粒pIRES中,插入位相正确,未改变阅读框架。转染的CHO细胞能够检测到目的基因的表达,在培养上清中也可以检测到蛋白质的表达。结论 本实验成功构建了牙龈卟啉单胞菌FimA蛋白和IL-15的真核共表达质粒pIRES-fimA:IL15,为研制增强免疫应答的抗牙龈卟啉单胞菌DNA疫苗奠定了基础。     

CBCT与RVG对下颌恒切牙根管形态评估的对比研究

目的:对比CBCT法与数字化X线成像法(RVG)对下颌恒切牙根管形态评估的差异。方法:对101颗离体牙进行唇舌向及近远中向数字化X线片拍摄和CBCT高清牙列模式扫描,对图像进行根管数目、Vertucci根管分型、根管钙化和根尖口开口位置的评估,并通过CBCT图像建立偏角度投照的三角函数模型,计算偏向投照的角度。结果:CBCT法显示有30颗为双根管,RVG近远中向投照显示有36颗为双根管,卡方检验显示对根管数目和Vertucci根管分型两种方法均具有显著性差异(P<0.05)。两种方法对根管钙化和根尖口开口位置的评估均不具有显著性差异。30颗双根管牙中下颌恒切牙唇舌向双根管的最大距离(LaL)在0~1.5 mm之间的,85.7%的为Vertucci III型,LaL在1.5~2 mm之间的,66.7%为Vertucci IV和V型。以根尖片上双根管显示距离为1 mm计算,LaL在1~2 mm之间的,偏向投照角度为26.6°~45.0°,LaL在0.1~1 mm之间的,偏向投照角度为45.0°~84.3°。结论:CBCT高清牙列模式扫描和RVG投照对于根管数目和Vertucci根管分型的评估具有显著性差异,对于根管钙化和根尖口开口位置的评估不具有显著性差异。LaL在1~2 mm之间时,偏角度投照的临床可操作性较强。     

Integrating other therapies with radiation for head neck cancer: search for optimal solution

Purpose: To review the role of concurrent chemoradiotherapy protocols with special emphasis on selection of appropriate chemotherapy drug, its timing and schedule with respect to radiation fractionation.     

Clinical and pathological aspects on some odontogenic tumors

Odontogenic tumors constitute a very diverse group of lesions that reflects the complex processes of odontogenesis. Controversies over their classification/subtyping, terminology and diagnosis have been persisted, which has direct bearings on therapeutic and/or prognostic implications.     

Can you be too old for oral implants? An update on ageing and plasticity in the oro‐facial sensorimotor system

This review focuses on the capacity of the brain for plasticity and the utility and efficacy of oral implants in helping to restore oro‐facial sensorimotor functions, especially in elderly patients. The review first outlines the components of the oro‐facial sensorimotor system which encompasses both oro‐facial tissues and a number of brain regions. One such region is the sensorimotor cortex that controls the activity of the numerous oro‐facial skeletal muscles. These muscles are involved in a number of functions including reflexes and the more complex sensorimotor functions of mastication, swallowing and speech. The review outlines the use by the brain of sensory inputs from oro‐facial receptors in order to provide for exquisite sensorimotor control of the activity of the oro‐facial muscles. It highlights the role in this sensorimotor control played by periodontal mechanoreceptors and their sensory inputs to the brain, and how oral implants in concert with the plastic capacity of the brain may, at least in part, compensate for reduced sensorimotor functioning when teeth are lost. It outlines findings of ageing‐related decrements in oro‐facial sensorimotor functions and control. The changes in oro‐facial tissues and the brain that underlie these ageing‐related functional alterations are also considered, along with adaptive and compensatory processes that utilise the brain's capacity for plasticity. The review also notes the evidence t hat rehabilitation that incorporates adjunctive approaches such as sensorimotor training paradigms in addition to oral prostheses such as implants may enhance these processes and help maintain or facilitate recovery of sensorimotor functioning in the elderly.     

Chemotherapy using new superselective intra-arterial infusion for advanced oral cancer

Purpose: We developed a new method of superselective intra-arterial infusion via the superficial temporal artery (HFT method: Hattori, Fuwa and Tohnai reported) and preoperatively performed daily concurrent radiotherapy and chemotherapy with docetaxel (DOC) and cisplatin (CDDP) using this method for 46 patients with stage Ⅲ, Ⅳ oral cancer.     

Navigation-guided oral and maxillofacial surgery (reconstructive and plastic, orthognathic, oncology)

Purpose: Advance in the field of compeer assisted surgery enables the surgical procedures to be less invasive and more accurate for the support of diagnosis imaging, pre-operative simulation and intraoperative navigation.     

