口腔黏膜白斑恶变差异蛋白质组学研究以及大学生龋病流行病学调查分析报告

发布时间：2018-06-08 08:38

本文选题：口腔白斑恶变 + 口腔鳞癌　；参考：《大连医科大学》2014年硕士论文

【摘要】：口腔黏膜白斑（oral leukoplakia，OLK）是口腔黏膜上的白色斑块或白色斑片，不能以临床和组织病理学的方法诊断为其他任何疾病者。OLK恶变率高，可恶变成严重危害人类生命健康的口腔黏膜鳞状细胞癌（oral squamous cellcarcinoma，OSCC)，其中非均质型白斑恶变率约为13%-17.5%，甚至在某些地区OLK恶变率可高达50%-70%。口腔黏膜鳞状细胞癌是世界十大肿瘤之一，在全身恶性肿瘤中排第六位。有研究结果显示，口腔鳞状细胞癌中有17%-35%是由口腔白斑恶变而来，因此研究口腔白斑恶变分子机制，筛选恶变相关蛋白，寻找恶变分子标志物，对口腔白斑恶变的早期发现、早期诊断及治疗，有效改善预后具有重要的科学意义和临床应用前景。目的：本项研究通过蛋白质组学检测技术筛检口腔黏膜白斑恶变相关蛋白，并分析OLK恶变前后差异蛋白的相关信息，为深入研究口腔黏膜白斑恶变分子机制，探寻口腔白斑恶变分子标志物及关键治疗靶点提供重要的研究基础。方法：本实验采用我们自行建立的口腔黏膜白斑恶变细胞模型，即人口腔黏膜白斑细胞系DOK及其诱导恶变细胞系口腔鳞状上皮癌细胞OSCC-B+D为实验对象，运用相对定量蛋白质组学方法高通量筛检DOK及OSCC-B+D细胞间蛋白质组的表达差异。首先采用稳定同位素二甲基标记技术，将CH2O和CD2O分别标记进入人口腔白斑细胞DOK-G及其恶变细胞OSCC-B+D的蛋白酶解肽段，然后将两种细胞样品混合通过高效液相色谱进行分离，最后样品进入具有高灵敏性、可微量上样的Orbitrap Elite质谱仪进行多肽检测，并通过MaxQuant软件（v1.1.2.5），在IPIHuman.（v3.87）数据库中对质谱检测结果进行蛋白质鉴定。结果：口腔黏膜白斑细胞DOK及其恶变细胞OSCC-B+D样品经过HPLC-MS/MS检测分析后，共鉴定出1459个蛋白，取三次重复实验的共同鉴定结果，筛检出931个蛋白。对获得的筛检结果进行进一步过滤，控制相对标准偏差RSD＜35%，最终共获得621个蛋白。分析和比较DOK及其恶变细胞OSCC-B+D蛋白质组的表达变化，筛选出197个恶变前后的差异蛋白，其中125个蛋白在恶变后呈显著性上调表达（平均氘氢比值2），72个蛋白在恶变后呈显著性下调表达（平均氘氢比值0.5）。我们采用uniprotKB数据库将197个差异蛋白按照其细胞内定位，蛋白分子功能及其参与的生物学过程进行分类分析，获得差异蛋白相关信息。依据细胞内定位信息，提示差异蛋白中定位于细胞质（cytoplasm）和细胞核（nucleus）的蛋白最多，均占22%，其他依次递减定位于线粒体（mitochondrion，12%），细胞膜（plasma membrane，11%），内质网（endoplasmic reticulum，8%），integralto membrane（7%）；依据蛋白分子功能信息，提示DNA结合（DNA binding）蛋白最多，约占9%,其次是RNA结合（RNA binding）蛋白，约占6%,其他依次递减为载体活性（transporter activity）蛋白（5%）,GTP酶调节活性（GTPase activity，3%）蛋白，转录监管活性（transcription regulator activity，2%）蛋白等；依据参与的生物学过程信息，提示参与核糖核酸/脱氧核糖核酸/核苷酸/核小体的代谢调节（regulation of RNA/DNA/nucleotide/nucleosome metabolic process）蛋白最多约占23%，其次为蛋白代谢（protein metabolism）约占12%，其他依次递减为转运（transport，11%）蛋白，细胞生长（cell growth，9%），细胞凋亡（apoptosis，7%），信号转导（signal transduction，2%）以及免疫反应（immuneresponse，，2%）相关蛋白。结论：本项研究通过高通量筛检口腔黏膜白斑细胞恶变前后的差异蛋白质组，发现197个蛋白质呈差异表达。根据差异蛋白相关信息，初步推测OLK恶变发生的可能机制：参与OLK恶变发生的蛋白大多定位于细胞核与细胞质中，DNA/RNA结合蛋白及参与核糖核酸/脱氧核糖核酸/核苷酸/核小体代谢调节的蛋白质在OLK恶变进程中发挥重要作用，DNA/RNA功能及代谢异常可能是OLK恶变的重要机制。本项研究为进一步深入细致地探索OLK恶变分子机制，筛选口腔黏膜白斑恶变分子标志物和关键治疗靶点提供有力的研究基础和科学依据。目的：通过对大学生进行口腔健康检查，了解这一人群的龋病患病情况，为制订口腔保健治疗方案提供科学依据，同时充实大学生龋病流行病学资料。方法：参照WHO口腔健康调查基本方法以及第三次全国口腔健康流行病学调查方案制定口腔检查表格，对大连理工大学570名大学生进行龋齿患病情况研究，调查内容包括龋均、患龋率等,以视诊结合探诊的方式检查后将结果记录在表格内。结果：570名大学生患龋率为58.07%，龋失补牙数共1013颗牙（龋牙数766颗，因龋失牙数10颗，因龋补牙数237颗），龋均为1.78±2.335，龋齿充填率为23.40%。男生和女生在患龋率分别为54.3%和73.5%，两者差异具有统计学意义（P＜0.05）；男生和女生的龋均分别为1.54±2.130和2.73±2.845，两者差异具有统计学意义（P＜0.05）。城市生源和农村生源学生的患龋率分别为62.1%和53.2%，两者差异具有统计学意义（P＜0.05）；城市生源和农村生源的龋均分别为1.95±2.322和1.56±2.389，两者差异无统计学意义（P＞0.05）。不同年龄分组的患龋率分别为58.5%（18-20岁）、54.9%（21-23岁）、64.3%（＞24岁），三者差异无统计学意义（P＞0.05）；不同年龄分组的龋均分别为1.72±2.108（18-20岁）、1.63±2.325（21-23岁）、2.36±2.968（＞24岁），三者组间比较差异具有统计学意义（P＜0.05）。对不同年龄分组的龋均进行两两比较后显示18-20岁年龄段与21-23岁年龄段的龋均无统计学差异，但二者与＞24岁年龄组均有统计学差异，随着年龄增长，龋均也随之增加。进一步对患龋牙位进行分析比较之后，发现磨牙患龋水平明显高于其他牙齿，其中下颌磨牙患龋率高于上颌磨牙，二者具有统计学差异（P＜0.05）。结论：通过本次龋病流行病学调查，结果表明大学生龋齿患病率较高，但龋齿充填率较低。本次调查显示大学生的龋病流行特点为：龋齿患病率女性高于男性、城市高于乡村；龋均水平女性高于男性、24岁以上年龄组高于18-23岁年龄组。患龋牙位的流行病特点为：磨牙患龋水平高于其他牙齿，下颌磨牙患龋水平高于上颌磨牙。
