口腔扁平苔藓患者血清的蛋白质组学研究
发布时间：2018-08-21 11:33
【摘要】：口腔扁平苔藓（oral lichen planus, OLP）是一种可长期反复发作的慢性口腔黏膜病，因长期糜烂病损易癌变，WHO将其列入癌前状态。OLP的发生发展与心理因素、内分泌因素、免疫因素、感染因素等有关，但具体发病机制尚不明确。因对其发病机制研究不明，治疗一直没有显著的效果。因此对于口腔扁平苔藓发病机制的探寻成为目前急需解决的问题。近些年来，生命科学出现了一个崭新的时代——蛋白质组时代，蛋白质组学是继基因组研究基础上发展起来的一门科学，是通过对基因转录、合成后的产物蛋白质动态和整体水平上的研究，可以直接阐明生命在生理和病理条件下可能的发病机制。蛋白质组技术发展迅速，双向凝胶电泳是最主要的分离方法，双向荧光差异凝胶电泳（two-dimensional fluorescencedifference in gel electrophoresis，2-D DIGE）技术是在传统的双向凝胶电泳的基础上将对照组和实验组样品分别用荧光染料标记，然后混和在一起再进行双向凝胶电泳，根据不同荧光材料激发不同的波长，使差异蛋白点以不同的颜色表现在2D胶的图像上，从而检测到蛋白样品间微小表达差异，有更高的灵敏性、重复性、准确性。现在此技术已经广泛应用于国内外医学研究，尤其是应用此技术鉴定出与癌症相关的分子标记物，从而为抑制细胞恶性分化，为开发治疗药物奠定理论基础。但是目前国内外研究对于运用2-D DIGE技术研究口腔扁平苔藓的报道十分罕见，因此本研究采用此技术筛选并且鉴定口腔扁平苔藓患者和健康人血清中的差异蛋白，进一步探讨其可能的作用机制。 目的：应用2-D DIGE以及液相色谱-质谱联用技术筛选、鉴定口腔扁平苔藓患者及健康人血清差异蛋白，进而分析口腔扁平苔藓患者与健康人相关蛋白水平有哪些变化，差异蛋白的功能及可能与口腔扁平苔藓有哪些关系，使口腔扁平苔藓的早期诊断预防、生物蛋白靶向治疗、预后判断趋于可能，同时也可证实双向荧光差异凝胶电泳在口腔扁平苔藓研究中具有实用价值。 方法： 1两种提取人血清蛋白方法比较：采用甲醇、10mM碳酸氢铵沉淀蛋白法和苯酚抽提的方法分别提取健康志愿者的血清蛋白，Bradford法测蛋白含量，进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，考马斯亮蓝染色，脱色后对比两种方法得到的蛋白条带，选择适合人血清蛋白的提取方法。 2双向荧光差异凝胶电泳：随机选出6例口腔扁平苔藓初诊患者和6例健康志愿者并抽取静脉血，分离血清，提取血清蛋白，Bradford法测定蛋白含量，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测各样品蛋白重复性、蛋白降解情况。双向荧光差异凝胶电泳分离蛋白，Typhoon9410扫描仪对2-D DIGE胶进行扫描，然后Deep Purple染色，DeCyderTM差异分析软件6.5版搜索差异点,将差异表达量大于1.2倍的蛋白质点进行标记并生成挖点坐标文件，导出后用Ettan spot picker挖点。 3液相色谱-质谱联用技术鉴定差异蛋白：挖出蛋白胶粒清洗、Trypsin溶液酶解成多肽，LTQ XL增强型二维线性离子阱质谱仪鉴定，，应用Bioworks Browser3.3.1软件通过ESQUEST运算方法在蛋白数据库（NCBI中的human fasta数据2013.12.10）搜索差异蛋白的酶解肽段，以确定被测差异蛋白可能的名称，从而鉴定可能的蛋白质。 结果： 1选择人血清蛋白提取的最适合方法：本研究采用了甲醇、10mM碳酸氢铵沉淀蛋白法和苯酚抽提的方法分别提取血清蛋白，Bradford法测蛋白含量，进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，比较发现用苯酚抽提方法得到的电泳条带更清晰，蛋白纯化程度更高，因此选择苯酚抽提的方法用于本研究。 2十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示：检测各样品蛋白显示各样品蛋白条带清晰，蛋白重复性较好，无明显蛋白丢失现象。 3双向荧光凝胶电泳结果 口腔扁平苔藓患者血清与健康人血清因用不同颜色的荧光染料标记，在2-D DIGE分离时，一些蛋白点是两种颜色的重合，即呈现出黄色，说明在口腔扁平苔藓患者血清和健康人中均有相同程度的表达，而另外还有一些各自特异高表达的蛋白点通过各自荧光染色的颜色显现出来，即呈现出红色或者绿色。经6个生物学重复，从而测得差异蛋白点平均为（583±183）个,选出重复性较好的差异点20个鉴定，得到12种蛋白，其中7种蛋白在患者血清中表达量降低，5种蛋白表达量升高。 4液相色谱-质谱鉴定差异蛋白：鉴定出12种差异蛋白，分别是结合珠蛋白、铜蓝蛋白、血清补体C3、载脂蛋白A-Ⅰ、激肽原、维生素D、α-2-巨球蛋白、人抗凝血酶Ⅲ、分泌型IgA、血清补体成分C9、人类因子B、β-1-金属结合球蛋白。 结论： 1苯酚抽提的方法更适合人血清蛋白的提取。 2口腔扁平苔藓患者血清中存在差异蛋白,差异蛋白点平均为（583±183）个,重复性较好的差异蛋白12个并且表达量发生变化，其中7个在患者血清中表达量降低，5个表达量升高。 3口腔扁平苔藓患者血清中质谱鉴定到得蛋白12个，其中结合珠蛋白、铜蓝蛋白、血清补体C3、载脂蛋白A-Ⅰ、激肽原、维生素D、α-2-巨球蛋白、人抗凝血酶Ⅲ、分泌型IgA、血清补体成分C9、人类因子B可能与口腔扁平苔藓发病机制密切相关。
