本文選題：人皮膚角質(zhì)形成細(xì)胞 + 蛋白質(zhì)組��； 參考：《暨南大學(xué)》2007年博士論文

【摘要】： 本論文建立了人皮膚角質(zhì)形成細(xì)胞(Keratinocyte，KC)和人皮膚表皮蛋白質(zhì)組學(xué)研究的核心技術(shù)平臺，并在此基礎(chǔ)上，分別從時間、病理狀態(tài)(銀屑病)和角質(zhì)形成細(xì)胞特異的細(xì)胞因子(人成纖維細(xì)胞生長因子7和人成纖維細(xì)胞生長因子10，即hFGF-7和hFGF-10)刺激等三個層面對人皮膚角質(zhì)形成細(xì)胞蛋白質(zhì)組的影響進(jìn)行了初步研究，為闡明體外長期培養(yǎng)對人皮膚角質(zhì)形成細(xì)胞影響的可能機(jī)制、皮膚病(銀屑病)的發(fā)病機(jī)制、以及角質(zhì)形成細(xì)胞特異的細(xì)胞因子對人皮膚角質(zhì)形成細(xì)胞作用的可能機(jī)制等提供理論依據(jù)。 第一章長期培養(yǎng)對人皮膚角質(zhì)形成細(xì)胞的影響 目的：建立人皮膚角質(zhì)形成細(xì)胞的體外分離、培養(yǎng)和傳代的方法，并建立人皮膚角質(zhì)形成細(xì)胞蛋白質(zhì)組學(xué)研究的核心技術(shù)平臺，探討體外長期培養(yǎng)對人皮膚角質(zhì)形成細(xì)胞蛋白質(zhì)組的影響，為進(jìn)一步研究體外長期培養(yǎng)對人皮膚角質(zhì)形成細(xì)胞影響的可能機(jī)制奠定基礎(chǔ)。 方法：取幼兒環(huán)切術(shù)后包皮，用DispaseⅡ酶消化法分離表皮，用Defined K-SFM培養(yǎng)基進(jìn)行體外培養(yǎng)，胰酶和EDTA聯(lián)合消化傳代，相差顯微鏡下觀察培養(yǎng)細(xì)胞的形態(tài)，采用免疫細(xì)胞化學(xué)方法對培養(yǎng)的細(xì)胞進(jìn)行鑒定，并用細(xì)胞計數(shù)法測定原代到第7代細(xì)胞的群體倍增時間(PDT)。 制備原代和第7代體外培養(yǎng)的人皮膚角質(zhì)形成細(xì)胞的總蛋白樣品，采用非線性IPG預(yù)制膠條(pH3-10，17cm)進(jìn)行第一維等電聚焦，第二維分子量垂直電泳采用12％SDS-聚丙烯酰胺凝膠。對雙向凝膠電泳的條件進(jìn)行優(yōu)化。采用PDQuest圖像分析軟件對獲得的雙向凝膠電泳圖譜進(jìn)行比較分析。選取2個原代和第7代角質(zhì)形成細(xì)胞的差異表達(dá)蛋白質(zhì)點(diǎn)，進(jìn)行膠內(nèi)酶切、串聯(lián)飛行時間質(zhì)譜(ABI 4700 TOF-TOF)分析。利用MASCOT在線檢索工具，將質(zhì)譜檢測得到的數(shù)據(jù)在NCBInr數(shù)據(jù)庫進(jìn)行檢索，并通過半定量RT-PCR對2個差異表達(dá)蛋白質(zhì)的mRNA水平進(jìn)行檢測。 結(jié)果：體外培養(yǎng)的原代人皮膚角質(zhì)形成細(xì)胞，細(xì)胞形態(tài)為圓形或橢圓形，隨著細(xì)胞傳代次數(shù)的增加，細(xì)胞形態(tài)發(fā)生很大改變：細(xì)胞形狀不規(guī)則，胞核周圍出現(xiàn)許多空泡。熒光顯微鏡下培養(yǎng)細(xì)胞的胞漿呈黃綠色，為角蛋白陽性染色。原代細(xì)胞的增殖速率較慢，約需10天達(dá)到融合，PDT為116小時，傳代后細(xì)胞增殖迅速，第2、3、4代PDT顯著縮短，第5代以后PDT明顯延長，細(xì)胞增殖速度減慢，第8代細(xì)胞長時間不能融合。 建立并優(yōu)化了人皮膚角質(zhì)形成細(xì)胞總蛋白的雙向凝膠電泳方法，獲得了蛋白質(zhì)分離效果較好的雙向凝膠電泳圖譜。