人皮肤角质形成细胞的蛋白质组学研究

发布时间：2018-06-02 01:03

本文选题：人皮肤角质形成细胞 + 蛋白质组　；参考：《暨南大学》2007年博士论文

【摘要】： 本论文建立了人皮肤角质形成细胞(Keratinocyte，KC)和人皮肤表皮蛋白质组学研究的核心技术平台，并在此基础上，分别从时间、病理状态(银屑病)和角质形成细胞特异的细胞因子(人成纤维细胞生长因子7和人成纤维细胞生长因子10，即hFGF-7和hFGF-10)刺激等三个层面对人皮肤角质形成细胞蛋白质组的影响进行了初步研究，为阐明体外长期培养对人皮肤角质形成细胞影响的可能机制、皮肤病(银屑病)的发病机制、以及角质形成细胞特异的细胞因子对人皮肤角质形成细胞作用的可能机制等提供理论依据。第一章长期培养对人皮肤角质形成细胞的影响目的：建立人皮肤角质形成细胞的体外分离、培养和传代的方法，并建立人皮肤角质形成细胞蛋白质组学研究的核心技术平台，探讨体外长期培养对人皮肤角质形成细胞蛋白质组的影响，为进一步研究体外长期培养对人皮肤角质形成细胞影响的可能机制奠定基础。方法：取幼儿环切术后包皮，用DispaseⅡ酶消化法分离表皮，用Defined K-SFM培养基进行体外培养，胰酶和EDTA联合消化传代，相差显微镜下观察培养细胞的形态，采用免疫细胞化学方法对培养的细胞进行鉴定，并用细胞计数法测定原代到第7代细胞的群体倍增时间(PDT)。制备原代和第7代体外培养的人皮肤角质形成细胞的总蛋白样品，采用非线性IPG预制胶条(pH3-10，17cm)进行第一维等电聚焦，第二维分子量垂直电泳采用12％SDS-聚丙烯酰胺凝胶。对双向凝胶电泳的条件进行优化。采用PDQuest图像分析软件对获得的双向凝胶电泳图谱进行比较分析。选取2个原代和第7代角质形成细胞的差异表达蛋白质点，进行胶内酶切、串联飞行时间质谱(ABI 4700 TOF-TOF)分析。利用MASCOT在线检索工具，将质谱检测得到的数据在NCBInr数据库进行检索，并通过半定量RT-PCR对2个差异表达蛋白质的mRNA水平进行检测。结果：体外培养的原代人皮肤角质形成细胞，细胞形态为圆形或椭圆形，随着细胞传代次数的增加，细胞形态发生很大改变：细胞形状不规则，胞核周围出现许多空泡。荧光显微镜下培养细胞的胞浆呈黄绿色，为角蛋白阳性染色。原代细胞的增殖速率较慢，约需10天达到融合，PDT为116小时，传代后细胞增殖迅速，第2、3、4代PDT显著缩短，第5代以后PDT明显延长，细胞增殖速度减慢，第8代细胞长时间不能融合。建立并优化了人皮肤角质形成细胞总蛋白的双向凝胶电泳方法，获得了蛋白质分离效果较好的双向凝胶电泳图谱。采用PDQuest软件对原代和第7代人皮肤角质形成细胞总蛋白的图谱进行分析，得到原代人皮肤角质形成细胞凝胶的匹配蛋白质点数为751，而第7代人皮肤角质形成细胞凝胶的匹配蛋白质点数为806，匹配率分别为75.3％和80.8％。采用串联飞行时间质谱对选取的2个表达量在第7代角质形成细胞中显著下调且分辨率较好的蛋白质点进行质谱分析，获得了相应的肽质量指纹图谱(PMF)，通过MASCOT在线检索工具检索NCBInr数据库，鉴定差异蛋白质1为与脂肪酸运输和代谢相关的PA-FABP，差异蛋白质2为与细胞凋亡调控相关的Galectin-7。最后通过半定量RT-PCR确证第7代角质形成细胞中PA-FABP和Galectin-7的基因转录水平较原代角质形成细胞相应的基因转录水平显著降低，并对PA-FABP和Galectin-7的生物学功能及其在体外长期培养对人皮肤角质形成细胞影响中的可能作用进行了初步分析。结论：成功建立了人皮肤角质形成细胞的体外分离、培养和传代的方法，并建立了人皮肤角质形成细胞蛋白质组学研究的核心技术平台；与原代人皮肤角质形成细胞相比，第7代细胞的PA-FABP和Galectin-7表达下调，提示细胞的脂肪酸运输和(或)代谢可能发生了改变，细胞的凋亡调控能力可能下降。第二章正常和银屑病表皮的蛋白质组学研究目的：建立人皮肤表皮蛋白质组学研究的核心技术平台，应用蛋白质组学的方法研究正常和寻常型银屑病表皮蛋白质组的差异，进而由差异蛋白质推测银屑病发生和发展的可能机制。