比较蛋白组学在口腔鳞癌患者及健康人血清的初步研究

目的:筛选口腔鳞癌及健康人血清差异表达的蛋白质。方法:收集口腔鳞癌患者外周血清17例,健康人外周血清17例,通过固相pH梯度双向凝胶电泳以及质谱鉴定和生物信息学分析,寻找在口腔鳞癌患者血清中特异表达的蛋白质。结果:先后对口腔鳞癌和健康人血清表达差异明显的11个蛋白质点进行质谱鉴定和生物信息学分析,鉴定了其中的2个点为触珠蛋白(haptoglobin,Hp),在口腔鳞癌血清中均为高表达;1个点为载脂蛋白A(apolipoprotein A)在口腔鳞癌血清中为低表达。结论:触珠蛋白和载脂蛋白A在口腔鳞癌外周血清和健康人外周血清的表达发生了改变,但其确切的调控机制有待进一步研究。     

口腔鳞癌组织蛋白差异表达的蛋白组学测定

目的 应用蛋白质组学(proteomics)方法建立人口腔鳞癌组织与正常口腔黏膜组织中的差异蛋白质表达。方法 收集10例口腔鳞癌组织及正常黏膜组织,提取蛋白质,进行双向凝胶电泳,经图像分析软件分析识别差异表达,应用液相色谱-质谱分析鉴定差异蛋白质。结果 分析鉴定出S100A7、S100A8、S100A9在口腔鳞癌中高表达。结论 S100A7、S1000A8、S100A9在口腔鳞癌发生中发生了改变,为进一步筛选口腔鳞癌特异性的分子标志物打下了坚实的基础。     

口腔鳞癌患者与健康人外周血清差异表达蛋白的初步研究

目的通过口腔鳞癌患者血清及健康人血清中蛋白质组成的对比分析,获得口腔鳞癌患者血清中的蛋白变化。方法口腔鳞癌患者及健康人外周血清各20例,通过固相p H梯度双向凝胶电泳以及质谱鉴定和生物信息学分析,寻找在口腔鳞癌中差异表达的蛋白质。结果口腔鳞癌和正常人血清差异表达明显的7个点,对其进行质谱鉴定和生物信息学分析,鉴定了其中的一个点,为Chain B,T-To-T(High)Quaternary Transitions In Human Hemoglobin.在口腔鳞癌血清中均为低表达。结论Chain B,T-To-T(High)Quaternary Transitions In Human Hemoglobin:Deshis146beta Deoxy Low-Sal.在口腔鳞癌发生发展过程中可能起重要调控作用。     

口腔鳞癌组织与口腔正常黏膜组织蛋白质差异表达的初步研究

目的:筛选口腔鳞癌及正常口腔黏膜组织的差异表达蛋白质,为研究口腔鳞癌发生机制提供实验依据.方法: 收集10 例口腔鳞癌组织及正常口腔黏膜组织,进行二维电泳,选择在表达差异量较大的29 个点进行质谱和生物信息学分析,确定所分析的蛋白质类型. 结果: 口腔鳞癌及相应正常口腔黏膜组织凝胶的平均蛋白质点数分别为2 325±390和2 487±281.双向凝胶电泳图显示,口腔鳞癌及正常口腔黏膜组织的差异表达蛋白质点数为29 个,这29 个点在癌组织中均为低表达,对其进行了质谱(PMF)和生物信息学分析,鉴定了其中的3 个点,它们是:β纤维蛋白(fibrin beta)、磷酸丙糖异构酶(triosephosphate isomerase TIM)、unknown蛋白.结论: β纤维蛋白、磷酸丙糖异构酶、unknown蛋白在口腔鳞癌发生发展过程中发生了改变,其机制尚待进一步阐明.     

口腔鳞癌患者唾液cyfra 21-1含量与癌组织CK19表达的相关性研究

目的：检测口腔鳞癌患者唾液中cyfra21-1含量、组织中CK19蛋白质表达和基因转录水平,探讨其相互关系。方法：对30例口腔鳞癌患者和30例正常人的唾液采用ELISA法检测cyfra21-1含量,其中6例口腔鳞癌患者的癌组织和癌旁组织分别采用免疫组织化学和实时定量RT-PCR法检测CK19蛋白质表达强度和CK19mRNA水平,采用SPSS10.0软件包进行统计学分析。结果：口腔鳞癌患者唾液cyfra21-1含量为（85.95±78.00）μg/L,显著高于对照组的（42.27±40.84）μg/L;口腔鳞癌癌组织CK19蛋白质表达强度和CK19mRNA水平显著高于癌旁组织。CK19mRNA水平与CK19蛋白质表达强度呈显著正相关,癌组织CK19蛋白质与唾液cyfra21-1含量也显著相关。结论：口腔鳞癌患者唾液cyfra21-1含量和组织CK19蛋白表达及基因转录水平显著升高,组织CK19表达升高,对唾液cyfra21-1含量的上升起着重要作用。     