采用PDQuest軟件對原代和第7代人皮膚角質(zhì)形成細(xì)胞總蛋白的圖譜進(jìn)行分析，得到原代人皮膚角質(zhì)形成細(xì)胞凝膠的匹配蛋白質(zhì)點(diǎn)數(shù)為751，而第7代人皮膚角質(zhì)形成細(xì)胞凝膠的匹配蛋白質(zhì)點(diǎn)數(shù)為806，匹配率分別為75.3％和80.8％。采用串聯(lián)飛行時間質(zhì)譜對選取的2個表達(dá)量在第7代角質(zhì)形成細(xì)胞中顯著下調(diào)且分辨率較好的蛋白質(zhì)點(diǎn)進(jìn)行質(zhì)譜分析，獲得了相應(yīng)的肽質(zhì)量指紋圖譜(PMF)，通過MASCOT在線檢索工具檢索NCBInr數(shù)據(jù)庫，鑒定差異蛋白質(zhì)1為與脂肪酸運(yùn)輸和代謝相關(guān)的PA-FABP，差異蛋白質(zhì)2為與細(xì)胞凋亡調(diào)控相關(guān)的Galectin-7。最后通過半定量RT-PCR確證第7代角質(zhì)形成細(xì)胞中PA-FABP和Galectin-7的基因轉(zhuǎn)錄水平較原代角質(zhì)形成細(xì)胞相應(yīng)的基因轉(zhuǎn)錄水平顯著降低，并對PA-FABP和Galectin-7的生物學(xué)功能及其在體外長期培養(yǎng)對人皮膚角質(zhì)形成細(xì)胞影響中的可能作用進(jìn)行了初步分析。 結(jié)論：成功建立了人皮膚角質(zhì)形成細(xì)胞的體外分離、培養(yǎng)和傳代的方法，并建立了人皮膚角質(zhì)形成細(xì)胞蛋白質(zhì)組學(xué)研究的核心技術(shù)平臺；與原代人皮膚角質(zhì)形成細(xì)胞相比，第7代細(xì)胞的PA-FABP和Galectin-7表達(dá)下調(diào)，提示細(xì)胞的脂肪酸運(yùn)輸和(或)代謝可能發(fā)生了改變，細(xì)胞的凋亡調(diào)控能力可能下降。 第二章正常和銀屑病表皮的蛋白質(zhì)組學(xué)研究 目的：建立人皮膚表皮蛋白質(zhì)組學(xué)研究的核心技術(shù)平臺，應(yīng)用蛋白質(zhì)組學(xué)的方法研究正常和尋常型銀屑病表皮蛋白質(zhì)組的差異，進(jìn)而由差異蛋白質(zhì)推測銀屑病發(fā)生和發(fā)展的可能機(jī)制。 方法：取正常人環(huán)切術(shù)后包皮和尋常型銀屑病患者皮損區(qū)皮膚，用DispaseⅡ酶消化法分離表皮，分別制備正常和尋常型銀屑病患者皮損區(qū)表皮的總蛋白樣品，采用非線性IPG預(yù)制膠條(pH3-10，17cm)進(jìn)行第一維等電聚焦，第二維分子量垂直電泳采用12％SDS-聚丙烯酰胺凝膠。采用PDQuest圖像分析軟件對獲得的雙向凝膠電泳圖譜進(jìn)行比較分析。選取正常和尋常型銀屑病患者皮損區(qū)表皮的差異表達(dá)蛋白質(zhì)點(diǎn)，進(jìn)行膠內(nèi)酶切、串聯(lián)飛行時間質(zhì)譜(ABI 4700 TOF-TOF)分析。利用MASCOT在線檢索工具，將質(zhì)譜檢測得到的數(shù)據(jù)在NCBInr數(shù)據(jù)庫進(jìn)行檢索，并通過半定量RT-PCR對鑒定的差異表達(dá)蛋白質(zhì)aB-Crystallin的mRNA水平進(jìn)行檢測。 結(jié)果：建立了人皮膚表皮總蛋白的雙向凝膠電泳方法，獲得了蛋白質(zhì)分離效果較好的雙向凝膠電泳圖譜。采用PDQuest軟件對正常和尋常型銀屑病患者皮損區(qū)表皮總蛋白的圖譜進(jìn)行分析，得到正常表皮雙向電泳凝膠的匹配蛋白質(zhì)點(diǎn)數(shù)為876，而尋常型銀屑病皮損區(qū)表皮雙向電泳凝膠的匹配蛋白質(zhì)點(diǎn)數(shù)為794，匹配率分別為79.6％和72.2％。