Tricine-SDS-PAGE法分析腮腺唾液蛋白

多种口腔疾病或系统性疾病的发生、发展过程中,唾液成分的质和量都可能发生改变［１］。唾液蛋白作为唾液的主要有机成分,与唾液的多种生理功能密切相关［２］。因此研究唾液蛋白对于了解机体的生理及病理状况具有重要意义。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（ＳＤＳ－...     

猪发育期牙胚釉原蛋白的分离纯化及其多克隆抗体的制备

目的　制备猪釉质蛋白多克隆抗体,为釉原蛋白的检测提供条件。方法　选择1月龄乳猪,分离埋于上下颌骨内的牙胚,刮取牙胚表面干酪样尚未完全矿化的釉质基质,通过盐酸胍抽提及SephadexG-200柱层析分离纯化猪发育期牙胚釉原蛋白,联合免疫家兔,抗血清经DE-52纤维素纯化,并经ELISA测定抗体效价。结果　 SDS-PAGE电泳结果发现采用SephadexG-200葡聚糖凝胶过滤能达到较为理想的釉原蛋白分离纯化效果,用所提纯的猪釉原蛋白免疫家兔,成功地制得兔抗猪釉原蛋白抗血清,抗血清稀释达1∶32 000时,采用ELISA法仍有明显的抗原抗体反应。结论　本实验成功制备得到抗釉原蛋白多克隆抗体,为釉原蛋白的检测提供了条件。     

牙齿发育相关基因adam28多克隆抗体的制备及鉴定

目的制备及鉴定抗ADAM28的多克隆抗体。方法采用反转录-多聚酶链式反应(RT-PCR)扩增出adam28基因的蛋白编码区序列,克隆到中间载体pMD18-T Vector中,再经双酶切得到adam28片段定向克隆到pGEX-4T-1载体中,构建融合表达载体pGEX-4T-adam28,在大肠杆菌中用IPTG进行诱导表达,得到GST-ADAM28融合蛋白,经过初步纯化及SDS-PAGE电泳,在35300的新蛋白带处直接割胶,作为抗原免疫新西兰大白兔后获取抗体。结果成功构建融合表达载体pGEX-4T-adam28,得到初步纯化的GST-ADAM28融合蛋白,免疫兔子1个月后,获得免疫血清,盐析法纯化得到多克隆抗体。Western印迹结果显示所得到的抗体具有较高的特异性,ELISA分析证实其效价可达1∶16000。结论成功制备出高效价的抗ADAM28多克隆抗体,为进一步研究ADAM28在牙齿发育中的作用和表达分布奠定基础。     

牙龈卟啉单胞菌牙龈蛋白酶K催化结构域融合蛋白的纯化及其多克隆抗体的制备

目的纯化牙龈卟啉单胞菌牙龈蛋白酶K催化结构域（KGPcd）融合蛋白并制备其多克隆抗体,为下一步的实验提供条件。方法KGPcd蛋白与载体pET-16b中的His标签融合表达,用Ni—NTA亲和层析纯化融合蛋白,复性后用Westernblot检测。以重组、纯化的KGPcd蛋白为抗原,免疫新西兰大白兔,制备多克隆抗体,用间接ELASA法检测抗体效价,WesternBlot进行抗血清特异性试验。结果经亲和层析、复性得到纯化融合蛋白His．KGPcd,Westernblot进一步证实纯化的蛋白为His．KGPcd融合蛋白。抗血清稀释达1：3200时,ELASA法仍有明显的抗原抗体反应,Westernblot也进一步证实抗血清能够与KGPcd发生特异性反应,抗体仅特异识别目的蛋白。结论成功制备兔抗KGPcd多克隆抗体,为后续研究奠定基础。     

小鼠cbfal多克隆抗体制备及在骨和牙胚中的表达

目的 制备cbfal多克隆抗体并进行鉴定和效价分析，观察其在骨和牙胚中的表达情况。方法 用自制的融合蛋白免疫新西兰大白兔，制备多抗血清，提取小鼠牙胚、颅骨和肝脏组织的总蛋白进行western blot，同时采用免疫组化染色观察其组织表达特性。结果 cbfal蛋白在小鼠颅骨和牙胚组织中均有表达，在人骨肉瘤细胞系中呈强阳性表达。在免疫组化和western b1ot的有效作用滴度分别为1：400和1：1000。结论 成功地制备了兔抗小鼠cbfal多克隆抗体。     