方法：取正常人环切术后包皮和寻常型银屑病患者皮损区皮肤，用DispaseⅡ酶消化法分离表皮，分别制备正常和寻常型银屑病患者皮损区表皮的总蛋白样品，采用非线性IPG预制胶条(pH3-10，17cm)进行第一维等电聚焦，第二维分子量垂直电泳采用12％SDS-聚丙烯酰胺凝胶。采用PDQuest图像分析软件对获得的双向凝胶电泳图谱进行比较分析。选取正常和寻常型银屑病患者皮损区表皮的差异表达蛋白质点，进行胶内酶切、串联飞行时间质谱(ABI 4700 TOF-TOF)分析。利用MASCOT在线检索工具，将质谱检测得到的数据在NCBInr数据库进行检索，并通过半定量RT-PCR对鉴定的差异表达蛋白质aB-Crystallin的mRNA水平进行检测。结果：建立了人皮肤表皮总蛋白的双向凝胶电泳方法，获得了蛋白质分离效果较好的双向凝胶电泳图谱。采用PDQuest软件对正常和寻常型银屑病患者皮损区表皮总蛋白的图谱进行分析，得到正常表皮双向电泳凝胶的匹配蛋白质点数为876，而寻常型银屑病皮损区表皮双向电泳凝胶的匹配蛋白质点数为794，匹配率分别为79.6％和72.2％。采用串联飞行时间质谱对选取的在寻常型银屑病患者皮损区表皮中5个表达量显著上调和1个表达量显著下调的差异蛋白质点进行质谱分析，获得了相应的肽质量指纹图谱(PMF)，通过MASCOT在线检索工具检索NCBInr数据库，鉴定这些差异蛋白质为一些与细胞增殖、凋亡、分化和炎症浸润等相关的蛋白质，其中在寻常型银屑病患者皮损区表皮中表达上调的5个差异蛋白质点分别为：S100 calcium binding protein A7-1ike 1、Psoriasin、αB-Crystallin、Peroxiredoxin 2 isoform b、Transglutaminase 3，precursor，在寻常型银屑病患者皮损区表皮中表达下调的1个差异蛋白质点为溶酶体半胱氨酸蛋白酶抑制剂Cystatin B。最后通过半定量RT-PCR确证寻常型银屑病患者皮损区表皮中αB-Crystallin的基因转录水平较正常表皮相应的基因转录水平显著升高，并初步探讨了差异蛋白质在寻常型银屑病发生和发展中的可能作用。结论：成功建立了人皮肤表皮蛋白质组学研究的核心技术平台；通过蛋白质组学的方法鉴定的差异表达蛋白质可能与寻常型银屑病角质形成细胞的过渡增殖、自发凋亡减弱和异常分化等密切相关。目的：构建含角质形成细胞特异的细胞因子hFGF-7、hFGF-10基因的重组腺病毒，通过重组腺病毒感染，在人皮肤角质形成细胞株(HaCat)中分泌表达hFGF-7、hFGF-10，并在建立人皮肤角质形成细胞蛋白质组学研究的核心技术平台的基础上，应用蛋白质组学的方法研究hFGF-7、hFGF-10的表达对HaCat细胞蛋白质组的影响，进而由差异蛋白质推测hFGF-7、hFGF-10对HaCat细胞作用的可能机制。方法：用PCR方法合成hFGF-7、hFGF-10基因片断并克隆到pET3c载体上，获得重组质粒pET3c-hFGF-7和pET3c-hFGF-10。分别以获得的两种重组质粒为模板，PCR扩增得到5’末端含BglⅡ位点、3’末端含HindⅢ位点的hFGF-7、hFGF-10基因片断，与穿梭载体pAdTrack-CMV连接，获得的重组穿梭质粒pAdTrack-CMV-hFGF-7和pAdTrack-CMV-hFGF-10经PmeⅠ线性化后，通过电转法导入含腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183中进行同源重组，获得重组腺病毒质粒pAdEasy-hFGF-7和pAdEasy-hFGF-10，经酶切鉴定后转染HEK-293细胞，在HEK-293细胞中包装并扩增重组腺病毒rAd-hFGF-7和rAd-hFGF-10。用重组腺病毒感染HaCat细胞，Western blotting检测培养上清中的重组hFGF-7、hFGF-10；MTT法检测重组腺病毒对HaCat细胞增殖的影响：重组腺病毒对HaCat细胞周期的影响通过流式细胞术检测。制备四组细胞总蛋白(HaCat、空载体腺病毒Ad感染组、重组腺病毒rAd-hFGF-7感染组和重组腺病毒rAd-hFGF-10感染组)。采用非线性IPG预制胶条(pH3-10，17cm)进行第一维等电聚焦，第二维分子量垂直电泳采用12％SDS-聚丙烯酰胺凝胶。