本文選題：角質(zhì)形成細(xì)胞 + 皮膚老化��； 參考：《重慶醫(yī)科大學(xué)》2009年博士論文

【摘要】：目的皮膚老化的機(jī)制不明,且臨床實(shí)際中缺乏皮膚老化判斷的客觀指標(biāo)。本課題以青年、中年、老年人曝光部位及非曝光部位的皮膚為研究對(duì)象,探尋其中差異表達(dá)的蛋白或特異表達(dá)的蛋白有可能成為皮膚老化的標(biāo)志性蛋白,探討角質(zhì)形成細(xì)胞在皮膚老化過(guò)程中的作用及分子機(jī)制。為后續(xù)研究全面解釋皮膚老化的機(jī)制,揭示衰老的本質(zhì)奠定基礎(chǔ)。方法借助蛋白質(zhì)組學(xué)研究技術(shù)平臺(tái),采用Dispase Ⅱ分離出表皮,優(yōu)化改進(jìn)樣本處理方法,使用加入了cocktail,RNase和DNase的裂解液提取表皮組織總蛋白,經(jīng)等電聚焦和垂直電泳,銀染后獲得了表皮組織中角質(zhì)形成細(xì)胞2-DE圖像,建立和優(yōu)化了適合于表皮組織研究的雙向凝膠電泳方法。染色后的凝膠用GS800密度掃描儀掃描,并用PDQuest7.4圖像分析軟件進(jìn)行圖像分析,強(qiáng)度校正、背景消減、點(diǎn)檢測(cè)、匹配、ID校正,確定差異蛋白點(diǎn)。挑選其中的蛋白點(diǎn)進(jìn)一步行MALDI-TOF質(zhì)譜分析及數(shù)據(jù)庫(kù)查詢(xún),最后鑒定差異候選蛋白。結(jié)果獲得的雙向凝膠電泳圖譜較為清晰,蛋白質(zhì)點(diǎn)分離完全,各組重復(fù)3次后得到的圖譜非常相似,進(jìn)行3塊膠間的蛋白質(zhì)點(diǎn)的匹配,測(cè)量膠間蛋白質(zhì)點(diǎn)在第一向IEF方向的偏差為(1.798±1.232)mm,在第二向SDS-PAGE方向上的偏差為(1.063±1.031)mm,獲得了重復(fù)性較好匹配率高的表皮組織雙向凝膠電泳圖譜,由此成功建立了適合人表皮組織比較蛋白質(zhì)組研究的雙向凝膠電泳方法。角質(zhì)形成細(xì)胞蛋白在pH3-10的范圍均有分布,但主要集中分布在pH 5-8范圍內(nèi),相對(duì)分子質(zhì)量主要集中在19-110k0之間。在pH 5-8范圍內(nèi)蛋白點(diǎn)的檢測(cè)、量化和點(diǎn)的匹配結(jié)果顯示：青年組曝光部位角質(zhì)形成細(xì)胞的平均蛋白點(diǎn)數(shù)為387±21,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)81.6%。中年組曝光部位角質(zhì)形成細(xì)胞平均蛋白點(diǎn)數(shù)為380±31,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)83.7%。分析比較兩組2-DE圖譜共確定43個(gè)差異蛋白點(diǎn),其中12個(gè)蛋白點(diǎn)在青年組曝光部位2-DE圖譜中高表達(dá),17個(gè)蛋白點(diǎn)在中年組曝光部位2-DE圖譜中高表達(dá),14個(gè)蛋白點(diǎn)在兩者間有2倍以上顯著性量變。中年組曝光部位角質(zhì)形成細(xì)胞的平均蛋白點(diǎn)數(shù)為380±31,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)83.7%。老年組曝光部位角質(zhì)形成細(xì)胞平均蛋白點(diǎn)數(shù)為365±17,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)84.5%。分析比較兩組2-DE圖譜共確定38個(gè)差異蛋白點(diǎn),其中13個(gè)蛋白點(diǎn)在中年組曝光部位2-DE圖譜中高表達(dá),5個(gè)蛋白點(diǎn)在老年組曝光部位2-DE圖譜中高表達(dá),20個(gè)蛋白點(diǎn)在兩者間有2倍以上顯著性量變。青年組非曝光部位角質(zhì)形成細(xì)胞的平均蛋白點(diǎn)數(shù)為372±18,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)82.2%。中年組非曝光部位角質(zhì)形成細(xì)胞平均蛋白點(diǎn)數(shù)為363±19,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)85.8%。分析比較兩組2-DE圖譜共確定47個(gè)差異蛋白點(diǎn),其中16個(gè)蛋白點(diǎn)在青年組非曝光部位2-DE圖譜中高表達(dá),9個(gè)蛋白點(diǎn)在中年組非曝光部位2-DE圖譜中高表達(dá),22個(gè)蛋白點(diǎn)在兩者間有2倍以上顯著性量變。中年組非曝光部位角質(zhì)形成細(xì)胞平均蛋白點(diǎn)數(shù)為363±19,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)85.8%。老年組非曝光部位角質(zhì)形成細(xì)胞的平均蛋白點(diǎn)數(shù)為359±15,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)83.4%。分析比較兩組2-DE圖譜共確定40個(gè)差異蛋白點(diǎn),其中11個(gè)蛋白點(diǎn)在中年組非曝光部位2-DE圖譜中高表達(dá),8個(gè)蛋白點(diǎn)在老年組非曝光部位2-DE圖譜中高表達(dá),21個(gè)蛋白點(diǎn)在兩者間有2倍以上顯著性量變。青年組曝光部位角質(zhì)形成細(xì)胞的平均蛋白點(diǎn)數(shù)為387±21,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)81.6%。青年組非曝光部位角質(zhì)形成細(xì)胞平均蛋白點(diǎn)數(shù)為372±18,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)82.2%。分析比較兩組2-DE圖譜共確定48個(gè)差異蛋白點(diǎn),其中17個(gè)蛋白點(diǎn)在青年組曝光部位2-DE圖譜中高表達(dá),11個(gè)蛋白點(diǎn)在青年組非曝光部位2-DE圖譜中高表達(dá),20個(gè)蛋白點(diǎn)在兩者間有2倍以上顯著性量變。中年組曝光部位角質(zhì)形成細(xì)胞的平均蛋白點(diǎn)數(shù)為380±31,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)83.7%。中年組非曝光部位角質(zhì)形成細(xì)胞平均蛋白點(diǎn)數(shù)為363±19,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)85.8%。分析比較兩組2-DE圖譜共確定43個(gè)差異蛋白點(diǎn),其中12個(gè)蛋白點(diǎn)在中年組曝光部位2-DE圖譜中高表達(dá),13個(gè)蛋白點(diǎn)在中年組非曝光部位2-DE圖譜中高表達(dá),18個(gè)蛋白點(diǎn)在兩者間有2倍以上顯著性量變。老年組曝光部位角質(zhì)形成細(xì)胞的平均蛋白點(diǎn)數(shù)為365±17,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)84.5%。老年組非曝光部位角質(zhì)形成細(xì)胞平均蛋白點(diǎn)數(shù)為359±15,3塊膠間蛋白質(zhì)點(diǎn)的匹配率達(dá)83.4%。分析比較兩組2-DE圖譜共確定37個(gè)差異蛋白點(diǎn),其中9個(gè)蛋白點(diǎn)在老年組曝光部位2-DE圖譜中高表達(dá),14個(gè)蛋白點(diǎn)在老年組非曝光部位2-DE圖譜中高表達(dá),14個(gè)蛋白點(diǎn)在兩者間有2倍以上顯著性量變。選取其中的35個(gè)差異蛋白進(jìn)行MALDI-TOF質(zhì)譜分析及數(shù)據(jù)庫(kù)查詢(xún),成功鑒定了21個(gè)與皮膚老化相關(guān)的候選蛋白。