本文選題：毛發(fā)周期 + 蛋白質組學　； 參考：《南京醫(yī)科大學》2010年博士論文

【摘要】： 毛發(fā)疾病是臨床的常見病和多發(fā)病,但在治療上缺乏非常有效的方法。隨著人們生活水平的提高,人們對健康毛發(fā)的要求也日益強烈。毛發(fā)的生長與脫落取決于毛囊周期性的生長。毛發(fā)的生長周期分為三期:即生長期(anagen)、退行期(catagen)及休止期(telogen)。雄激素性脫發(fā)是一種具有遺傳傾向、漸進性頭發(fā)脫落,病理表現為毛囊體積及密度逐漸縮小,生長期與休止期毛囊的比例逐漸降低。如果能夠局部下調毛囊內雄激素受體的表達水平,將有望成功地治療或逆轉由雄激素介導的雄激素性脫發(fā)。 本研究構建了小鼠毛發(fā)周期的動物模型,用蛋白組學的方法研究差異表達的因子,通過Western blot和免疫組化來驗證;進一步研究毛發(fā)的生長周期中相關信號通路及MicroRNA的表達譜;選擇雄激素受體為靶標,用RNA干擾的方法來研究其在雄激素脫發(fā)基因治療中的作用。主要研究內容和結果如下: 一、小鼠毛發(fā)周期的蛋白質組學研究 構建小鼠毛發(fā)周期的動物模型,共篩選出95個差異表達蛋白,其中45個通過質譜分析和SwissProt蛋白數據庫檢索得到鑒定,如肌球蛋白輕鏈、原肌球蛋白1、膜聯蛋白A、核纖層蛋白A/C、延伸因子1、細胞色素b、磷酸甘油醛脫氫酶等,這些蛋白主要與細胞凋亡、細胞增殖與分化和信號轉導等有關。其中5個通過Western blot和免疫組化證實它們在毛發(fā)周期中有差異表達。 二、小鼠毛發(fā)周期相關信號轉導及MicroRNA表達譜的研究 用Western blot方法研究小鼠毛發(fā)周期中細胞周期及凋亡相關的信號通路,如細胞周期蛋白A、B、D、E,細胞周期調節(jié)、凋亡相關的P53、P21、P38、BCL2、Bax、Rb,信號轉導相關的TGF-β1等的表達。提取小鼠皮膚組織的RNA,進行miRNA微陣列芯片檢測并行數據分析,結果發(fā)現miR-690、miR-1308、miR-291a-5p、miR-212、miR-31有2倍以上的差異表達。 三、針對雄激素受體的RNA干擾對雄激素脫發(fā)基因治療的基礎研究 構建針對雄激素受體(androgen receptor,AR)RNA干擾的真核表達質粒,將其轉染人毛乳頭細胞(dermal papilla cell,DPC)后發(fā)現其能抑制AR的表達。MTT法及3H-TdR摻入法證明RNAi質粒轉染對人DPC增殖無促進作用,流式細胞儀檢測結果顯示轉染不會引起細胞的凋亡及細胞周期的改變。RNAi質粒能在轉染體外培養(yǎng)的毛囊,但對毛發(fā)的生長無明顯的促進作用。脂質體轉染的方法能成功將RNAi質粒轉染小鼠的皮膚。 綜上所述,本研究成功建立了小鼠毛發(fā)周期的蛋白質表達譜與miRNA表達譜,發(fā)現了差異表達的多個蛋白及miRNA,并深入研究了小鼠毛發(fā)周期中相關信號傳導的分子機制。選擇雄激素受體為靶標,采用RNA干擾的方法研究其轉染人毛乳頭細胞、人毛囊和小鼠皮膚的生物學效應。本實驗為今后研究毛發(fā)生長的宏觀調控網絡與毛發(fā)疾病的診斷和基因治療提供了實驗依據,從而為進一步開發(fā)防治脫發(fā)的新型藥物提供可能的方向。
[Abstract]:Hair disease is a common and frequent disease in clinic, but it lacks effective treatment. With the improvement of people's living standard, people's demand for healthy hair is becoming more and more intense. The growth and shedding of hair depends on the periodic growth of hair follicles. The growth cycle of hair can be divided into three stages: the growth period of anagenase, the receding stage of catagenesis, and the rest period of Thalogena. Androgen alopecia is a kind of hair loss with genetic tendency and progressive hair loss. The pathological manifestation is that the volume and density of hair follicles decrease gradually and the proportion of hair follicles between growth period and rest period decreases gradually. If the expression of androgen receptor in hair follicles can be down-regulated locally, androgen-induced alopecia may be successfully treated or reversed. In this study, an animal model of mouse hair cycle was established, and the differential expression factors were studied by proteomics, which were verified by Western blot and immunohistochemistry, and the related signal pathways and microRNAs expression profiles in hair growth cycle were further studied. The role of androgen receptor in androgen alopecia gene therapy was studied by RNA interference. The main contents and results are as follows: first, the proteomics of mouse hair cycle was used to construct the animal model of mouse hair cycle and 95 differentially expressed proteins were screened out. 45 of them were identified by mass spectrometry and SwissProt protein database, such as myosin light chain, promyosin 1, binding protein A, nuclear laminin A / C, extender 1, cytochrome b, glyceraldehyde phosphate dehydrogenase, etc. These proteins are mainly related to cell apoptosis, cell proliferation and differentiation and signal transduction. Five of them showed differential expression in hair cycle by Western blot and immunohistochemistry. Second, the study of mouse hair cycle related signal transduction and microRNA expression profile; Western blot method was used to study the cell cycle and apoptosis-related signaling pathways in mouse hair cycle, such as cyclin A, B, D, E, cell cycle regulation. Apoptosis-related expression of P53P21, P38, BCL2, Baxfen, Rb, signal transduction related TGF- 尾 1, etc. The RNAs from mouse skin were extracted, and the miRNA microarray analysis was performed. The results showed that miR-690 miR-1308 miR-291a-5pmmiR-212miR-31 had more than 2 times differential expression. Thirdly, the basic research of androgen receptor RNA interference on androgen alopecia gene therapy was to construct eukaryotic expression plasmid for androgen receptor ARN RNA interference. The expression of AR and 3H-TdR incorporation in human dermal papilla cells were inhibited by transfection of DPCs. The results showed that the transfection of RNAi plasmid could not promote the proliferation of human dermal papilla cells. The results of flow cytometry showed that transfection could not induce apoptosis and cell cycle change. RNAi plasmid could transfect hair follicles in vitro, but had no obvious effect on hair growth. The RNAi plasmid was successfully transfected into the skin of mice by liposome transfection. To sum up, we successfully established the protein expression profile and miRNA expression profile of mouse hair cycle, and found several differentially expressed proteins and miRNAs, and studied the molecular mechanism of related signal transduction in mouse hair cycle. The biological effects of androgen receptor on human dermal papilla cells, human hair follicles and mouse skin were studied by RNA interference. This experiment provides experimental basis for the future study on the macroscopical control network of hair growth and the diagnosis and gene therapy of hair diseases, thus providing a possible direction for the further development of new drugs for the prevention and treatment of hair loss.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R758.7

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本文編號：2010817