采用PDQuest图像分析软件对获得的双向凝胶电泳图谱进行比较分析。选择4个重组腺病毒感染后表达量增加的蛋白质点，进行胶内酶切、串联飞行时间质谱(ABI 4700 TOF-TOF)分析。利用MASCOT在线检索工具，将质谱检测得到的数据在NCBInr数据库进行检索，并通过半定量RT-PCR和Western blotting对其中的差异表达蛋白质VDAC2在四组细胞中的mRNA水平和蛋白质水平进行检测。结果：成功构建了含hFGF-7、hFGF-10基因的重组腺病毒rAd-hFGF-7和rAd-hFGF-10。重组腺病毒可高效感染HaCat细胞，培养上清与hFGF-7、hFGF-10抗体呈阳性反应。重组腺病毒可促进HaCat细胞的增殖，并可改变HaCat的细胞周期，，进入S期和G2期的细胞比率增加。获得了蛋白质分离效果较好的双向凝胶电泳图谱。采用PDQuest软件对四组细胞总蛋白的图谱进行分析，得到对照组HaCat细胞和Ad感染组双向电泳凝胶的匹配蛋白质点数为516和535，匹配率分别为67.5％和70.0％；而重组腺病毒rAd-hFGF-7、rAd-hFGF-10感染组双向电泳凝胶的匹配蛋白质点数为602和587，匹配率分别为78.8％和76.8％。采用串联飞行时间质谱对选取的4个重组腺病毒感染后表达量增加的蛋白质点进行质谱分析，获得了相应的肽质量指纹图谱(PMF)，通过MASCOT在线检索工具检索NCBInr数据库，鉴定这些差异蛋白质为一些与细胞凋亡、细胞骨架调控、蛋白质降解等相关的蛋白质，包括：VDAC2、Proteasome alpha 1 subunit，isoform 2、Gelsolin-like capping protein和Protein disulfide isomerase-associated 3 precursor。最后通过半定量RT-PCR和Western blotting在mRNA和蛋白质水平上验证了差异表达蛋白质VDAC2在重组腺病毒rAd-hFGF-7、rAd-hFGF-10感染的HaCat细胞中表达上调，并初步探讨了差异蛋白质VDAC2在hFGF-7和hFGF-10生物学功能中的可能作用。结论：通过细菌内同源重组成功构建了重组腺病毒rAd-hFGF-7和rAd-hFGF-10，制备出的重组腺病毒感染HaCat细胞后分泌表达hFGF-7、hFGF-10，并可促进HaCat细胞的增殖，改变HaCat细胞周期。通过蛋白质组学的方法鉴定的差异表达蛋白质VDAC2可能与hFGF-7，hFGF-10的抗细胞凋亡生物学功能相关。
[Abstract]:In this paper, the core technology platform of human skin keratinocyte (Keratinocyte, KC) and human skin epidermis was established, and on this basis, from time, pathological state (psoriasis) and keratinocyte specific cytokine (human fibroblast growth factor 7 and human fibroblast growth factor 10, namely hFGF-) 7 and hFGF-10) the effects of three layers of stimulation on human keratinocyte proteome were preliminarily studied to elucidate the possible mechanism of the effect of long-term culture on human keratinocytes, the pathogenesis of dermatosis (psoriasis), and the formation of keratinocytes with keratinocyte specific cytokine on human skin keratinocytes. The possible mechanism is provided for theoretical basis.