我們的實(shí)驗(yàn)獲得了一些有價(jià)值的發(fā)現(xiàn)：在不同的年齡組、不同的部位檢測(cè)到了相同的蛋白,3次檢測(cè)到膜聯(lián)蛋白A2 (annexin A2)、2次檢測(cè)到角蛋白K2C80、2次檢測(cè)到熱休克蛋白、2次檢測(cè)到谷胱甘肽s-轉(zhuǎn)移酶(GSTs)、2次檢測(cè)到白蛋白(albumin),提示這幾種蛋白可能為皮膚老化中的重要蛋白質(zhì)；通過(guò)組間的比較分析,在青年組和中年組曝光部位HSP高表達(dá),在青年組和老年組曝光部位K2C80高表達(dá),提示HSP和K2C80可能是光老化的特異蛋白；在青年組和中年組的非暴光部位都有膜聯(lián)蛋白A2 (annexin A2)及谷胱甘肽s-轉(zhuǎn)移酶(GSTs)的表達(dá),在中年組和老年組的非暴光部位都有白蛋白(albumin)的表達(dá),可能與自然老化密切相關(guān)；而P21可能與自然老化直接相關(guān),HSP70與紫外線導(dǎo)致的皮膚光老化關(guān)系密切。結(jié)論利用蛋白質(zhì)組的技術(shù)平臺(tái),建立了適合皮膚老化研究的比較蛋白質(zhì)組的方法。通過(guò)比較不同部位、不同年齡人群中皮膚角質(zhì)形成細(xì)胞在自然、連續(xù)、動(dòng)態(tài)的成長(zhǎng)過(guò)程中存在差異表達(dá)蛋白。鑒定的21個(gè)候選蛋白功能各異,它們通過(guò)參與細(xì)胞的基礎(chǔ)能量代謝；影響蛋白質(zhì)的合成、折疊、降解過(guò)程；調(diào)控細(xì)胞的生長(zhǎng)、增殖、分化；參與細(xì)胞骨架蛋白的構(gòu)建；參與細(xì)胞的凋亡、免疫調(diào)控。使細(xì)胞增殖能力降低,加速細(xì)胞的凋亡；損傷細(xì)胞之間的連接,分泌各種細(xì)胞因子,通過(guò)各種通路,加速膠原的降解,導(dǎo)致氧化和抗氧化的失衡,形成了錯(cuò)綜復(fù)雜的網(wǎng)絡(luò)體系,在自然老化和(或)光老化的過(guò)程發(fā)揮一定作用。我們探尋到其中差異表達(dá)的蛋白或特異表達(dá)的蛋白有可能成為皮膚老化的標(biāo)志性蛋白,初步探討了角質(zhì)形成細(xì)胞在皮膚老化過(guò)程中的作用及分子機(jī)制,為揭示衰老的本質(zhì)奠定了基礎(chǔ)。但要確定某種蛋白是皮膚老化的標(biāo)志性蛋白,尚需進(jìn)一步驗(yàn)證。
[Abstract]:Objective the mechanism of skin aging is unknown, and the objective index of skin aging judgment is lack in clinical practice. This subject takes the skin of young, middle-aged, aged and unexposed parts as the research object, exploring the proteins expressed differently and the specific protein can become the marker protein of skin aging, and explore horny. The role and molecular mechanism of the formation of cells in the process of skin aging. It lays the foundation for the comprehensive explanation of the mechanism of skin aging and revealing the essence of aging. Methods using the proteomics technology platform, Dispase II was used to separate the epidermis, optimize the sample processing method, and use the crack of cocktail, RNase and DNase. The solution extracts the total protein of epidermal tissue, after isoelectric focusing and vertical electrophoresis, after silver staining, the 2-DE images of keratinocytes in the epidermis are obtained. The two-dimensional gel electrophoresis method suitable for the study of epidermal tissue is established and optimized. The dyed gel is scanned with GS800 density scanner, and the image is divided by PDQuest7.4 image analysis software. Analysis, intensity correction, background subtraction, point detection, matching, ID correction, and determination of differential protein points. Select the protein points in a walking MALDI-TOF mass spectrometry analysis and database query, and finally identify the difference candidate proteins. The results obtained are clear, the protein points are separated completely, and the atlas obtained after 3 times is repeated. It is often similar to match the protein points between 3 pieces of glue. The deviation of the protein point in the first IEF direction is (1.798 + 1.232) mm, and the deviation in the direction of second to SDS-PAGE is (1.063 + 1.031) mm, and the duplex better matching rate of the epidermal tissue two direction gel electrophoresis atlas is obtained. Thus, the suitable skin group is successfully established. The keratinocyte protein was distributed in the range of pH3-10, but mainly concentrated in the range of pH 5-8, and the relative molecular mass was mainly concentrated in the 19-110k0. The detection of protein points in the pH 5-8 range, quantizing and point matching results showed that the exposed parts of the young group were horny. The average protein point of the formation of cells was 387 + 21,3 blocks, and the matching rate of protein points was reached in 81.6%. middle age group. The average protein points of keratinocytes in the exposed parts