[Abstract]:Oral leukoplakia (oral leukoplakia, OLK) is a white plaque or white plaque on the oral mucosa. It can not be diagnosed as any other disease by clinical and histopathological methods, and the rate of malignant transformation of.OLK is high, and the evil becomes the oral squamous cellcarcinoma (OSCC), which is serious harm to human life and health (oral squamous cellcarcinoma, OSCC). The malignant change rate of white leukoplakia is about 13%-17.5%. Even in some areas, the rate of OLK malignant change can be as high as 50%-70%. oral mucosa squamous cell carcinoma is one of the ten largest tumors in the world, and it is ranked sixth in the malignant tumor of the whole body. The results of research show that the 17%-35% is malignant in oral squamous cell carcinoma, and therefore, the malignant change of oral leukoplakia is studied. Submechanism, screening of malignant change related proteins and finding malignant molecular markers, early detection of oral leukoplakia malignancy, early diagnosis and treatment, and effective improvement of prognosis have important scientific significance and clinical application prospects.
Objective: This study screened oral leukoplakia malignancy related proteins by proteomic technology and analyzed the related information of differential proteins before and after OLK malignancy, and provided an important research basis for the in-depth study of the molecular mechanism of oral leukoplakia and the exploration of biomarkers and key therapeutic targets for oral leukoplakia.
Methods: in this experiment, the oral leukoplakic leukoplakia malignant cell model, the human oral leukoplakic leukoplakia cell line DOK and the oral squamous cell carcinoma cell line OSCC-B+D induced by the malignant cell line, were used as the experimental object. The relative quantitative proteomics method was used to detect the differential expression of the protein group between DOK and OSCC-B+D cells. Firstly, CH2O and CD2O were used to label the CH2O and CD2O respectively into the protease peptide segments of the human oral leukoplakia cell DOK-G and its malignant cell OSCC-B+D, and then the samples were mixed by high performance liquid chromatography, and the final samples were highly sensitive, and the Orbitrap Elite could be found in the sample. The peptide was detected by mass spectrometer and identified by MaxQuant software (v1.1.2.5) in the IPIHuman. (v3.87) database.
