ITRAQ技术分析静压力作用下人牙龈成纤维细胞差异表达蛋白

发布时间：2018-06-12 22:55

本文选题：HGFs + PLGA支架　；参考：《广西医科大学》2017年硕士论文

【摘要】：目的:(1)使用PLGA支架对人牙龈成纤维细胞(HGFs)进行三维培养,构建HGFs-PLGA力学加载模型。(2)采用i TRAQ标记联合二维液相色谱-串联质谱技术(2D-LC-MS/MS)对三维培养及压应力作用下HGFs标本进行蛋白质组学定量分析,筛选静压力作用下HGFs差异表达蛋白。方法:(1)HGFs原代培养及来源鉴定。(2)用第4代的HGFs,制备细胞密度为1×105个/ml的细胞悬液,在1 cm~2PLGA支架上接种1ml悬液。荧光电镜观察HGFs-PLGA复合培养5天后HGFs生长情况,使用CCK8法进行细胞增殖活性检测。(3)建立HGFs-PLGA支架三维复合培养模型,培养4天。实验组分为A、B、C组,对其施加大小为25g/cm~2的压应力,作用时间分别为24、48、72h;对照组分为A0、B0、C0组,分别培养24、48、72h,与实验组对应,不加力处理。每组设置2个重复组。(4)提取各组HGFs总蛋白,Bradford法测定蛋白浓度。(5)i TRAQ定量蛋白质组学技术分析HGFs中差异表达的蛋白质,根据生物信息学对筛选出的差异蛋白进行深层次分析。结果:(1)成功培养HGFs原代并传代纯化。(2)荧光电镜结果显示,聚焦PLGA支架不同平面时,均有观察到HGFs密集生长。CCK-8检测显示细胞种植在PLGA支架培养3-5天细胞处于对数生长期,5-7天处于平台期,符合细胞生长规律。(3)i TRAQ技术鉴定到总蛋白2449个,定量蛋白2438个。GO分析表明,差异表达的蛋白主要参与代谢和细胞信号传导的生物学过程;在细胞位置中主要附着于细胞器、细胞膜上、细胞外基质、突触等位置;在分子功能中主要参与催化活性、转运、酶活性调节、抗氧化等功能。信号通路富集分析发现,差异表达的蛋白主要参与能量代谢通路以及信号转导通路。以i TRAQ技术研究机械压应力作用下HGFs差异表达蛋白,KEGG富集分析结果显示,处在互作关键节点的蛋白多达20多种,其中有TGF-β、细胞色素P450、谷胱甘肽-S-转移酶、血小板衍生生长因子、Ⅰ型胶原蛋白、基质金属蛋白酶1、MAP激酶p38、IL-6、转录因子p65、转录因子p50、纤维连结蛋白、α2-巨球蛋白、纤溶酶原激活因子抑制因子等。结论:(1)PLGA支架能成功构建HGFs三维培养及力学加载模型。(2)i TRAQ技术筛选出静压力作用下HGFs差异表达蛋白,经分析处在蛋白质相互作用网络的节点上的关键蛋白重要的10个是核因NF-k B p65、p50,MAP激酶P38,TGF-β,基质金属蛋白酶抑制剂-1(TIMP-1),α2巨球蛋白,Ⅰ型胶原蛋白,纤维连结蛋白,IL-6,血小板衍生生长因子受体,血小板反应蛋白-1。
[Abstract]:Objective: to culture human gingival fibroblasts HGFs using PLGA scaffold. A mechanical loading model of HGFs-PLGA was constructed. The differential expression proteins of HGFs under static pressure were screened by using I-TRAQ labeling and two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS) technique. Methods the cell suspensions with cell density of 1 脳 105 / ml were prepared by primary culture and identification of the origin of HGFs from 1: 10 ~ (1) 1ml suspension was inoculated on 1 cm ~ (2) PLGA scaffold with HGFs of the fourth passage. The cell density of HGFs was 1 脳 10 ~ (5) / ml and the cell density was 1 脳 10 ~ (5) / ml. The growth of HGFs was observed by fluorescence electron microscope after 5 days of HGFs-PLGA co-culture. The cell proliferation activity of HGFs-PLGA was detected by CCK8. The model of HGFs-PLGA scaffold was established by using CCK8. The HGFs-PLGA scaffold was cultured for 4 days. The experimental group was divided into two groups: group A (n = 24) and group C (n = 24), respectively. The control group was divided into two groups: group A (n = 24) and group C (n = 24). The control group was divided into two groups: group A (n = 24) and group C (n = 24). Two repeats were set up in each group.) the total protein of HGFs in each group was extracted by Bradford method to determine the protein concentration. The protein in HGFs was analyzed by quantitative proteomics. The differentially expressed proteins in HGFs were analyzed by bioinformatics, and the differentially expressed proteins were further analyzed by bioinformatics. Results (1) HGFs were cultured successfully and purified by passage. 2) the results of fluorescence electron microscopy showed that when PLGA scaffolds were focused on different planes, HGFs dense growth. CCK-8 assay showed that the cells planted in PLGA scaffold for 3-5 days were in logarithmic growth phase and 5 to 7 days on the platform stage. The total protein was 2449 and the quantitative protein 2438. Go analysis showed that the HGFs were in the logarithmic growth phase at 5-7 days, and the total protein was 2449 in accordance with the cell growth rule. The differentially expressed proteins are mainly involved in the biological processes of metabolism and cell signal transduction; they are mainly attached to organelle, cell membrane, extracellular matrix, synapse and other sites in the cellular position; they are mainly involved in the catalytic activity and transport in the molecular function. Enzyme activity regulation, antioxidant and other functions. Signal pathway enrichment analysis showed that differentially expressed proteins were mainly involved in energy metabolism pathway and signal transduction pathway. KEGG enrichment analysis of HGFs differentially expressed proteins under mechanical compressive stress by iTRAQ technique showed that there were more than 20 proteins at the key nodes of interaction, including TGF- 尾, cytochrome P450, glutathione -Stransferase and platelet-derived growth factor. Type I collagen, matrix metalloproteinase-1 map kinase p38 / IL-6, transcription factor p65, transcription factor p50, fibronectin, 伪 2-macroglobulin, plasminogen activator inhibitor, etc. Conclusion the three dimensional culture of HGFs and the mechanical loading model of HGFs can be successfully constructed by the PLGA scaffold. The differentially expressed proteins of HGFs under static pressure can be screened by using the method of .jou2i TRAQ. Ten of the key proteins at the node of the protein-protein interaction network were nuclear NF-k B p65 p50 map kinase P38 TGF- 尾, matrix metalloproteinase inhibitor -1, 伪 2 macroglobulin, type I collagen. Fibronectin IL-6, platelet-derived growth factor receptor, platelet-reactive protein-1.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.5

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