本文選題：分化 + HaCaT��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：目的 F1F0-ATP(合成)酶是一種廣泛分布于各種組織細胞內(nèi)的酶復(fù)合體,是線粒體氧化磷酸化體系的重要組成部分,主要在細胞內(nèi)或細胞膜發(fā)揮合成或水解ATP的功能。本研究著眼于F1F0-ATP(合成)酶在角質(zhì)形成細胞分化過程中表達的變化,及其可能發(fā)揮的作用。為進一步研究角質(zhì)形成細胞分化與細胞內(nèi)外ATP水平及能量代謝的關(guān)系奠定基礎(chǔ)。 方法 1、采用免疫組化方法檢測和比較分析了正常皮膚、銀屑病、角化棘皮瘤、皮膚鱗癌皮損中F1F0-ATP(合成)酶β亞基(ATP5B)的表達模式； 2、在建立體外培養(yǎng)密度依賴的HaCaT細胞分化模型基礎(chǔ)上,用免疫印跡、免疫熒光和RT-PCR的方法檢測F1F0-ATP(合成)酶p亞基(ATP5B)表達的變化； 3、應(yīng)用ATP熒光定量檢測方法檢測了F1F0-ATP(合成)酶小分子抑制劑——寡霉素和白皮杉醇,對于HaCaT細胞內(nèi)外ATP水平的影響,并用免疫印跡方法檢測了其對于HaCaT細胞分化標志(involucrin)的影響。 結(jié)果 1、在正常皮膚、銀屑病皮損和角化棘皮瘤中, ATP5B隨表皮分化而表達增高；在銀屑病皮損和角化棘皮瘤中ATP5B較正常皮膚表達下降；鱗癌癌灶中ATP5B較周邊相對正常皮膚表達下降； 2、建立了體外培養(yǎng)的密度依賴的HaCaT細胞分化模型,在mRNA水平和蛋白水平驗證了分化指標的表達隨培養(yǎng)時間延長而增加； 3、在密度依賴的HaCaT分化模型中,ATP5B在mRNA水平上,第6d和第8d可見顯著上調(diào)(p0.05),在蛋白水平上未見明顯改變(p0.05)； 4、常規(guī)培養(yǎng)狀態(tài)下,寡霉素可降低HaCaT細胞內(nèi)ATP的水平和involucrin蛋白表達水平；白皮杉醇主要降低HaCaT細胞外ATP的水平,對胞內(nèi)ATP水平和involucrin蛋白表達水平無明顯抑制作用； 5、在密度依賴的分化模型中,白皮杉醇可顯著降低分化模型后期細胞內(nèi)ATP的水平和involucrin的蛋白表達水平(p0.05)。 結(jié)論 1、F1F0-ATP(合成)酶可能隨著角質(zhì)形成細胞的分化而表達增高,在銀屑病等幾類常見表皮增殖性疾病皮損中表達下調(diào)。 2、F1F0-ATP(合成)酶抑制劑可以抑制HaCaT細胞的分化,并且其作用可能與細胞內(nèi)ATP水平的下調(diào)有關(guān)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R751

【共引文獻】

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本文編號：1997529