皮肤老化过程中角质形成细胞比较蛋白质组分析与鉴定

发布时间：2018-07-03 12:57

本文选题：角质形成细胞 + 皮肤老化　；参考：《重庆医科大学》2009年博士论文

【摘要】：目的皮肤老化的机制不明,且临床实际中缺乏皮肤老化判断的客观指标。本课题以青年、中年、老年人曝光部位及非曝光部位的皮肤为研究对象,探寻其中差异表达的蛋白或特异表达的蛋白有可能成为皮肤老化的标志性蛋白,探讨角质形成细胞在皮肤老化过程中的作用及分子机制。为后续研究全面解释皮肤老化的机制,揭示衰老的本质奠定基础。方法借助蛋白质组学研究技术平台,采用Dispase Ⅱ分离出表皮,优化改进样本处理方法,使用加入了cocktail,RNase和DNase的裂解液提取表皮组织总蛋白,经等电聚焦和垂直电泳,银染后获得了表皮组织中角质形成细胞2-DE图像,建立和优化了适合于表皮组织研究的双向凝胶电泳方法。染色后的凝胶用GS800密度扫描仪扫描,并用PDQuest7.4图像分析软件进行图像分析,强度校正、背景消减、点检测、匹配、ID校正,确定差异蛋白点。挑选其中的蛋白点进一步行MALDI-TOF质谱分析及数据库查询,最后鉴定差异候选蛋白。结果获得的双向凝胶电泳图谱较为清晰,蛋白质点分离完全,各组重复3次后得到的图谱非常相似,进行3块胶间的蛋白质点的匹配,测量胶间蛋白质点在第一向IEF方向的偏差为(1.798±1.232)mm,在第二向SDS-PAGE方向上的偏差为(1.063±1.031)mm,获得了重复性较好匹配率高的表皮组织双向凝胶电泳图谱,由此成功建立了适合人表皮组织比较蛋白质组研究的双向凝胶电泳方法。角质形成细胞蛋白在pH3-10的范围均有分布,但主要集中分布在pH 5-8范围内,相对分子质量主要集中在19-110k0之间。在pH 5-8范围内蛋白点的检测、量化和点的匹配结果显示：青年组曝光部位角质形成细胞的平均蛋白点数为387±21,3块胶间蛋白质点的匹配率达81.6%。中年组曝光部位角质形成细胞平均蛋白点数为380±31,3块胶间蛋白质点的匹配率达83.7%。分析比较两组2-DE图谱共确定43个差异蛋白点,其中12个蛋白点在青年组曝光部位2-DE图谱中高表达,17个蛋白点在中年组曝光部位2-DE图谱中高表达,14个蛋白点在两者间有2倍以上显著性量变。中年组曝光部位角质形成细胞的平均蛋白点数为380±31,3块胶间蛋白质点的匹配率达83.7%。老年组曝光部位角质形成细胞平均蛋白点数为365±17,3块胶间蛋白质点的匹配率达84.5%。分析比较两组2-DE图谱共确定38个差异蛋白点,其中13个蛋白点在中年组曝光部位2-DE图谱中高表达,5个蛋白点在老年组曝光部位2-DE图谱中高表达,20个蛋白点在两者间有2倍以上显著性量变。青年组非曝光部位角质形成细胞的平均蛋白点数为372±18,3块胶间蛋白质点的匹配率达82.2%。中年组非曝光部位角质形成细胞平均蛋白点数为363±19,3块胶间蛋白质点的匹配率达85.8%。分析比较两组2-DE图谱共确定47个差异蛋白点,其中16个蛋白点在青年组非曝光部位2-DE图谱中高表达,9个蛋白点在中年组非曝光部位2-DE图谱中高表达,22个蛋白点在两者间有2倍以上显著性量变。中年组非曝光部位角质形成细胞平均蛋白点数为363±19,3块胶间蛋白质点的匹配率达85.8%。老年组非曝光部位角质形成细胞的平均蛋白点数为359±15,3块胶间蛋白质点的匹配率达83.4%。分析比较两组2-DE图谱共确定40个差异蛋白点,其中11个蛋白点在中年组非曝光部位2-DE图谱中高表达,8个蛋白点在老年组非曝光部位2-DE图谱中高表达,21个蛋白点在两者间有2倍以上显著性量变。