ELISA检测抗变形链球菌的单克隆抗体和多克隆抗体与人心脏…

变形链球菌与人类龋病发生具有最密切的关系。在龋病（变链菌）的免疫学研究中，备受关注的问题之一是抗变链菌的抗体与人心脏组织是否存在免疫交叉反应。本文采用酶联免疫吸附试验（ＥＬＩＳＡ），分别检测了抗变链菌和多克隆抗体和单克隆抗体，与正常人胚心脏匀浆，正常成人心脏匀匀和乙型溶血链球菌的免疫交叉反应性。结果表明：抗变链菌Ｃ血清型全菌的多克隆抗体，与上述三种抗原均存在一定程度的免疫交叉反应；而抗变链菌１８５     

ELISA检测抗变形链球菌的单克隆抗体和多克隆抗体与人心脏组织的免疫交叉反应

变形链球菌与人类龋病发生具有最密切的关系。在龋病（变链菌）的免疫学研究中，备受关注的问题之一是抗变链菌的抗体与人心脏组织是否存在免疫交叉反应。本文采用酶联免疫吸附试验（ＥＬＩＳＡ），分别检测了抗变链菌和多克隆抗体和单克隆抗体，与正常人胚心脏匀浆、正常成人心脏匀浆和乙型溶血链球菌的免疫交叉反应性。结果表明：抗变链菌Ｃ血清型全菌的多克隆抗体，与上述三种抗原均存在一定程度的免疫交叉反应；而抗变链菌１８５—ＳＡ的单克隆抗体，则未检测出免疫交叉反应。     

牙龈卟啉单胞菌和具核梭杆菌多克隆抗体的制备及应用效果评价

目的    制备高效价高纯度的牙龈卟啉单胞菌（P. gingivalis）和具核梭杆菌（F. nucleatum）多克隆抗体,并评价其应用效果。方法    将新鲜培养的P. gingivalis和F. nucleatum菌液灭活后与佐剂乳化混匀,作为抗原皮下多点注射免疫雄性新西兰大白兔,定期免疫并在抗体效价达到预期后采用颈动脉取血获得抗血清。采用间接ELISA法测定多克隆抗体交叉反应。应用硫酸铵沉淀法纯化多克隆抗体,并通过Western-blot方法鉴定抗体的纯度。荧光显微镜观察多克隆抗体应用于免疫荧光实验的效果。结果    免疫6周后,血清抗体效价达到1∶320 000 ~ 1∶640 000,相互之间未出现交叉反应,且纯度较高。免疫荧光实验中可观察到明显的荧光,并可见P. gingivalis的球杆状形态和F. nucleatum的短杆状形态。结论    成功制备了P. gingivalis和F. nucleatum多克隆抗体,其特异性良好,为后续的免疫学相关实验奠定了基础。     

腭裂相关基因MCPR1在大肠杆菌中的融合表达及抗体制备

目的 :MCPR1是我室利用消减杂交技术克隆出来的新基因 ,本研究利用融合蛋白制备MCPR1的多克隆抗体。方法 :采用多聚酶链式反应 (PCR)扩增出MCPR1基因蛋白编码序列 ,进一步将该基因片段克隆到中间载体pGEX -T -easy ,再酶切得到MCPR1片段定向克隆到 pGEX -4T -1载体中 ,构建融合表达载体pGEX -4T-MCPR1,在大肠杆菌中用IPTG进行诱导表达 ,得到GST -MCPR1融合蛋白 ,进行初步纯化及SDS -PAGE电泳 ,直接割胶 ,将其作为抗原免疫兔子。结果 :成功构建融合表达载体pGEX -4T -MCPR1,得到初步纯化的GST-MCPR1融合蛋白 ,免疫兔子 1个月后 ,获得免疫血清 ,初步纯化得到多克隆抗体。Western印迹分析表明所得到的抗体具有较强的特异性 ,ELISA分析证实其效价为 10 6。结论 :MCPR1多克隆抗体的获得性为在蛋白水平研究新MCPR1的功能提供了实验基础 ,后续实验可以利用免疫分析技术研究MCPR1蛋白与其它蛋白质的相互作用 ,从而为阐明MCPR1在胚胎发育及腭裂形成中所发挥的作用提供实验手段。     

EDA蛋白多克隆抗体的制备

目的 :制备EDA蛋白的多克隆抗体。方法 :用含EDA蛋白的蛋白凝胶免疫兔子 ,用ELISA和West ernblotting方法检测抗血清效价。结果 :两只动物中一只产生了高效价的抗血清 ,另一只产生的效价较低。结论 :用含EDA蛋白的蛋白凝胶免疫兔子可获得了高效价的抗血清 ,为深入研究该蛋白在牙齿发育中的具体作用奠定了基础     

Suppression of reflex saliva from rat parotid gland following intracerebroventricular injection of hypertonic NaCl and sucrose.

These effects were studied in conscious rats. Salivary volume and flow rate induced by eating solid diet were decreased by both the hypertonic solutions, compared with the effects of normal saline. This finding suggests that central osmotic perception affects parotid salivary secretion in rats.     