应用双向电泳和质谱技术筛选口腔扁平苔藓差异蛋白

目的:筛选口腔扁平苔藓及正常黏膜组织的差异表达蛋白,为口腔扁平苔藓的发病机制及诊断治疗的研究提供实验室依据.方法:收集口腔扁平苔藓组织及正常口腔黏膜组织,提取组织蛋白,蛋白定量,进行双向电泳,图象分析寻找差异点,基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析鉴定表达差异量较大的蛋白质点,确定所分析的蛋白质类型和功能.结果:(1)人口腔扁平苔藓及正常黏膜组织的双向电泳图谱的平均蛋白质点数分别为1 576±67和1 608±73,同一组织凝胶在蛋白质点位置上重复性较好.(2)通过比较人口腔扁平苔藓与正常黏膜组织的双向凝胶电泳图谱,得到差异表达蛋白质点数为13个,其中7个点在口腔扁平苔藓中为高表达,6个点在口腔扁平苔藓中为低表达.选择1O个表达差异量较大的蛋白质点进行质谱和生物信息学分析,鉴定了其中的4个点,包括有锰超氧化物歧化酶(Mn-SOD),膜联蛋白I,波形蛋白,未知蛋白等.结论:锰超氧化物歧化酶,膜联蛋白I,波形蛋白,未知蛋白可能参与了口腔扁平苔藓的发生和发展,其相关机制尚待进一步阐明.双向电泳-质谱分析技术为口腔扁平苔藓发生发展过程中差异表达蛋白的研究提供了有效的技术手段.     

应用蛋白质组学研究白斑差异表达蛋白

目的:通过筛选口腔白斑及正常黏膜组织的差异表达蛋白,为研究白斑发生机制提供实验依据。方法:收集口腔白斑组织及正常口腔黏膜组织,提取组织蛋白,进行二维电泳,选择在表达差异量较大的11个点进行质谱和生物信息学分析,确定所分析的蛋白质类型。结果:人口腔白斑及正常黏膜组织的二维电泳图谱的平均蛋白质点数分别为1726±67和1608±73,蛋白质点重复性较好。人口腔白斑与正常黏膜组织的双向凝胶电泳图谱,差异表达蛋白质点数为38个,其中16个点在白斑中为高标达,22个点在白斑中为低表达,选择11个表达差异量较大的蛋白质点进行质谱和生物信息学分析,鉴定出其中的5个点,分别为膜联蛋白A2,角蛋白8,角蛋白1,II型角蛋白亚基,免疫球蛋白(Ig)κ型轻链的恒定区(C区)片段等。结论:膜联蛋白A2,角蛋白8,角蛋白1,II型角蛋白亚基,Igκ型轻链的C区片段等在口腔白斑发生发展过程中发生了改变。     

蛋白质组学在口腔医学中的研究进展

蛋白质组学是在人类基因组计划研究发展的基础上形成的新兴学科,它的主要任务是识别、鉴定细胞、组织或机体的全部蛋白质,并分析蛋白质的功能及其模式.随着蛋白分离,鉴定技术和生物信息学的完善,蛋白质组学在各个基础和临床领域得到了长足的发展.在口腔医学领域从鳞癌蛋白质含量对比分析开始,其高通量、高灵敏度的技术特点已逐渐应用于口腔牙体组织、唾液的蛋白质全表达、口腔内环境菌群蛋白功能研究、口腔肿瘤发生发展的蛋白机制表达和生物标记等方面,并取得了相应的成果,我们就蛋白质组学在口腔医学中应用的相关技术及其研究进展作一阐述.     

慢性牙周炎患者和健康者非刺激性全唾液中的差异蛋白比较

目的 比较慢性牙周炎患者和健康对照者非刺激性全唾液(WUS)中的差异表达蛋白.方法 采集5名慢性牙周炎患者和5名健康者的WUS,利用双向凝胶电泳(2-DE)分离蛋白,将二者之间的差异蛋白质点进行基质辅助激光解析电离串联飞行时间质谱鉴定.结果 通过软件对2组样品的双向胶上的点进行对比分析,发现17个差异点,经质谱鉴定明确...     

