本文選題：HGFs + PLGA支架　； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:(1)使用PLGA支架對(duì)人牙齦成纖維細(xì)胞(HGFs)進(jìn)行三維培養(yǎng),構(gòu)建HGFs-PLGA力學(xué)加載模型。(2)采用i TRAQ標(biāo)記聯(lián)合二維液相色譜-串聯(lián)質(zhì)譜技術(shù)(2D-LC-MS/MS)對(duì)三維培養(yǎng)及壓應(yīng)力作用下HGFs標(biāo)本進(jìn)行蛋白質(zhì)組學(xué)定量分析,篩選靜壓力作用下HGFs差異表達(dá)蛋白。方法:(1)HGFs原代培養(yǎng)及來(lái)源鑒定。(2)用第4代的HGFs,制備細(xì)胞密度為1×105個(gè)/ml的細(xì)胞懸液,在1 cm~2PLGA支架上接種1ml懸液。熒光電鏡觀察HGFs-PLGA復(fù)合培養(yǎng)5天后HGFs生長(zhǎng)情況,使用CCK8法進(jìn)行細(xì)胞增殖活性檢測(cè)。(3)建立HGFs-PLGA支架三維復(fù)合培養(yǎng)模型,培養(yǎng)4天。實(shí)驗(yàn)組分為A、B、C組,對(duì)其施加大小為25g/cm~2的壓應(yīng)力,作用時(shí)間分別為24、48、72h;對(duì)照組分為A0、B0、C0組,分別培養(yǎng)24、48、72h,與實(shí)驗(yàn)組對(duì)應(yīng),不加力處理。每組設(shè)置2個(gè)重復(fù)組。(4)提取各組HGFs總蛋白,Bradford法測(cè)定蛋白濃度。(5)i TRAQ定量蛋白質(zhì)組學(xué)技術(shù)分析HGFs中差異表達(dá)的蛋白質(zhì),根據(jù)生物信息學(xué)對(duì)篩選出的差異蛋白進(jìn)行深層次分析。結(jié)果:(1)成功培養(yǎng)HGFs原代并傳代純化。(2)熒光電鏡結(jié)果顯示,聚焦PLGA支架不同平面時(shí),均有觀察到HGFs密集生長(zhǎng)。CCK-8檢測(cè)顯示細(xì)胞種植在PLGA支架培養(yǎng)3-5天細(xì)胞處于對(duì)數(shù)生長(zhǎng)期,5-7天處于平臺(tái)期,符合細(xì)胞生長(zhǎng)規(guī)律。(3)i TRAQ技術(shù)鑒定到總蛋白2449個(gè),定量蛋白2438個(gè)。GO分析表明,差異表達(dá)的蛋白主要參與代謝和細(xì)胞信號(hào)傳導(dǎo)的生物學(xué)過(guò)程;在細(xì)胞位置中主要附著于細(xì)胞器、細(xì)胞膜上、細(xì)胞外基質(zhì)、突觸等位置;在分子功能中主要參與催化活性、轉(zhuǎn)運(yùn)、酶活性調(diào)節(jié)、抗氧化等功能。信號(hào)通路富集分析發(fā)現(xiàn),差異表達(dá)的蛋白主要參與能量代謝通路以及信號(hào)轉(zhuǎn)導(dǎo)通路。以i TRAQ技術(shù)研究機(jī)械壓應(yīng)力作用下HGFs差異表達(dá)蛋白,KEGG富集分析結(jié)果顯示,處在互作關(guān)鍵節(jié)點(diǎn)的蛋白多達(dá)20多種,其中有TGF-β、細(xì)胞色素P450、谷胱甘肽-S-轉(zhuǎn)移酶、血小板衍生生長(zhǎng)因子、Ⅰ型膠原蛋白、基質(zhì)金屬蛋白酶1、MAP激酶p38、IL-6、轉(zhuǎn)錄因子p65、轉(zhuǎn)錄因子p50、纖維連結(jié)蛋白、α2-巨球蛋白、纖溶酶原激活因子抑制因子等。結(jié)論:(1)PLGA支架能成功構(gòu)建HGFs三維培養(yǎng)及力學(xué)加載模型。(2)i TRAQ技術(shù)篩選出靜壓力作用下HGFs差異表達(dá)蛋白,經(jīng)分析處在蛋白質(zhì)相互作用網(wǎng)絡(luò)的節(jié)點(diǎn)上的關(guān)鍵蛋白重要的10個(gè)是核因NF-k B p65、p50,MAP激酶P38,TGF-β,基質(zhì)金屬蛋白酶抑制劑-1(TIMP-1),α2巨球蛋白,Ⅰ型膠原蛋白,纖維連結(jié)蛋白,IL-6,血小板衍生生長(zhǎng)因子受體,血小板反應(yīng)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, 偽 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, 偽 2 macroglobulin, type I collagen. Fibronectin IL-6, platelet-derived growth factor receptor, platelet-reactive protein-1.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R783.5

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本文編號(hào)：2011383