[Abstract]:Oral lichen planus (OLP) is a chronic oral mucosal disease which can recur for a long time. It is easy to be cancerous because of long-term erosion. The occurrence and development of OLP is related to psychological factors, endocrine factors, immune factors, infection factors and so on, but the specific pathogenesis is not clear. In recent years, a new era has emerged in the life sciences - proteomics era. Proteomics is a science developed on the basis of genomic research, through the study of the pathogenesis of oral lichen planus. The study of gene transcription, protein dynamics and overall levels of the synthesized products can directly elucidate the possible pathogenesis of life under physiological and pathological conditions. Based on the traditional two-dimensional gel electrophoresis, the control group and the experimental group were labeled with fluorescent dyes respectively, and then mixed together for two-dimensional gel electrophoresis. It has been widely used in medical research at home and abroad, especially in the identification of molecular markers related to cancer, so as to inhibit the malignant differentiation of cells and lay a theoretical foundation for the development of therapeutic drugs. Domestic and foreign studies on the use of 2-D DIGE technology to study oral lichen planus are very rare, so this study used this technology to screen and identify the differences in serum proteins in patients with oral lichen planus and healthy people, to further explore its possible mechanism.
Objective: To identify the serum differential proteins in patients with oral lichen planus (OLP) and healthy people by 2-D DIGE and liquid chromatography-mass spectrometry (LC-MS), and then to analyze the changes of the levels of related proteins in patients with OLP and healthy people, the function of the differential proteins and the possible relationship between the different proteins and OLP. Early diagnosis and prevention, targeted therapy of biological protein, and prognosis judgment tend to be possible. At the same time, two-dimensional fluorescence differential gel electrophoresis has practical value in the study of oral lichen planus.
Method:
1 Comparison of two methods for extracting human serum protein: methanol, 10 mM ammonium bicarbonate precipitation protein and phenol extraction were used to extract serum protein from healthy volunteers, Bradford method was used to determine the protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, decolorization and comparison of the eggs obtained by the two methods. White strip, choose suitable extraction method for human serum protein.