S100A4基因与口腔鳞癌相关性分析

目的：研究S100A4mRNA表达与口腔鳞癌分化、转移关系。方法：分别提取40例口腔鳞癌组织及远端正常口腔黏膜组织的RNA,用半定量逆转录一聚合酶链反应（RT—PCR）法测定S100A4基因在口腔鳞癌和配对组织mRNA水平的差异表达情况。并结合临床资料,对S100A4基因差异表达与口腔鳞癌病人临床病理参数进行相关性分析。结果：S100A4mRNA在口腔鳞癌中高表达,与癌旁正常口腔黏膜组织比较,具有显著性差异（P〈0．01）。S100A4mRNA的表达水平与口腔鳞癌的分化、淋巴结转移成负相关。S100A4mRNA与患者的性别、年龄、肿瘤大小无关。结论：S100A4基因表达和口腔鳞癌的发生发展、分化、侵袭转移密切关}S100A4mRNA可作为判定口腔鳞癌恶性程度及评价预后的重要指标,也可作为新的生物标志物及治疗口腔鳞癌浸润转移的潜在靶目标。     

Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.     

Identification of protein differences between two clinical isolates of Streptococcus mutans by proteomic analysis

INTRODUCTION: Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Different strains of S. mutans may display different virulence mechanisms, so the isolation of the differential proteins is illuminating. METHODS: S. mutans strains 9-1 and 9-2, which both colonized the same oral cavity, were selected after screening for the possession of suspected virulence traits. The soluble cellular proteins were extracted from steady-state planktonic cells of strains 9-1 and 9-2 and were analyzed using high-resolution two-dimensional gel electrophoresis. Then, replicate maps of proteins from the two strains were generated. Proteins expressed only in strain 9-1 or 9-2 were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry, by which peptide mass fingerprints were generated, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. RESULTS: There were 12 proteins only expressed in strain 9-1 and three proteins only expressed in strain 9-2. They were involved in protein biosynthesis, protein folding, cell wall biosynthesis, fatty acid biosynthesis, nucleotide biosynthesis, repair of DNA damage, carbohydrate metabolism, signal transduction, and translation. CONCLUSION: The identification of proteins differentially expressed between strains 9-1 and 9-2 provides new information concerning the mechanisms of cariogenesis     

Analysis of differentially expressed proteins in oral squamous cell carcinoma by MALDI‐TOF MS

J Oral Pathol Med (2010) 40 : 369–379 Purpose: To explore the presence of differentially expressed proteins in OSCC for discrimination of tumour and normal mucosa to establish potential biomarkers and therapeutic targets. Experimental Design: Paired protein samples of 12 individuals (tongue cancer and non‐cancerous mucosa) were separated by two‐dimensional polyacrylamid gel electrophoresis. The protein patterns were compared pairwise and protein spots were quantified. We identified about 70 regulated proteins which we subsequently identified by MALDI‐TOF mass spectrometry. Results: Cancerous and non‐cancerous tissues could be most precisely distinguished by a panel of proteins. They include the heat shock proteins (hsp)70 and 90, keratins (ck) 5, 6, 13, 14, 16, 17 and 19, beta globin, alpha‐2‐actin, stratifin, tropomyosin, calreticulin precursor, beta‐2‐tubulin, galectin7, thioredoxin, involucrin, adenylyl‐cyclase‐associated protein, disulfide isomerase‐associated protein, thyrosine 3‐monooxygenase, MYL2 and the s100 calcium binding protein. MYL3, cardiac muscle alpha actin 1 proprotein and transferrin were under‐represented in OSCC. Six biomarkers, ck6 und ck13, beta globin, alpha‐2‐actin, hsp70 and hsp90 discriminated best between cancerous and non‐cancerous oral tissues. All over‐expressed proteins were analysed by STRING‐analysis to highlight experimentally determined and computationally predicted interactions between the proteins. Especially involucrin, hsp70, calreticulin precursor, stratifin, (ck) 5, 6, 14, 19, tyrosine 3‐monooxygenase, beta‐2‐tubulin and disulfide isomerase associated protein showed multiple relations. Conclusion: We identified six proteins which are differentially expressed in most OSCC compared to healthy tissues. Of those, by string analysis, multiple interaction partners are assumed for hsp70. This protein is supposed to be the most promising candidate as marker molecule and target for OSCC therapy.     