Results: after HPLC-MS/MS detection and analysis of oral leukoplakia leukoplakia DOK and its malignant cell OSCC-B+D samples, 1459 proteins were identified and 931 proteins were screened out from three repeated experiments. The results of screening were further filtered, and the standard deviation was RSD < 35%, and 621 proteins were obtained. By analyzing and comparing the changes in the expression of DOK and its malignant cell OSCC-B+D proteome, 197 differential proteins were screened, of which 125 proteins were significantly up-regulated after the malignant change (the average deuterium hydrogen ratio 2), and the 72 proteins decreased significantly after the malignant change (the ratio of deuterium hydrogen was 0.5).
We used the uniprotKB database to classify the 197 differentially proteins according to their intracellular localization, protein molecular function and their biological processes to obtain differential protein related information. According to the intracellular location information, the most proteins located in the cytoplasm (cytoplasm) and the nucleus (nucleus) in the differential proteins were the most, which accounted for 22%. Others descended in sequence in mitochondria (mitochondrion, 12%), cell membrane (plasma membrane, 11%), endoplasmic reticulum (endoplasmic reticulum, 8%), integralto membrane (7%). According to protein molecular function information, DNA binding (DNA binding) protein was most, accounting for about 9%, followed by RNA binding (RNA) protein, accounting for 6%, and the others descended successively in turn. Carrier activity (transporter activity) protein (5%), GTP enzyme regulatory activity (GTPase activity, 3%) protein, transcriptional regulatory activity (transcription regulator activity, 2%), etc., and in accordance with the biological process information involved in the metabolic regulation of ribonucleic acid / deoxynuclear nucleic acid / nucleosome (regulation of RNA/DNA/nucl) Eotide/nucleosome metabolic process) protein accounted for about 23%, followed by protein metabolism (protein metabolism) accounting for about 12%, others descended in turn to transport (transport, 11%) protein, cell growth (cell growth, 9%), apoptosis (apoptosis, 7%), signal transduction (signal transduction, 2%), and immune response (immuneresponse, 2%) related proteins.
Conclusion: in this study, 197 proteins were found to be differentially expressed in the differential proteome before and after the malignant transformation of leukoplakia cells in the oral mucosa. According to the information of differential protein, the possible mechanism of OLK malignant transformation was preliminarily deduced: most of the proteins involved in the OLK malignant transformation were located in the nucleus and cytoplasm, and DNA/RNA combined with eggs. The protein that Rhizoma Bletillae participates in RNA / deoxyribonucleic acid / nucleosome metabolic regulation plays an important role in the OLK malignant transformation process. The DNA/RNA function and metabolic abnormalities may be an important mechanism for the malignant transformation of OLK. This study is to further explore the OLK malignant molecular mechanism and screen the biomarkers of oral leukoplakia malignant molecules. The material and key therapeutic targets provide strong basis for research and scientific basis.
Objective: to understand the prevalence of dental caries in this population by oral health examination to college students, to provide a scientific basis for the formulation of oral health care treatment scheme and to enrich the epidemiological data of dental caries for college students.
Methods: according to the basic methods of the WHO oral health survey and the third national oral health epidemiological survey, 570 college students in Dalian University of Technology were studied for dental caries, including caries and caries. The results were recorded in the form after inspection. Inside.
Results: the caries rate of 570 college students was 58.07%, the number of dental caries was 1013 teeth (766 teeth, 10 teeth, 237 dental caries), and the caries were 1.78 + 2.335. The dental caries rate of 23.40%. boys and girls was 54.3% and 73.5%, respectively, and the difference was statistically significant (P < 0.05), and the caries of boys and girls were both. The differences were 1.54 + 2.130 and 2.73 + 2.845 respectively (P < 0.05). The rate of dental caries of urban and rural students were 62.1% and 53.2% respectively (P < 0.05), and the caries of urban and rural sources were 1.95 + 2.322 and 1.56 + 2.389, respectively (P > 0.05) the incidence of caries in different age groups was 58.5% (18-20 years old), 54.9% (21-23 years old) and 64.3% (> 24 years), and there was no significant difference in three (P > 0.05). The average caries in different age groups were 1.72 + 2.108 (18-20 years old), 1.63 + 2.325 (> 54.9% years old), and the differences were statistically significant (P <). There was no significant difference in caries between the age group of 18-20 years and the age group at the age of 21-23, but there was a statistical difference between the two and the 21-23 years old age group, but the caries increased with the age of 24. After the analysis and comparison of the dental caries, it was found that the level of dental caries was significantly higher than that of the 21-23. The incidence of dental caries in mandibular molars was higher than that in maxillary molars, and the difference between the two groups was statistically significant (P < 0.05).
Conclusion: through the epidemiological survey of caries, the prevalence rate of dental caries of college students is higher, but the rate of dental caries filling is low. This survey shows that the prevalence of dental caries in college students is higher than that of men, the city is higher than that of the countryside, the average caries level in women is higher than that of men, and the age group over 24 years old is higher than the age group of 18-23 years. The epidemiological characteristics of dental caries are: the level of molar caries is higher than that of other teeth, and the level of caries of mandibular molars is higher than that of maxillary molars.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.85;R781.1

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