of the middle age group were 380 + 31,3 blocks, and the matching rate of protein points was 83.7%. analysis and compared two groups of 2-DE atlas. The 12 protein points were in the youth group exposure department. The high expression of the 17 protein points in the 2-DE Atlas of the middle-aged group was high, and the 14 protein points were more than 2 times of the significant quantitative change between the two. The average protein points of the keratinocytes at the exposed parts of the middle-aged group were 380 + 31,3 blocks, and the matching rate of the protein points was reached to the level of the keratinocytes at the exposure site of the aged group. The matching rate of protein points between 365 + 17,3 blocks was 84.5%. analysis and compared with the two groups of 2-DE maps, 38 different protein points were determined. Among them, 13 protein points were highly expressed in the 2-DE Atlas of the exposed parts of the middle age group. The 5 protein points were high in the 2-DE Atlas of the exposed parts of the aged group, and the 20 protein points were more than 2 times more significant between the two groups. The average protein points of the keratinocytes in the unexposed parts of the young group were 372 + 18,3 blocks. The matching rate of the protein points of the keratinocytes in the unexposed parts of the middle aged group 82.2%. was 363 + 19,3 blocks, and the matching rate of the protein points was 85.8%. analysis and compared the two groups of 2-DE atlas to determine 47 difference protein points, 16 protein points were highly expressed in the 2-DE Atlas of the unexposed parts of the young group, 9 protein points were highly expressed in the 2-DE Atlas of the unexposed parts of the middle age group, and the 22 protein points were more than 2 times significant. The average protein points of the keratinocytes in the non exposed parts of the middle-aged group were 363 + 19,3 blocks, and the matching rate of the protein points was 85.. The average protein points of the keratinocytes in the non exposed parts of the aged group of 8%. were 359 + 15,3 blocks with 83.4%. analysis and compared with the two groups of 2-DE maps, 40 different protein points were determined, of which 11 protein points were highly expressed in the 2-DE Atlas of the unexposed parts of the middle age group, and the 8 protein points were in the 2-DE Atlas of the unexposed parts of the elderly group. The average protein points of the keratinocytes at the exposed parts of the young group were 387 + 21,3 blocks, and the matching rate of the protein points of the keratinocytes in the young group was 387 + 21,3. The average protein points of the keratinocytes in the unexposed parts of the young group were 372 + 18,3 blocks, and the matching rate of the protein points of 372 + 18,3 blocks was up to 82.2%. points. Two groups of 2-DE atlas were compared to determine 48 difference protein points, of which 17 protein points were highly expressed in the 2-DE Atlas of young group exposure, and 11 protein points were highly expressed in the unexposed part of the young group, and the 20 protein points were more than 2 times significant quantitative change between the two. The average protein points of the keratinocytes at the exposed parts of the middle age group were found. The matching rate of protein points between 380 + 31,3 blocks was reached to the average protein points of the keratinocytes in the unexposed parts of the middle age group of 83.7%.. The matching rate of protein points of 363 + 19,3 blocks was 85.8%. analysis and compared with the two groups of 2-DE maps, 43 different protein points were determined, of which 12 protein points were