Chapter 1 the effect of long-term culture on human keratinocytes
Objective: to establish a method for the isolation, culture and generation of human keratinocytes in vitro, and to establish a key technical platform for the proteomics research of human keratinocytes in vitro, and to explore the effect of long-term culture on the proteome of human keratinocytes in vitro, so as to study the formation of human skin keratinocyte in vitro for a further period. The possible mechanism of cell impact lays the foundation.
Methods: after circumcision of children, the epidermis was separated by Dispase II enzyme digestion and cultured in vitro with Defined K-SFM medium. Trypsin and EDTA were combined to digest the passage. The morphology of the cultured cells was observed under the phase contrast microscope. The cultured cells were identified by immunocytochemical method, and the cell count method was used to determine the original seventh. The group doubling time of the cells (PDT).
The total protein samples of human skin keratinocytes cultured in the original and seventh generation in vitro were prepared by using the nonlinear IPG prefabricated adhesive strip (pH3-10,17cm) for the first isoelectric focusing. The second two-dimensional molecular weight vertical electrophoresis was used to use 12%SDS- polyacrylamide gel. The two dimensional gel electrophoresis strips were optimized. The PDQuest image analysis software was used. A comparative analysis of two-dimensional gel electrophoresis atlas was obtained. 2 original and seventh generation keratinocytes were selected for the differential expression of protein points, in gel enzyme digestion, series of time of flight mass spectrometry (ABI 4700 TOF-TOF) analysis. By using MASCOT online retrieval tool, the data obtained by mass spectrometry were retrieved in the NCBInr database and half determined. RT-PCR was used to detect the mRNA level of 2 differentially expressed proteins.
Results: the original human keratinocytes were cultured in vitro. The cell morphology was round or oval. With the increase of the number of cells, the cell morphology changed greatly: the cell shape was irregular and many vacuoles appeared around the nucleus. The cytoplasm of the cells under the fluorescence microscope was yellowish green, which was a keratin positive stain. The cell proliferation rate is slow, it takes about 10 days to reach the fusion, PDT is 116 hours, the cell proliferation is fast after the passage, the 2,3,4 generation PDT is shortened significantly. After fifth generations, the PDT is obviously prolonged, the cell proliferation speed is slow, and the eighth generation of cells can not fuse for a long time.
The bi-directional gel electrophoresis of the total protein of human keratinocyte was established and optimized, and a better two-dimensional gel electrophoresis Atlas of protein separation was obtained. The PDQuest software was used to analyze the total protein of the original and seventh generation human keratinocytes, and the matching eggs of the original human keratinocyte gel were obtained. The number of white matter points was 751, while the number of matching protein points of the seventh generation skin keratinocyte gel was 806, the matching rate was 75.3% and 80.8%. was analyzed by tandem time of flight mass spectrometry for the significant reduction and better resolution of the 2 expressed protein particles in the seventh generation keratinocytes. The peptide mass fingerprint (PMF) was used to retrieve the NCBInr database by MASCOT online retrieval tool. The differential protein 1 was identified as the PA-FABP associated with the transport and metabolism of fatty acids. The differential protein 2 was the Galectin-7. related to the regulation of apoptosis. Finally, the genes of PA-FABP and Galectin-7 in the seventh generation keratinocytes were confirmed by semi quantitative RT-PCR. The transcriptional level of the transcriptional level is significantly lower than that of the primary keratinocyte, and the possible role in the biological function of PA-FABP and Galectin-7 and its effect on human skin keratinocytes in the long term culture in vitro has been analyzed.