Continuous analysis of parotid saliva during resting and short-duration simulated chewing

Objective

Parotid saliva flow is increased by mastication and its composition is also modified. The aim of this work was to clarify the relationships between flow rate, pH and protein concentration, during resting and short-duration simulated chewing, using continuous and fractional saliva collections.

Design

Parotid saliva flow rate, pH and protein concentration, as it exits the Stensen's duct, were determined on seven subjects in response to one and 30 ipsilateral jaw clenches. To achieve this, we have developed a system able to collect parotid saliva and to measure continuously flow rate, pH and protein concentration and synchronised to the values at the exit of the duct.

Results

With increase in flow rate, pH increased linearly and its protein concentration decreased linearly with the logarithm of the flow rate. With an increase in flow rate from 50 to 500 μl/min, the pH increased from 5.8 to 7.0 and the protein concentration decreased from 1.0 to 0.7 g/l.Measurements made on parotid saliva fractions confirmed the variations in pH and protein concentration with flow rate and showed that α-amylase concentration was significantly related to both salivary conductivity and protein concentration.

Conclusions

Continuous measurements of salivary flow and composition offer a simple and convenient way to determine the precise relationships between different types of oral stimulation and parotid salivary flow and composition.     

Enzyme activities in parotid saliva of patients with the restrictive type of anorexia nervosa

ObjectiveIn patients with anorexia nervosa (AN) specific signs may occur in the oral cavity, but there are conflicting reports about their significance, especially concerning changes in salivary composition.The aim of this clinical study was to evaluate the resting parotid flow rate (PFR) and the activity of the following enzymes in parotid saliva: amylase, aspartate amino transferase (AST), lysozyme, peroxidase, serine and acidic proteases in the acute phase of the restrictive type of AN and to compare the findings with those in healthy controls.DesignForty-one subjects participated (20 patients with AN, 21 matched healthy controls), parotid saliva was collected using a modified Lashley cap at rest. Enzyme activities were measured with fluorimetric and photometric assays.ResultsThe unstimulated PFR was significantly lower than in the controls, lysozyme and AST activity was significantly lower, and amylase showed a high inter-individual variability. A positive correlation for amylase and lysozyme and negative ones for lysozyme and BMI, lysozyme and IBW%, serine protease and salivary flow were observed.ConclusionsThe reduced PFR and enzyme activities levels suggest that AN does not only affect the quantity of the saliva but also its quality and, its biological functions. The results obtained should help to provide a better understanding of the effect of AN disease on the pathogenesis of at least some oral diseases. Further research is needed on any possible role of reduced lysozyme and transaminase activity in maintaining oral protection against external toxic agents and bacteria.     

腮腺肿瘤患者唾液CEA及CA125的ROC分析

目的 通过ROC分析方法,观察并对比唾液样本及血液样本的CA125及CEA水平变化对腮腺肿瘤的临床诊断效能.方法 检测正常对照组(30例)和腮腺肿瘤患者(101例)的腮腺液、混合唾液及血液的CA125及CEA水平,通过ROC分析,观察两种标志物单独及联合应用时,诊断腮腺肿瘤的敏感度(SE)、特异度(SP)、AUC值及界值(cutoff).结果 ROC分析结果显示,混合唾液CA125及CEA对腮腺肿瘤的诊断效能最高(AUC分别为80.4％,78.4％),二者独立应用的诊断效能与联合应用无显著差异.腮腺液及血液样本CA125及CEA的诊断效能处于较低水平(AUC＜ 65％).结论 混合唾液样本CA125与CEA水平的变化可以较好地反映腮腺肿瘤的存在,其临床效能优于腮腺液及血液样本.     

儿童唾液富组蛋白含量与患龋状况的分析

目的:分析富组蛋白含量与患龋状况间的关系,为唾液蛋白的研究及龋病病因的探讨提供基础资料。方法:随机抽取42名3~5岁儿童,按患龋状况分为2组:龋病高危组23例(dft≥5且CSI≥10),无龋组19例(dft=0,CSI=0)。用高效液相色谱仪分离测定儿童刺激性、非刺激性全唾液中3种主要富组蛋白(histadine-rich proteins,HRPs)HRP-1、HRP-3、HRP-5的含量,计算总HRPs含量,并分析唾液中HRP-1、HRP-3、HRP-5、总HRPs的浓度与机体患龋状况的关系。结果:①非刺激性全唾液中HRP-1、HRP-3、HRP-5的含量及总HRPs含量分别是(8.56±3.42)、(13.91±6.59)、(7.35±3.23)、(29.65±8.69)μg/ml,刺激性全唾液中HRP-1、HRP-3、HRP-5和总HRPs含量分别为(10.85±3.71)、(15.92±5.94)、(7.68±3.28)、(34.69±9.41)μg/ml。刺激性全唾液中HRP-1、HRP-3和总HRPs含量高于非刺激性全唾液,差异有统计学意义(P<0.05)。②不同性别间全唾液中HRP-1、HRP-3、HRP-5及总HRPs含量之差异无统计学意义。③龋病高危组和无龋组刺激性全唾液HRPs含量差别无统计学意义,而非刺激性全唾液总HRPs的含量在龋病高危组和无龋组之间的差别有统计学意义(P<0.05),但与患龋状况无相关关系。结论:①刺激性全唾液中HRPs的含量高于非刺激性全唾液中的含量。②龋病高危组非刺激性全唾液之HRPs含量明显低于无龋组,但与机体的患龋程度无密切相关关系。     