highly expressed in the 2-DE Atlas of the exposed parts of the middle age group, 13 The protein points were highly expressed in the 2-DE Atlas of the unexposed parts of the middle-aged group, and the 18 protein points were more than 2 times significant. The average protein points of the keratinocytes at the exposed parts of the aged group were 365 + 17,3 blocks, the average protein points of the non exposed keratinocytes in the elderly group were 359 +. The matching rate of protein points between 15,3 blocks reached 83.4%. analysis and compared two groups of 2-DE maps to determine 37 difference protein points, of which 9 protein points were highly expressed in the 2-DE Atlas of the exposed parts of the aged group, and the 14 protein points were highly expressed in the 2-DE Atlas of the unexposed parts of the aged group, and the 14 protein points had more than 2 times significant quantitative variation between the two. 35 of the 35 differential proteins were analyzed by mass spectrometry and database query, and 21 candidate proteins associated with skin aging were successfully identified. Our experiments obtained some valuable findings: the same protein was detected in different age groups, different parts of the protein, and the 3 times detected the annexin A2 (annexin A2) and the 2 detection of the angle. The protein K2C80,2 detected the heat shock protein at the 2 time and detected the glutathione s- transferase (GSTs) and the 2 times the albumin (albumin), suggesting that these proteins may be important proteins in the skin aging. Through comparison and analysis among the groups, the HSP in the exposed parts of the young and middle-aged groups was highly expressed, and the exposure site was K2C8 in the young and the elderly groups. 0 high expression, suggesting that HSP and K2C80 may be specific proteins of light aging; the unexposed parts of young and middle-aged groups are expressed in unexposed parts of A2 (annexin A2) and glutathione s- transferase (GSTs). The expression of albumin (albumin) in the unexposed parts of the middle age group and the elderly group may be closely related to natural aging, and P21. It may be directly related to natural aging. HSP70 and UV induced skin photoaging are closely related. Conclusion using the technical platform of protein group, a comparative protein group suitable for skin aging is established. By comparing different parts of age, the skin keratinocytes in different age groups are natural, continuous and dynamic. There are differential expression proteins in the process. The 21 candidate proteins have different functions. They participate in the basic energy metabolism of the cells; affect the synthesis, folding and degradation of protein; regulate the growth, proliferation and differentiation of cells; participate in the construction of cytoskeleton protein; participate in cell apoptosis and immunoregulation. Low, accelerating cell apoptosis; damaging the connections between cells, secreting a variety of cytokines, accelerating collagen degradation through various pathways, resulting in oxidation and antioxidant imbalance, forming a complex network system that plays a certain role in natural aging and / or photoaging. Specifically expressed proteins may become the marker proteins of skin aging, preliminarily discuss the role and molecular mechanism of keratinocytes in the aging process of the skin, which lay the foundation for revealing the essence of aging. But to determine a certain protein as a marker protein of skin aging, further verification is needed.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R339.11

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