本文關(guān)鍵詞： NO HaCaT細(xì)胞 人角質(zhì)形成細(xì)胞 雙向電泳 質(zhì)譜分析　出處：《大連醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:研究一氧化氮(NO)對(duì)體外培養(yǎng)的永生化角質(zhì)形成細(xì)胞(HaCaT細(xì)胞)及人原代培養(yǎng)的角質(zhì)形成細(xì)胞(KC)蛋白質(zhì)表達(dá)的影響,在蛋白水平上尋找NO作用的靶位。進(jìn)一步探討NO在銀屑病發(fā)病中的地位和作用。 方法:體外培養(yǎng)HaCaT細(xì)胞及人原代KC,通過(guò)CCK-8法檢測(cè)不同濃度外源性NO供體S-亞硝基谷胱甘肽(GSNO)作用后,在不同時(shí)間點(diǎn)的細(xì)胞增殖率,繪制濃度與細(xì)胞增值率曲線,篩選最佳GSNO濃度。向處于指數(shù)增長(zhǎng)期的HaCaT細(xì)胞及人原代KC中加入最佳濃度的GSNO進(jìn)行刺激。對(duì)照組不予處理。加藥處理的細(xì)胞和對(duì)照組用細(xì)胞裂解液分別進(jìn)行充分裂解,提取細(xì)胞全蛋白。應(yīng)用2-D純化試劑盒進(jìn)行蛋白純化,蛋白定量。純化后的蛋白中加入上樣緩沖液制成樣本,首先進(jìn)行第一向等電聚焦電泳,膠條的平衡,再進(jìn)行第二向聚丙烯酰胺凝膠電泳。電泳完成的凝膠進(jìn)行硝酸銀染色,圖像掃描,用PD-QUEST8.0軟件進(jìn)行分析,篩選出加入GSNO前后的差異點(diǎn),切下進(jìn)行質(zhì)譜分析,對(duì)蛋白質(zhì)種類(lèi)進(jìn)行鑒定。 結(jié)果: (1)應(yīng)用CCK-8法,繪制GSNO濃度與細(xì)胞增值率曲線顯示,隨著GSNO濃度的增加,細(xì)胞增殖呈下降趨勢(shì),與時(shí)間無(wú)顯著關(guān)系。(2)HaCaT細(xì)胞組及人KC組進(jìn)行雙向電泳后出點(diǎn)良好;加入GSNO前、后HaCaT細(xì)胞組平均蛋白質(zhì)點(diǎn)數(shù)分別為482±31,,495±28,組內(nèi)匹配率達(dá)82.1%和81.9%;加入GSNO前、后人KC組平均蛋白質(zhì)點(diǎn)數(shù)分別為543±27和529±29,匹配率為80.8%和81.4%。(3)差異蛋白點(diǎn)數(shù)總共為29個(gè),其中,2個(gè)點(diǎn)僅在加入GSNO后表達(dá);14個(gè)點(diǎn)在加入GSNO后高表達(dá);13個(gè)點(diǎn)在加入GSNO后地表達(dá)或消失;進(jìn)行質(zhì)譜分析,檢測(cè)到17種已知蛋白,3種未知蛋白。 結(jié)論:(1)該研究建立了NO作用前、后HaCaT細(xì)胞及人KC的雙向電泳圖譜,發(fā)現(xiàn)二者之間存在差異表達(dá)蛋白; (2)本研究通過(guò)NO作用前后HaCaT細(xì)胞及人KC的雙向電泳及質(zhì)譜分析,發(fā)現(xiàn)抑制素、角蛋白、肌動(dòng)蛋白結(jié)合蛋白等17種差異表達(dá)的蛋白; (3)NO通過(guò)調(diào)節(jié)多種靶向蛋白的表達(dá),調(diào)節(jié)KC增殖周期、分化或凋亡,介導(dǎo)早期銀屑病的炎性反應(yīng)。
[Abstract]:Objective: to study the effects of nitric oxide (no) on the protein expression of immortalized keratinocytes (HaCaT cells) and human primary cultured keratinocytes (KC) in vitro. To explore the role of no in the pathogenesis of psoriasis. Methods: HaCaT cells and human KC cells were cultured in vitro. The effects of different concentrations of exogenous no donor, Snitroglutathione (Snitroglutathione), were detected by CCK-8 assay. At different time points, the cell proliferation rate, concentration and cell proliferation rate curve were drawn. Select the best concentration of GSNO. Add the best concentration of GSNO to the HaCaT cells in exponential growth stage and human KC. The control group is not treated. The cells treated with drugs are used in the control group. The cell lytic solution was fully lysed respectively. The whole cell protein was extracted. The protein was purified by 2-D purification kit and the protein was quantification.After the purified protein, the sample was prepared by adding the sample buffer solution, and the first isoelectric focusing electrophoresis was carried out, and the equilibrium of the rubber strip was carried out. Then the second polyacrylamide gel electrophoresis was carried out. The gel was stained with silver nitrate, scanned by image and analyzed by PD-QUEST8.0 software. The difference points before and after the addition of GSNO were screened, and the protein species were identified by mass spectrometry. Results: using CCK-8 method, the curves of GSNO concentration and cell proliferation rate showed that the proliferation of cells decreased with the increase of GSNO concentration. There was no significant relationship between the time and the time. The exit point of HaCaT cell group and human KC group were good after two dimensional electrophoresis. Before and after the addition of GSNO, the average protein number of HaCaT cells was 482 鹵31, 495 鹵28, and the matching rates were 82.1% and 81.9 respectively. Before the addition of GSNO, the average protein points in KC group were 543 鹵27 and 529 鹵29, respectively, and the matching rates were 80.8% and 81.4, respectively. Among them, 2 dots were expressed only after the addition of GSNO. 14 dots were overexpressed after adding GSNO. 13 dots were expressed or disappeared after adding GSNO. 17 known proteins and 3 unknown proteins were detected by mass spectrometry. Conclusion 1) Two-dimensional electrophoretic patterns of HaCaT cells and human KC before and after no treatment were established in this study. It was found that there were differentially expressed proteins between them. In this study, 17 differentially expressed proteins, such as inhibin, keratin and actin binding protein, were found by two-dimensional electrophoresis and mass spectrometry analysis of HaCaT cells and human KC before and after no treatment. No mediates the inflammatory response of early psoriasis by regulating the expression of many target proteins, regulating the proliferation cycle, differentiation or apoptosis of KC.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R758.63

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本文編號(hào)：1488408