采用串聯(lián)飛行時間質(zhì)譜對選取的在尋常型銀屑病患者皮損區(qū)表皮中5個表達(dá)量顯著上調(diào)和1個表達(dá)量顯著下調(diào)的差異蛋白質(zhì)點(diǎn)進(jìn)行質(zhì)譜分析，獲得了相應(yīng)的肽質(zhì)量指紋圖譜(PMF)，通過MASCOT在線檢索工具檢索NCBInr數(shù)據(jù)庫，鑒定這些差異蛋白質(zhì)為一些與細(xì)胞增殖、凋亡、分化和炎癥浸潤等相關(guān)的蛋白質(zhì)，其中在尋常型銀屑病患者皮損區(qū)表皮中表達(dá)上調(diào)的5個差異蛋白質(zhì)點(diǎn)分別為：S100 calcium binding protein A7-1ike 1、Psoriasin、αB-Crystallin、Peroxiredoxin 2 isoform b、Transglutaminase 3，precursor，在尋常型銀屑病患者皮損區(qū)表皮中表達(dá)下調(diào)的1個差異蛋白質(zhì)點(diǎn)為溶酶體半胱氨酸蛋白酶抑制劑Cystatin B。最后通過半定量RT-PCR確證尋常型銀屑病患者皮損區(qū)表皮中αB-Crystallin的基因轉(zhuǎn)錄水平較正常表皮相應(yīng)的基因轉(zhuǎn)錄水平顯著升高，并初步探討了差異蛋白質(zhì)在尋常型銀屑病發(fā)生和發(fā)展中的可能作用。 結(jié)論：成功建立了人皮膚表皮蛋白質(zhì)組學(xué)研究的核心技術(shù)平臺；通過蛋白質(zhì)組學(xué)的方法鑒定的差異表達(dá)蛋白質(zhì)可能與尋常型銀屑病角質(zhì)形成細(xì)胞的過渡增殖、自發(fā)凋亡減弱和異常分化等密切相關(guān)。 目的：構(gòu)建含角質(zhì)形成細(xì)胞特異的細(xì)胞因子hFGF-7、hFGF-10基因的重組腺病毒，通過重組腺病毒感染，在人皮膚角質(zhì)形成細(xì)胞株(HaCat)中分泌表達(dá)hFGF-7、hFGF-10，并在建立人皮膚角質(zhì)形成細(xì)胞蛋白質(zhì)組學(xué)研究的核心技術(shù)平臺的基礎(chǔ)上，應(yīng)用蛋白質(zhì)組學(xué)的方法研究hFGF-7、hFGF-10的表達(dá)對HaCat細(xì)胞蛋白質(zhì)組的影響，進(jìn)而由差異蛋白質(zhì)推測hFGF-7、hFGF-10對HaCat細(xì)胞作用的可能機(jī)制。 方法：用PCR方法合成hFGF-7、hFGF-10基因片斷并克隆到pET3c載體上，獲得重組質(zhì)粒pET3c-hFGF-7和pET3c-hFGF-10。分別以獲得的兩種重組質(zhì)粒為模板，PCR擴(kuò)增得到5’末端含BglⅡ位點(diǎn)、3’末端含HindⅢ位點(diǎn)的hFGF-7、hFGF-10基因片斷，與穿梭載體pAdTrack-CMV連接，獲得的重組穿梭質(zhì)粒pAdTrack-CMV-hFGF-7和pAdTrack-CMV-hFGF-10經(jīng)PmeⅠ線性化后，通過電轉(zhuǎn)法導(dǎo)入含腺病毒骨架質(zhì)粒pAdEasy-1的大腸桿菌BJ5183中進(jìn)行同源重組，獲得重組腺病毒質(zhì)粒pAdEasy-hFGF-7和pAdEasy-hFGF-10，經(jīng)酶切鑒定后轉(zhuǎn)染HEK-293細(xì)胞，在HEK-293細(xì)胞中包裝并擴(kuò)增重組腺病毒rAd-hFGF-7和rAd-hFGF-10。用重組腺病毒感染HaCat細(xì)胞，Western blotting檢測培養(yǎng)上清中的重組hFGF-7、hFGF-10；MTT法檢測重組腺病毒對HaCat細(xì)胞增殖的影響：重組腺病毒對HaCat細(xì)胞周期的影響通過流式細(xì)胞術(shù)檢測。 制備四組細(xì)胞總蛋白(HaCat、空載體腺病毒Ad感染組、重組腺病毒rAd-hFGF-7感染組和重組腺病毒rAd-hFGF-10感染組)。采用非線性IPG預(yù)制膠條(pH3-10，17cm)進(jìn)行第一維等電聚焦，第二維分子量垂直電泳采用12％SDS-聚丙烯酰胺凝膠。