Conclusion: the method of isolation, culture and passage of human keratinocytes in vitro has been successfully established, and the core technical platform for the proteomics of human keratinocytes is established. Compared with the original human keratinocytes, the expression of PA-FABP and Galectin-7 in the seventh generation cells is down, suggesting the transport of fatty acids in the cells. And (or) metabolism may change, and the ability of cell apoptosis regulation may decrease.
The second chapter is the proteomics study of normal and psoriatic epidermis.
Objective: to establish a core technology platform for the study of human skin epidermis, and to study the difference between normal and vulgaris psoriasis epidermis by proteomics, and then to speculate on the possible mechanism of the occurrence and development of psoriasis by differentially protein.
Methods: the skin of the skin lesions of the patients with circumcision and psoriasis vulgaris were obtained. The epidermis was separated by Dispase II enzyme digestion. The total protein samples of the epidermis of normal and psoriasis vulgaris patients were prepared respectively. The first dimensional isoelectric focusing was performed with the nonlinear IPG prefabricated glue strips (pH3-10,17cm), and the first two-dimensional molecular weight was vertical. 12%SDS- polyacrylamide gel was used in swimming. PDQuest image analysis software was used to compare the obtained bi-directional gel electrophoresis. The protein points were expressed in the epidermis of skin lesions of normal and normal psoriasis patients. Enzyme digestion, tandem time of flight mass spectrometry (ABI 4700 TOF-TOF), and MASCOT on-line detection were used. The data detected by mass spectrometry were retrieved in the NCBInr database, and the mRNA level of the differential expression of protein aB-Crystallin was detected by semi quantitative RT-PCR.
Results: a bi-directional gel electrophoresis method was established for the skin total protein of human skin. The two-dimensional gel electrophoresis Atlas of protein separation was obtained. The total protein of epidermis of normal and psoriasis vulgaris patients was analyzed by PDQuest software, and the number of matched protein points of normal surface skin gel electrophoresis gel was 8. 76, the number of matched protein points in the epidermis of psoriasis vulgaris area was 794, the matching rate was 79.6% and 72.2%. was used in tandem mass spectrometry to significantly increase 5 expressions in the epidermis of skin lesions of psoriatic patients with psoriasis vulgaris and the difference of protein points of 1 expressions in the epidermis of psoriasis vulgaris patients. The corresponding peptide mass fingerprints (PMF) were obtained, and the NCBInr database was retrieved by MASCOT online retrieval tools to identify the proteins related to cell proliferation, apoptosis, differentiation and inflammatory infiltration, in which 5 differential protein points were expressed in the epidermis of the skin lesions of patients with psoriasis vulgaris. S100 calcium binding protein A7-1ike 1, Psoriasin, alpha B-Crystallin, Peroxiredoxin 2 isoform B, Transglutaminase 3, precursor, 1 differentially expressed protein points in the epidermis of psoriasis vulgaris patients are lysosomal cysteine protease inhibitors The gene transcriptional level of alpha B-Crystallin in epidermis of skin lesions of patients with normal psoriasis is significantly higher than that of normal epidermis, and the possible role of differential protein in the occurrence and development of psoriasis vulgaris is preliminarily discussed.
Conclusion: the key technical platform for the study of human skin epidermal proteomics has been successfully established, and the differentially expressed proteins identified by proteomics may be closely related to the transition and proliferation of keratinocytes of psoriasis vulgaris, the decrease of spontaneous apoptosis and abnormal differentiation.
Objective: to construct a recombinant adenovirus containing cytokeratinocyte specific cytokine hFGF-7, hFGF-10 gene, and to express hFGF-7 and hFGF-10 in human skin keratinocyte strain (HaCat) by recombinant adenovirus infection, and on the basis of the core technical platform for the establishment of human keratinocyte proteomic research. The method of white matter study studies the effect of hFGF-7, hFGF-10 expression on the protein group of HaCat cells, and then the possible mechanism of the effect of hFGF-7 and hFGF-10 on HaCat cells by differentially protein.