2-Dimensional Fluorescence Differential Gel Electrophoresis: Six patients with oral lichen planus and six healthy volunteers were randomly selected and venous blood samples were taken. Serum protein was isolated and extracted. Protein content was determined by Bradford method. Protein repeatability and protein degradation were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein was separated by isogel electrophoresis. Typhoon 9410 scanner scanned 2-D DIGE gel. Deep Purple staining and DeCyderTM difference analysis software version 6.5 searched for the difference points. Protein points with different expression more than 1.2 times were labeled and digged coordinate files were generated. Then the digging points were deduced by Ettan spot picker.
3. Identification of differentially expressed proteins by liquid chromatography-mass spectrometry (LC-MS): dig-out protein colloidal cleaning, hydrolysis of Trypsin solution into peptides, identification by LTQ-XL enhanced two-dimensional linear ion trap mass spectrometer, ESQUEST operation method using Bioworks Browser 3.3.1 software in the protein database (human FASTA data in NCBI 2013.12.10) to search for differentially expressed proteins of enzymes The peptide segment was determined to identify the possible name of the differential protein and identify the possible protein.
Result:
1. Choose the most suitable method to extract human serum protein. Methanol, 10 mM ammonium bicarbonate precipitation method and phenol extraction were used to extract serum protein. Bradford method was used to determine the protein content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out. The results showed that the electrophoretic bands obtained by phenol extraction method were clearer. It is clear that the protein is more purified, so phenol extraction method is selected for this study.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein bands of each sample were clear, the protein repeatability was good, and there was no obvious protein loss.
3 two way fluorescence gel electrophoresis results
Serum of patients with oral lichen planus and serum of healthy persons were labeled with fluorescent dyes of different colors. When 2-D DIGE was separated, some protein spots were overlapped by two colors, that is, yellow, indicating the same level of expression in serum of patients with oral lichen planus and serum of healthy persons. In addition, there were some eggs with specific high expression. The white dots showed red or green color by fluorescence staining. After six biological repetitions, the average number of differential protein spots was (583
4. Identification of differentially expressed proteins by liquid chromatography-mass spectrometry: 12 differentially expressed proteins were identified as binding globin, ceruloplasmin, serum complement C3, apolipoprotein A-I, kininogen, vitamin D, alpha-2-macroglobulin, human antithrombin III, secretory IgA, serum complement C9, human factor B, beta-1-metal binding globulin.
Conclusion:
1 phenol extraction method is more suitable for the extraction of human serum protein.
2 There were differential proteins in the serum of patients with oral lichen planus. The average number of differential protein spots was (583 65
3 Twelve proteins were identified in the serum of patients with oral lichen planus by mass spectrometry. The binding globin, ceruloplasmin, serum complement C3, apolipoprotein A-I, kininogen, vitamin D, alpha-2-macroglobulin, human antithrombin III, secretory IgA, serum complement C9 and human factor B may be closely related to the pathogenesis of oral lichen planus.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.5
本文编号：2195586
[Abstract]:Oral lichen planus (OLP) is a chronic oral mucosal disease which can recur for a long time. It is easy to be cancerous because of long-term erosion. The occurrence and development of OLP is related to psychological factors, endocrine factors, immune factors, infection factors and so on, but the specific pathogenesis is not clear. In recent years, a new era has emerged in the life sciences - proteomics era. Proteomics is a science developed on the basis of genomic research, through the study of the pathogenesis of oral lichen planus. The study of gene transcription, protein dynamics and overall levels of the synthesized products can directly elucidate the possible pathogenesis of life under physiological and pathological conditions. Based on the traditional two-dimensional gel electrophoresis, the control group and the experimental group were labeled with fluorescent dyes respectively, and then mixed together for two-dimensional gel electrophoresis. It has been widely used in medical research at home and abroad, especially in the identification of molecular markers related to cancer, so as to inhibit the malignant differentiation of cells and lay a theoretical foundation for the development of therapeutic drugs. Domestic and foreign studies on the use of 2-D DIGE technology to study oral lichen planus are very rare, so this study used this technology to screen and identify the differences in serum proteins in patients with oral lichen planus and healthy people, to further explore its possible mechanism.