Clinical evaluation of demineralized bone allograft in a hyaluronic acid carrier for sinus lift augmentation in humans: a computed tomography and histomorphometric study

OBJECTIVES: Natural and synthetic graft materials are used routinely in sinus floor augmentations to help support implants in atrophic maxillary ridges. This clinical study was based on the hypothesis that the clinical effectiveness of demineralized freeze-dried bone allograft/demineralized bone matrix (DFDBA/DBM) in sinus lifts varies when used in combination bone graft substitute materials. To test this hypothesis, DFDBA was used together with one of three materials: in saline plus anorganic bone (DFDBA: Bio-Oss; in hyaluronic acid (DFDBA: HY, 32 : 68, w/w; DBX alone; DBX plus Bio-Oss; and DBX plus tricalcium phosphate granules (beta-TCP). MATERIAL AND METHODS: Thirty-two sinus lift procedures, eight per group, were performed on 26 patients. Before surgery and at 8 months post-surgery when implants were placed, ridge heights were visualized by computed tomography (CT) and measured by morphometric analysis. Cores of bone were removed by trephine at the sites of implant placement; these biopsies from the graft sites were used for histomorphometric analysis. RESULTS: All 32 sinus lift elevations were successful when measured by CT, increasing from an average 2.84+/-0.2 mm before treatment to 15.2+/-0.6 mm after treatment. The percent of each biopsy that was occupied by new bone and incorporated bone graft materials varied with each treatment: DFDBA+Bio-Oss, DBX+Bio-Oss, or DBX alone was higher than that for DBX+beta-TCP by approximately 10%. When comparing only newly formed bone, DBX+beta-TCP treatment resulted in 50% less bone than the other three preparations. All grafted sites received implants as per the treatment plan for each patient. CONCLUSIONS: This study confirmed the hypothesis that new bone formation is dependent on the DFDBA formulation used and demonstrated that DBX, alone or in combination with other materials, can be used successfully for sinus floor elevation.     

氟化泡沫对正畸固定矫治患者牙周指数及牙釉质脱矿的影响

目的:探讨氟化泡沫对正畸固定矫患者牙周指数及牙釉质脱矿的影响。方法:选取2013年6月~2014年6月30例舌侧正畸治疗患者(600颗牙)为研究对象,根据入院时间分为对照组15例(297颗牙)和观察组15例(293颗牙),对照组采取常规口腔卫生措施,观察组在对照组的基础上定期使用1.23%氟化泡沫。随访12 个月,观察两组牙釉质脱矿牙位分布、脱矿指数(EDI)、菌斑指数(PLI)和出血指数(BI)。结果:牙釉质脱矿主要分布于上颌侧切牙、上颌中切牙及下颌前磨牙等;观察组牙釉质釉质脱矿率11.26%明显低于对照组17.51%(χ²=4.666,P<0.05);观察组EDI、PLI、BI等均明显低于对照组(t=9.947, 4.435, 7.789,P<0.05)。结论:舌侧正畸治疗中牙釉质脱矿发生率较高,氟化泡沫有助于减少正畸治疗中的牙釉质脱矿发生,降低牙周指数。     

Tumor microenvironment in oral cancer: novel approaches for immunotherapy

Squamous cell carcinoma of the head and neck (SCCHN), which arises from the squamous mucosal epithelium of the oral cavity, pharynx and larynx, is a major health problem in the US and other parts of the world, especially in developing countries.     

Cone beam imaging: is this the ultimate imaging modality?

This review article provides an overview of cone beam (CB) imaging technology and its role in orofacial imaging, including comparison with two-dimensional (2D) radiography and multislice computed tomography (MCT). The radiation dose levels of CB systems are discussed, with reference to those delivered by MCT and common dental 2D views. The large variation in dose levels delivered by CB systems and the importance of using ultra low-dose CB units are emphasized. Low-dose MCT protocols can be used. CB and MCT image quality are compared. CB is an essential technique that all dental and orofacial clinicians must be familiar with. Where ultra low-dose systems and protocols are used, CB imaging should be considered in day-to-day clinical practice. However, CB imaging is not the technique of choice in many clinical scenarios. Rather than replacing other modalities, CB imaging complements intraoral 2D radiography, panoramic radiography, MCT and other techniques including magnetic resonance imaging, ultrasound and nuclear medicine. MCT is a much more powerful and flexible modality and presently remains the technique of choice over CB imaging in many clinical scenarios. All radiologic examinations, including CB and MCT, should be comprehensively evaluated in entirety. The responsibilities and the radiological skill levels of clinicians involved in imaging as well as the associated ethical and medico-legal implications require consideration.