青年组曝光部位角质形成细胞的平均蛋白点数为387±21,3块胶间蛋白质点的匹配率达81.6%。青年组非曝光部位角质形成细胞平均蛋白点数为372±18,3块胶间蛋白质点的匹配率达82.2%。分析比较两组2-DE图谱共确定48个差异蛋白点,其中17个蛋白点在青年组曝光部位2-DE图谱中高表达,11个蛋白点在青年组非曝光部位2-DE图谱中高表达,20个蛋白点在两者间有2倍以上显著性量变。中年组曝光部位角质形成细胞的平均蛋白点数为380±31,3块胶间蛋白质点的匹配率达83.7%。中年组非曝光部位角质形成细胞平均蛋白点数为363±19,3块胶间蛋白质点的匹配率达85.8%。分析比较两组2-DE图谱共确定43个差异蛋白点,其中12个蛋白点在中年组曝光部位2-DE图谱中高表达,13个蛋白点在中年组非曝光部位2-DE图谱中高表达,18个蛋白点在两者间有2倍以上显著性量变。老年组曝光部位角质形成细胞的平均蛋白点数为365±17,3块胶间蛋白质点的匹配率达84.5%。老年组非曝光部位角质形成细胞平均蛋白点数为359±15,3块胶间蛋白质点的匹配率达83.4%。分析比较两组2-DE图谱共确定37个差异蛋白点,其中9个蛋白点在老年组曝光部位2-DE图谱中高表达,14个蛋白点在老年组非曝光部位2-DE图谱中高表达,14个蛋白点在两者间有2倍以上显著性量变。选取其中的35个差异蛋白进行MALDI-TOF质谱分析及数据库查询,成功鉴定了21个与皮肤老化相关的候选蛋白。我们的实验获得了一些有价值的发现：在不同的年龄组、不同的部位检测到了相同的蛋白,3次检测到膜联蛋白A2 (annexin A2)、2次检测到角蛋白K2C80、2次检测到热休克蛋白、2次检测到谷胱甘肽s-转移酶(GSTs)、2次检测到白蛋白(albumin),提示这几种蛋白可能为皮肤老化中的重要蛋白质；通过组间的比较分析,在青年组和中年组曝光部位HSP高表达,在青年组和老年组曝光部位K2C80高表达,提示HSP和K2C80可能是光老化的特异蛋白；在青年组和中年组的非暴光部位都有膜联蛋白A2 (annexin A2)及谷胱甘肽s-转移酶(GSTs)的表达,在中年组和老年组的非暴光部位都有白蛋白(albumin)的表达,可能与自然老化密切相关；而P21可能与自然老化直接相关,HSP70与紫外线导致的皮肤光老化关系密切。结论利用蛋白质组的技术平台,建立了适合皮肤老化研究的比较蛋白质组的方法。通过比较不同部位、不同年龄人群中皮肤角质形成细胞在自然、连续、动态的成长过程中存在差异表达蛋白。鉴定的21个候选蛋白功能各异,它们通过参与细胞的基础能量代谢；影响蛋白质的合成、折叠、降解过程；调控细胞的生长、增殖、分化；参与细胞骨架蛋白的构建；参与细胞的凋亡、免疫调控。使细胞增殖能力降低,加速细胞的凋亡；损伤细胞之间的连接,分泌各种细胞因子,通过各种通路,加速胶原的降解,导致氧化和抗氧化的失衡,形成了错综复杂的网络体系,在自然老化和(或)光老化的过程发挥一定作用。我们探寻到其中差异表达的蛋白或特异表达的蛋白有可能成为皮肤老化的标志性蛋白,初步探讨了角质形成细胞在皮肤老化过程中的作用及分子机制,为揭示衰老的本质奠定了基础。但要确定某种蛋白是皮肤老化的标志性蛋白,尚需进一步验证。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R339.11

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