Methods: PCR method was used to synthesize hFGF-7, hFGF-10 gene fragment and cloned to pET3c vector. The recombinant plasmid pET3c-hFGF-7 and pET3c-hFGF-10. were obtained with two recombinant plasmids as templates, and PCR amplification was used to amplify hFGF-7, hFGF-10 gene fragment at the end of the 5 'terminal and Hind III loci at the end of 3', and the shuttle carrier pAdTrack-CMV. The recombinant shuttle plasmid pAdTrack-CMV-hFGF-7 and pAdTrack-CMV-hFGF-10 were linearized by Pme I, and the recombinant adenovirus plasmid pAdEasy-hFGF-7 and pAdEasy-hFGF-10 were obtained by electrotransfer into the Escherichia coli BJ5183 containing the adenoviral skeleton plasmid pAdEasy-1. The recombinant adenovirus plasmid pAdEasy-hFGF-7 and pAdEasy-hFGF-10 were transfected to HEK-293 cells after enzyme digestion and in HEK. The recombinant adenovirus rAd-hFGF-7 and rAd-hFGF-10. were packed and amplified in -293 cells to infect HaCat cells with recombinant adenovirus, and Western blotting was used to detect the recombinant hFGF-7, hFGF-10, and MTT method to detect the effect of recombinant adenovirus on the proliferation of HaCat cells: the effects of recombinant adenovirus on the HaCat cell cycle were detected by flow cytometry.
Four groups of total cell proteins (HaCat, adenovirus Ad infection group, recombinant adenovirus rAd-hFGF-7 infection group and recombinant adenovirus rAd-hFGF-10 infection group) were used for first dimensional isoelectric focusing with nonlinear IPG prefabricated adhesive (pH3-10,17cm), and 12%SDS- polyacrylamide gel was used for two-dimensional molecular weight vertical electrophoresis. PDQuest images were used. Compare and analyze the obtained two-dimensional gel electrophoresis atlas. Select the protein points of the 4 recombinant adenoviruses that increase after infection, carry out the gel internal enzyme cutting, the tandem time of flight mass spectrometry (ABI 4700 TOF-TOF) analysis. Using the MASCOT online retrieval tool, the data obtained by the mass spectrometry are retrieved in the NCBInr database and passed. Semi quantitative RT-PCR and Western blotting were used to detect the mRNA level and protein level of the differentially expressed protein VDAC2 in four groups of cells.
Results: the recombinant adenovirus containing hFGF-7, hFGF-10 gene and recombinant adenovirus rAd-hFGF-7 and rAd-hFGF-10. recombinant adenovirus could efficiently infect HaCat cells. The culture supernatant was positive with hFGF-7 and hFGF-10 antibody. Recombinant adenovirus could promote the proliferation of HaCat cells, and the cell cycle of HaCat could be changed, and the ratio of the cells to S and G2 phase increased. Add.
Bi-directional gel electrophoresis Atlas of protein separation was obtained. The total protein atlas of four groups of cells was analyzed by PDQuest software. The number of matched protein points of the two-dimensional gel electrophoresis gel of the control group HaCat cells and Ad infected groups was 516 and 535, the matching rate was 67.5% and 70%, while the recombinant adenovirus rAd-hFGF-7, rAd-hFGF The number of matching protein points of the two-dimensional gel electrophoresis gel in the -10 infection group was 602 and 587, the matching rate was 78.8% and the 76.8%. was analyzed by mass spectrometry with tandem time of flight mass spectrometry, and the corresponding peptide mass fingerprints (PMF) were obtained. The MASCOT online retrieval tool was used. The NCBInr database was retrieved to identify the proteins related to apoptosis, cytoskeleton regulation and protein degradation, including VDAC2, Proteasome alpha 1 subunit, isoform 2, Gelsolin-like capping protein and Protein disulfide isomerase-associated 3 finally passed through
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341

【引证文献】