口腔扁平苔蘚患者血清的蛋白質(zhì)組學(xué)研究
發(fā)布時(shí)間：2018-08-21 11:33
【摘要】：口腔扁平苔蘚（oral lichen planus, OLP）是一種可長期反復(fù)發(fā)作的慢性口腔黏膜病，因長期糜爛病損易癌變，WHO將其列入癌前狀態(tài)。OLP的發(fā)生發(fā)展與心理因素、內(nèi)分泌因素、免疫因素、感染因素等有關(guān)，但具體發(fā)病機(jī)制尚不明確。因?qū)ζ浒l(fā)病機(jī)制研究不明，治療一直沒有顯著的效果。因此對于口腔扁平苔蘚發(fā)病機(jī)制的探尋成為目前急需解決的問題。近些年來，生命科學(xué)出現(xiàn)了一個(gè)嶄新的時(shí)代——蛋白質(zhì)組時(shí)代，蛋白質(zhì)組學(xué)是繼基因組研究基礎(chǔ)上發(fā)展起來的一門科學(xué)，是通過對基因轉(zhuǎn)錄、合成后的產(chǎn)物蛋白質(zhì)動(dòng)態(tài)和整體水平上的研究，可以直接闡明生命在生理和病理?xiàng)l件下可能的發(fā)病機(jī)制。蛋白質(zhì)組技術(shù)發(fā)展迅速，雙向凝膠電泳是最主要的分離方法，雙向熒光差異凝膠電泳（two-dimensional fluorescencedifference in gel electrophoresis，2-D DIGE）技術(shù)是在傳統(tǒng)的雙向凝膠電泳的基礎(chǔ)上將對照組和實(shí)驗(yàn)組樣品分別用熒光染料標(biāo)記，然后混和在一起再進(jìn)行雙向凝膠電泳，根據(jù)不同熒光材料激發(fā)不同的波長，使差異蛋白點(diǎn)以不同的顏色表現(xiàn)在2D膠的圖像上，從而檢測到蛋白樣品間微小表達(dá)差異，有更高的靈敏性、重復(fù)性、準(zhǔn)確性�，F(xiàn)在此技術(shù)已經(jīng)廣泛應(yīng)用于國內(nèi)外醫(yī)學(xué)研究，尤其是應(yīng)用此技術(shù)鑒定出與癌癥相關(guān)的分子標(biāo)記物，從而為抑制細(xì)胞惡性分化，為開發(fā)治療藥物奠定理論基礎(chǔ)。但是目前國內(nèi)外研究對于運(yùn)用2-D DIGE技術(shù)研究口腔扁平苔蘚的報(bào)道十分罕見，因此本研究采用此技術(shù)篩選并且鑒定口腔扁平苔蘚患者和健康人血清中的差異蛋白，進(jìn)一步探討其可能的作用機(jī)制。 目的：應(yīng)用2-D DIGE以及液相色譜-質(zhì)譜聯(lián)用技術(shù)篩選、鑒定口腔扁平苔蘚患者及健康人血清差異蛋白，進(jìn)而分析口腔扁平苔蘚患者與健康人相關(guān)蛋白水平有哪些變化，差異蛋白的功能及可能與口腔扁平苔蘚有哪些關(guān)系，使口腔扁平苔蘚的早期診斷預(yù)防、生物蛋白靶向治療、預(yù)后判斷趨于可能，同時(shí)也可證實(shí)雙向熒光差異凝膠電泳在口腔扁平苔蘚研究中具有實(shí)用價(jià)值。 方法： 1兩種提取人血清蛋白方法比較：采用甲醇、10mM碳酸氫銨沉淀蛋白法和苯酚抽提的方法分別提取健康志愿者的血清蛋白，Bradford法測蛋白含量，進(jìn)行十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳，考馬斯亮藍(lán)染色，脫色后對比兩種方法得到的蛋白條帶，選擇適合人血清蛋白的提取方法。 2雙向熒光差異凝膠電泳：隨機(jī)選出6例口腔扁平苔蘚初診患者和6例健康志愿者并抽取靜脈血，分離血清，提取血清蛋白，Bradford法測定蛋白含量，十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳檢測各樣品蛋白重復(fù)性、蛋白降解情況。雙向熒光差異凝膠電泳分離蛋白，Typhoon9410掃描儀對2-D DIGE膠進(jìn)行掃描，然后Deep Purple染色，DeCyderTM差異分析軟件6.5版搜索差異點(diǎn),將差異表達(dá)量大于1.2倍的蛋白質(zhì)點(diǎn)進(jìn)行標(biāo)記并生成挖點(diǎn)坐標(biāo)文件，導(dǎo)出后用Ettan spot picker挖點(diǎn)。 