唾液富脯蛋白的分离提纯

收集刺激性腮腺唾液１０００ｍｌ，利用先进的生化技术分离提纯出唾液富腈蛋白（ＰＲＰｓ）。经硫酸铵浓度差超速离心后得到初步纯化的ＰＲＰｓ，再经葡聚糖凝胶（Ｓｅｐｈａｄｅｘ）Ｇ－７５过滤和ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析后可得到ＰＲＰｓ的６个组份。其分子量大小分别为１４３００、１０８００、１１０００、１２３５０、１３０００和１２５８０ｕ；等电点范围在４．１８￣４．６０之间。６种组分的ＰＲ     

Histatins 2 and 4 are autoproteolytic degradation products of human parotid saliva

Freshly collected human parotid saliva contains 8 cationic proteins, as demonstrated by capillary electrophoresis. These proteins include lysozyme, histatin 6 and the 6 salivary histidine-rich polypeptides (HRPs 1-6). Neither histatin 2 nor histatin 4 are present in native undegraded parotid saliva but appear only after autoproteolytic degradation of the saliva. Histatin 2 appears to arise through slow degradation of HRP-1, and histatin 4 is mainly produced as a rapid breakdown product of HRP-3.     

Levels of pre-kallikrein in resting and stimulated human parotid and submandibular saliva

Salivary tissue kallikrein is stored in an active form in human salivary glands. Pre-kallikrein has been demonstrated in mixed saliva, but it is not clear if the various salivary glands contribute equally. This study set out to determine if pre-kallikrein is present in human parotid and submandibular salivas at rest, whether levels change during stimulation, and to compare the pattern of pre-kallikrein and kallikrein secretion with that of total protein. Resting and citric acid-stimulated parotid and submandibular, and gum-stimulated parotid saliva samples were collected from 6 healthy subjects. Salivary flows were determined gravimetrically. Total protein concentration and kallikrein enzymic activity were assayed using standard techniques. Pre-kallikrein was assayed following trypsinisation of duplicate samples. Pre-kallikrein was present in parotid and submandibular ductal saliva. Proportions of pre-kallikrein and active kallikrein were similar in salivas secreted at rest and during stimulation, and both outputs mirrored protein output in both major glands. Gum-stimulated parotid saliva showed lower activity than resting, and no differences were seen between resting and stimulated submandibular samples.     

腮腺肿瘤患者唾液和血液中CEA、CA125含量的检测及其在肿瘤组织中的表达

目的：检测腮腺肿瘤患者唾液和血液中CA125、CEA的含量及其在腮腺肿瘤组织中的表达。方法选择腮腺良性肿瘤患者83例,腮腺恶性肿瘤患者18例,对照组为30名健康志愿者。采用化学发光法( CLIA)检测唾液及血液样本中CEA和CA125含量。采用免疫组化法检测腮腺肿瘤组织中CEA和CA125的表达。结果腮腺肿瘤患者混合唾液中CEA和CA125水平明显高于对照组。腮腺肿瘤患者与对照组血液中CEA和CA125水平无显著差异。良、恶性肿瘤患者唾液中CEA和CA125含量无显著差异。 CEA及CA125的分布特点为,混合唾液中含量最高,血液中含量最低。相关性分析显示,混合唾液中CEA与CA125呈现明显正相关性( r=0.652-0.913)。在大部分腮腺肿瘤组织中,CEA和CA125均为阳性表达,而且部分病例的肿瘤组织周围正常腺体也可见CEA及CA125的阳性表达。结论唾液中CEA和CA125水平的变化可以反映腮腺肿瘤的存在,但无法区分腮腺肿瘤的良恶性。血液中CEA和CA125不能反映腮腺肿瘤的存在。腮腺肿瘤患者的混合唾液样本中CEA与CA125呈现同步变化的趋势。唾液中CEA与CA125可能主要来源于涎腺腺体及肿瘤组织,而不是来自血液。