采用PDQuest圖像分析軟件對獲得的雙向凝膠電泳圖譜進(jìn)行比較分析。選擇4個重組腺病毒感染后表達(dá)量增加的蛋白質(zhì)點(diǎn)，進(jìn)行膠內(nèi)酶切、串聯(lián)飛行時間質(zhì)譜(ABI 4700 TOF-TOF)分析。利用MASCOT在線檢索工具，將質(zhì)譜檢測得到的數(shù)據(jù)在NCBInr數(shù)據(jù)庫進(jìn)行檢索，并通過半定量RT-PCR和Western blotting對其中的差異表達(dá)蛋白質(zhì)VDAC2在四組細(xì)胞中的mRNA水平和蛋白質(zhì)水平進(jìn)行檢測。 結(jié)果：成功構(gòu)建了含hFGF-7、hFGF-10基因的重組腺病毒rAd-hFGF-7和rAd-hFGF-10。重組腺病毒可高效感染HaCat細(xì)胞，培養(yǎng)上清與hFGF-7、hFGF-10抗體呈陽性反應(yīng)。重組腺病毒可促進(jìn)HaCat細(xì)胞的增殖，并可改變HaCat的細(xì)胞周期，，進(jìn)入S期和G2期的細(xì)胞比率增加。 獲得了蛋白質(zhì)分離效果較好的雙向凝膠電泳圖譜。采用PDQuest軟件對四組細(xì)胞總蛋白的圖譜進(jìn)行分析，得到對照組HaCat細(xì)胞和Ad感染組雙向電泳凝膠的匹配蛋白質(zhì)點(diǎn)數(shù)為516和535，匹配率分別為67.5％和70.0％；而重組腺病毒rAd-hFGF-7、rAd-hFGF-10感染組雙向電泳凝膠的匹配蛋白質(zhì)點(diǎn)數(shù)為602和587，匹配率分別為78.8％和76.8％。采用串聯(lián)飛行時間質(zhì)譜對選取的4個重組腺病毒感染后表達(dá)量增加的蛋白質(zhì)點(diǎn)進(jìn)行質(zhì)譜分析，獲得了相應(yīng)的肽質(zhì)量指紋圖譜(PMF)，通過MASCOT在線檢索工具檢索NCBInr數(shù)據(jù)庫，鑒定這些差異蛋白質(zhì)為一些與細(xì)胞凋亡、細(xì)胞骨架調(diào)控、蛋白質(zhì)降解等相關(guān)的蛋白質(zhì)，包括：VDAC2、Proteasome alpha 1 subunit，isoform 2、Gelsolin-like capping protein和Protein disulfide isomerase-associated 3 precursor。最后通過半定量RT-PCR和Western blotting在mRNA和蛋白質(zhì)水平上驗(yàn)證了差異表達(dá)蛋白質(zhì)VDAC2在重組腺病毒rAd-hFGF-7、rAd-hFGF-10感染的HaCat細(xì)胞中表達(dá)上調(diào)，并初步探討了差異蛋白質(zhì)VDAC2在hFGF-7和hFGF-10生物學(xué)功能中的可能作用。 結(jié)論：通過細(xì)菌內(nèi)同源重組成功構(gòu)建了重組腺病毒rAd-hFGF-7和rAd-hFGF-10，制備出的重組腺病毒感染HaCat細(xì)胞后分泌表達(dá)hFGF-7、hFGF-10，并可促進(jìn)HaCat細(xì)胞的增殖，改變HaCat細(xì)胞周期。 通過蛋白質(zhì)組學(xué)的方法鑒定的差異表達(dá)蛋白質(zhì)VDAC2可能與hFGF-7，hFGF-10的抗細(xì)胞凋亡生物學(xué)功能相關(guān)。
[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
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R341

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 汪靜;卡泊三醇軟膏皮膚滲透性研究[D];吉林大學(xué);2013年

本文編號：1966568