Objective: To identify the serum differential proteins in patients with oral lichen planus (OLP) and healthy people by 2-D DIGE and liquid chromatography-mass spectrometry (LC-MS), and then to analyze the changes of the levels of related proteins in patients with OLP and healthy people, the function of the differential proteins and the possible relationship between the different proteins and OLP. Early diagnosis and prevention, targeted therapy of biological protein, and prognosis judgment tend to be possible. At the same time, two-dimensional fluorescence differential gel electrophoresis has practical value in the study of oral lichen planus.
Method:
1 Comparison of two methods for extracting human serum protein: methanol, 10 mM ammonium bicarbonate precipitation protein and phenol extraction were used to extract serum protein from healthy volunteers, Bradford method was used to determine the protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, decolorization and comparison of the eggs obtained by the two methods. White strip, choose suitable extraction method for human serum protein.
2-Dimensional Fluorescence Differential Gel Electrophoresis: Six patients with oral lichen planus and six healthy volunteers were randomly selected and venous blood samples were taken. Serum protein was isolated and extracted. Protein content was determined by Bradford method. Protein repeatability and protein degradation were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein was separated by isogel electrophoresis. Typhoon 9410 scanner scanned 2-D DIGE gel. Deep Purple staining and DeCyderTM difference analysis software version 6.5 searched for the difference points. Protein points with different expression more than 1.2 times were labeled and digged coordinate files were generated. Then the digging points were deduced by Ettan spot picker.
3. Identification of differentially expressed proteins by liquid chromatography-mass spectrometry (LC-MS): dig-out protein colloidal cleaning, hydrolysis of Trypsin solution into peptides, identification by LTQ-XL enhanced two-dimensional linear ion trap mass spectrometer, ESQUEST operation method using Bioworks Browser 3.3.1 software in the protein database (human FASTA data in NCBI 2013.12.10) to search for differentially expressed proteins of enzymes The peptide segment was determined to identify the possible name of the differential protein and identify the possible protein.
Result:
1. Choose the most suitable method to extract human serum protein. Methanol, 10 mM ammonium bicarbonate precipitation method and phenol extraction were used to extract serum protein. Bradford method was used to determine the protein content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out. The results showed that the electrophoretic bands obtained by phenol extraction method were clearer. It is clear that the protein is more purified, so phenol extraction method is selected for this study.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein bands of each sample were clear, the protein repeatability was good, and there was no obvious protein loss.
3 two way fluorescence gel electrophoresis results
Serum of patients with oral lichen planus and serum of healthy persons were labeled with fluorescent dyes of different colors. When 2-D DIGE was separated, some protein spots were overlapped by two colors, that is, yellow, indicating the same level of expression in serum of patients with oral lichen planus and serum of healthy persons. In addition, there were some eggs with specific high expression. The white dots showed red or green color by fluorescence staining. After six biological repetitions, the average number of differential protein spots was (583
4. Identification of differentially expressed proteins by liquid chromatography-mass spectrometry: 12 differentially expressed proteins were identified as binding globin, ceruloplasmin, serum complement C3, apolipoprotein A-I, kininogen, vitamin D, alpha-2-macroglobulin, human antithrombin III, secretory IgA, serum complement C9, human factor B, beta-1-metal binding globulin.
Conclusion:
1 phenol extraction method is more suitable for the extraction of human serum protein.
2 There were differential proteins in the serum of patients with oral lichen planus. The average number of differential protein spots was (583 65
3 Twelve proteins were identified in the serum of patients with oral lichen planus by mass spectrometry. The binding globin, ceruloplasmin, serum complement C3, apolipoprotein A-I, kininogen, vitamin D, alpha-2-macroglobulin, human antithrombin III, secretory IgA, serum complement C9 and human factor B may be closely related to the pathogenesis of oral lichen planus.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.5
【引证文献】
相关硕士学位论文 前1条
1 张梅洁;对化湿行瘀清热方剂治疗口腔扁平苔藓前后血清中差异蛋白的研究[D];河北医科大学;2016年
本文编号：2195586
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