2型糖尿病大鼠下颌骨的蛋白质组学研究

目的:通过蛋白质组学研究,检测2型糖尿病大鼠下颌骨与正常大鼠下颌骨表达差异的蛋白。方法:选取8周龄Wistar大鼠和2型糖尿病大鼠Goto-Kakizaki(GK)大鼠,分别作为对照组和实验组。测量2组大鼠的体重和血糖,同期处死后切取下颌骨。提取各组下颌骨蛋白后,经双向凝胶电泳(2-DE)及MALDI-TOF/TOF质谱分析,鉴定筛选出2组大鼠下颌骨组织差异表达的蛋白。采用SPSS15.0软件包对数据进行统计学分析。结果:2组大鼠体重无显著差异,GK大鼠血糖显著高于Wistar大鼠(P<0.05)。经蛋白质组学研究,成功鉴定出20个2组间差异3倍以上的蛋白,其中5个蛋白点差异倍数在20倍以上。5个蛋白主要涉及代谢、蛋白结合以及信号转导3大功能。结论:2型糖尿病大鼠与正常大鼠下颌骨组织间存在的差异蛋白,有助于探讨2型糖尿病对下颌骨的影响机制。     

Identification of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform

New and more consistent biomarkers of oral squamous cell carcinoma (OSCC) are needed to improve early detection of the disease and to monitor patient management. The aim of this study was to detect new OSCC tumor markers in saliva. Unstimulated saliva, collected from patients with primary stage I OSCC as matched pre-and post-treatment samples, was used in the analysis. A surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in the saliva samples. This analysis revealed 26 proteins with significantly different expression levels in the pre-and post-treatment samples (P < 0.05). A 14 kDa protein detected in pre-treatment saliva from the OSCC patients was identified as a truncated cystatin SA-I, with deletion of three amino acids from the N-terminus. The authors propose that ProteinChip analysis may provide a reliable screening test for early diagnosis of OSCC and that truncated cystatin SA-I might be a useful tumor biomarker for OSCC.     

Deep sequencing salivary proteins for periodontitis using proteomics

Objectives

Saliva is a bodily fluid transuded from gingival crevice fluid and blood and contains many proteins. Proteins in saliva have been studied as markers for periodontal diseases. Mass spectrometric analysis is applied to investigate biomarker proteins that are related to periodontitis.

Material and methods

Saliva samples were collected from 207 participants including 36 pairs matched for age, sex, and smoking who joined Yangpyeong health cohort. Periodontitis was defined by 2005 5th European guideline. Shotgun proteomics was applied to detect proteins from saliva samples. Principal component analysis and Ingenuity Pathway Analysis for canonical pathway and protein pathway were applied. Protein-protein interaction was also applied. Enzyme-linked immunosorbent assay (ELISA) was used to verify the candidate protein markers among another matched participants (n = 80).

Results

Shotgun proteomics indicated that salivary S100A8 and S100A9 were candidate biomarkers for periodontitis. ELISA confirmed that both salivary S100A8 and S100A9 were higher in those with periodontitis compared to those without periodontitis (paired-t test, p < 0.05).

Conclusion

Our proteomics data showed that S100A8 and S100A9 in saliva could be candidate biomarkers for periodontitis. The rapid-test-kit using salivary S100A8 and S100A9 will be a practical tool for reducing the risk of periodontitis and promotion of periodontal health.

Clinical relevance

A rapid-test-kit using salivary biomarkers, S100A8 and S100A9, could be utilized by clinicians and individuals for screening periodontitis, which might reduce the morbidity of periodontitis and promote periodontal health.

     

重组人白细胞介素-1β诱导人牙髓细胞蛋白质组的差异分析

目的分析重组人白细胞介素-1β（rhIL-1β）诱导前后牙髓细胞蛋白质的差异,鉴定2组间差异表达的蛋白质。方法采用双向凝胶电泳技术分离牙髓细胞全蛋白,获得对照组和诱导组牙髓细胞蛋白质图谱,Image Master2D Elit 5.0软件分析确认差异表达蛋白。通过基质辅助的激光解吸飞行时间质谱对差异表达的蛋白质点进行质谱鉴定,得到肽质量指纹图谱。结果比较对照组和诱导组蛋白质图谱,发现了39个蛋白质点差异明显。其中15个蛋白质点在诱导组高表达,新增13个蛋白质点,7个蛋白质点低表达,4个蛋白质点仅在对照组中表达;质谱鉴定后,10个蛋白得到确认。结论牙髓细胞对rhIL-1β的应答反应是一个非常复杂的过程,多种蛋白质分子涉及其中。鉴定了牙髓细胞中与rhIL-1β作用密切相关的2个差异蛋白,为探索早期牙髓炎的应答机制提供了新的线索和思路。