3液相色譜-質(zhì)譜聯(lián)用技術(shù)鑒定差異蛋白：挖出蛋白膠粒清洗、Trypsin溶液酶解成多肽，LTQ XL增強(qiáng)型二維線性離子阱質(zhì)譜儀鑒定，，應(yīng)用Bioworks Browser3.3.1軟件通過ESQUEST運(yùn)算方法在蛋白數(shù)據(jù)庫（NCBI中的human fasta數(shù)據(jù)2013.12.10）搜索差異蛋白的酶解肽段，以確定被測差異蛋白可能的名稱，從而鑒定可能的蛋白質(zhì)。 結(jié)果： 1選擇人血清蛋白提取的最適合方法：本研究采用了甲醇、10mM碳酸氫銨沉淀蛋白法和苯酚抽提的方法分別提取血清蛋白，Bradford法測蛋白含量，進(jìn)行十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳，比較發(fā)現(xiàn)用苯酚抽提方法得到的電泳條帶更清晰，蛋白純化程度更高，因此選擇苯酚抽提的方法用于本研究。 2十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳顯示：檢測各樣品蛋白顯示各樣品蛋白條帶清晰，蛋白重復(fù)性較好，無明顯蛋白丟失現(xiàn)象。 3雙向熒光凝膠電泳結(jié)果 口腔扁平苔蘚患者血清與健康人血清因用不同顏色的熒光染料標(biāo)記，在2-D DIGE分離時(shí)，一些蛋白點(diǎn)是兩種顏色的重合，即呈現(xiàn)出黃色，說明在口腔扁平苔蘚患者血清和健康人中均有相同程度的表達(dá)，而另外還有一些各自特異高表達(dá)的蛋白點(diǎn)通過各自熒光染色的顏色顯現(xiàn)出來，即呈現(xiàn)出紅色或者綠色。經(jīng)6個(gè)生物學(xué)重復(fù)，從而測得差異蛋白點(diǎn)平均為（583±183）個(gè),選出重復(fù)性較好的差異點(diǎn)20個(gè)鑒定，得到12種蛋白，其中7種蛋白在患者血清中表達(dá)量降低，5種蛋白表達(dá)量升高。 4液相色譜-質(zhì)譜鑒定差異蛋白：鑒定出12種差異蛋白，分別是結(jié)合珠蛋白、銅藍(lán)蛋白、血清補(bǔ)體C3、載脂蛋白A-Ⅰ、激肽原、維生素D、α-2-巨球蛋白、人抗凝血酶Ⅲ、分泌型IgA、血清補(bǔ)體成分C9、人類因子B、β-1-金屬結(jié)合球蛋白。 結(jié)論： 1苯酚抽提的方法更適合人血清蛋白的提取。 2口腔扁平苔蘚患者血清中存在差異蛋白,差異蛋白點(diǎn)平均為（583±183）個(gè),重復(fù)性較好的差異蛋白12個(gè)并且表達(dá)量發(fā)生變化，其中7個(gè)在患者血清中表達(dá)量降低，5個(gè)表達(dá)量升高。 3口腔扁平苔蘚患者血清中質(zhì)譜鑒定到得蛋白12個(gè)，其中結(jié)合珠蛋白、銅藍(lán)蛋白、血清補(bǔ)體C3、載脂蛋白A-Ⅰ、激肽原、維生素D、α-2-巨球蛋白、人抗凝血酶Ⅲ、分泌型IgA、血清補(bǔ)體成分C9、人類因子B可能與口腔扁平苔蘚發(fā)病機(jī)制密切相關(guān)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R781.5
本文編號(hào)：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R781.5
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 張梅潔;對化濕行瘀清熱方劑治療口腔扁平苔蘚前后血清中差異蛋白的研究[D];河北醫(yī)科大學(xué);2016年
本文編號(hào)：2195586
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