F1F0-ATP（合成）酶在角质形成细胞分化过程中的表达及其功能研究

发布时间：2018-06-08 22:49

本文选题：分化 + HaCaT　；参考：《中南大学》2012年博士论文

【摘要】：目的 F1F0-ATP(合成)酶是一种广泛分布于各种组织细胞内的酶复合体,是线粒体氧化磷酸化体系的重要组成部分,主要在细胞内或细胞膜发挥合成或水解ATP的功能。本研究着眼于F1F0-ATP(合成)酶在角质形成细胞分化过程中表达的变化,及其可能发挥的作用。为进一步研究角质形成细胞分化与细胞内外ATP水平及能量代谢的关系奠定基础。方法 1、采用免疫组化方法检测和比较分析了正常皮肤、银屑病、角化棘皮瘤、皮肤鳞癌皮损中F1F0-ATP(合成)酶β亚基(ATP5B)的表达模式； 2、在建立体外培养密度依赖的HaCaT细胞分化模型基础上,用免疫印迹、免疫荧光和RT-PCR的方法检测F1F0-ATP(合成)酶p亚基(ATP5B)表达的变化； 3、应用ATP荧光定量检测方法检测了F1F0-ATP(合成)酶小分子抑制剂——寡霉素和白皮杉醇,对于HaCaT细胞内外ATP水平的影响,并用免疫印迹方法检测了其对于HaCaT细胞分化标志(involucrin)的影响。结果 1、在正常皮肤、银屑病皮损和角化棘皮瘤中, ATP5B随表皮分化而表达增高；在银屑病皮损和角化棘皮瘤中ATP5B较正常皮肤表达下降；鳞癌癌灶中ATP5B较周边相对正常皮肤表达下降； 2、建立了体外培养的密度依赖的HaCaT细胞分化模型,在mRNA水平和蛋白水平验证了分化指标的表达随培养时间延长而增加； 3、在密度依赖的HaCaT分化模型中,ATP5B在mRNA水平上,第6d和第8d可见显著上调(p0.05),在蛋白水平上未见明显改变(p0.05)； 4、常规培养状态下,寡霉素可降低HaCaT细胞内ATP的水平和involucrin蛋白表达水平；白皮杉醇主要降低HaCaT细胞外ATP的水平,对胞内ATP水平和involucrin蛋白表达水平无明显抑制作用； 5、在密度依赖的分化模型中,白皮杉醇可显著降低分化模型后期细胞内ATP的水平和involucrin的蛋白表达水平(p0.05)。结论 1、F1F0-ATP(合成)酶可能随着角质形成细胞的分化而表达增高,在银屑病等几类常见表皮增殖性疾病皮损中表达下调。 2、F1F0-ATP(合成)酶抑制剂可以抑制HaCaT细胞的分化,并且其作用可能与细胞内ATP水平的下调有关。
[Abstract]:objective
F1F0-ATP (synthetic) enzyme is an enzyme complex that is widely distributed in various tissue cells. It is an important part of the mitochondrial oxidative phosphorylation system. It plays the function of synthesizing or hydrolyzing ATP mainly in cell or cell membrane. This study focuses on the changes in the expression of F1F0-ATP (synthetic) enzyme in the process of keratinocytic differentiation, and it can be used in this study. It can lay a foundation for further studying the relationship between keratinocyte differentiation and intracellular ATP level and energy metabolism.
Method
1, the expression patterns of F1F0-ATP (synthetic) enzyme beta subunit (ATP5B) in normal skin, psoriasis, keratoderma, and skin squamous cell carcinoma were detected and compared by immunohistochemical method.
2, on the basis of the establishment of a density dependent HaCaT cell differentiation model in vitro, the changes in the expression of F1F0-ATP (synthetic) enzyme P subunit (ATP5B) were detected by immunoblotting, immunofluorescence and RT-PCR.
3, the effect of F1F0-ATP (synthetic) enzyme small molecule inhibitor, oligomycin and paclitaxel, on the ATP level inside and outside HaCaT cells was detected by ATP fluorescence quantitative detection method, and the effect of its effect on the HaCaT cell differentiation marker (involucrin) was detected by immunoblotting.
Result
1, in normal skin, psoriasis and keratoderma, the expression of ATP5B was increased with the differentiation of epidermis, and the expression of ATP5B in psoriatic lesions and keratoderma was lower than that in normal skin, and the expression of ATP5B in the squamous cell carcinoma was lower than that of the peripheral normal skin.
2, the density dependent HaCaT cell differentiation model was established in vitro, and the expression of differentiation index increased with the prolongation of culture time at mRNA level and protein level.
3, in the density dependent HaCaT differentiation model, ATP5B was significantly up-regulated at mRNA level (6D and 8D) (P0.05), and no significant change was found in protein level (P0.05).
4, under conventional culture, oligomycin could reduce the level of ATP in HaCaT cells and the expression level of involucrin protein, and the level of ATP in HaCaT cells was decreased mainly by paclitaxel, and there was no obvious inhibitory effect on the intracellular ATP level and the expression of involucrin protein.
5, in a density dependent differentiation model, taxol significantly decreased the intracellular ATP level and involucrin protein expression in the later stage of differentiation (P0.05).
conclusion
1, F1F0-ATP (synthetic) enzymes may be increased with keratinocyte differentiation and downregulated in psoriasis and other common epidermic proliferative diseases.
2, F1F0-ATP (synthetic) enzyme inhibitors can inhibit the differentiation of HaCaT cells, and its effect may be related to the downregulation of ATP level in cells.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R751

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