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

【摘要】： 毛發(fā)疾病是臨床的常見(jiàn)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(duì)健康毛發(fā)的要求也日益強(qiáng)烈。毛發(fā)的生長(zhǎng)與脫落取決于毛囊周期性的生長(zhǎng)。毛發(fā)的生長(zhǎng)周期分為三期:即生長(zhǎng)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(jìn)性頭發(fā)脫落,病理表現(xiàn)為毛囊體積及密度逐漸縮小,生長(zhǎng)期與休止期毛囊的比例逐漸降低。如果能夠局部下調(diào)毛囊內(nèi)雄激素受體的表達(dá)水平,將有望成功地治療或逆轉(zhuǎn)由雄激素介導(dǎo)的雄激素性脫發(fā)。 本研究構(gòu)建了小鼠毛發(fā)周期的動(dòng)物模型,用蛋白組學(xué)的方法研究差異表達(dá)的因子,通過(guò)Western blot和免疫組化來(lái)驗(yàn)證;進(jìn)一步研究毛發(fā)的生長(zhǎng)周期中相關(guān)信號(hào)通路及MicroRNA的表達(dá)譜;選擇雄激素受體為靶標(biāo),用RNA干擾的方法來(lái)研究其在雄激素脫發(fā)基因治療中的作用。主要研究?jī)?nèi)容和結(jié)果如下: 一、小鼠毛發(fā)周期的蛋白質(zhì)組學(xué)研究 構(gòu)建小鼠毛發(fā)周期的動(dòng)物模型,共篩選出95個(gè)差異表達(dá)蛋白,其中45個(gè)通過(guò)質(zhì)譜分析和SwissProt蛋白數(shù)據(jù)庫(kù)檢索得到鑒定,如肌球蛋白輕鏈、原肌球蛋白1、膜聯(lián)蛋白A、核纖層蛋白A/C、延伸因子1、細(xì)胞色素b、磷酸甘油醛脫氫酶等,這些蛋白主要與細(xì)胞凋亡、細(xì)胞增殖與分化和信號(hào)轉(zhuǎn)導(dǎo)等有關(guān)。其中5個(gè)通過(guò)Western blot和免疫組化證實(shí)它們?cè)诿l(fā)周期中有差異表達(dá)。 二、小鼠毛發(fā)周期相關(guān)信號(hào)轉(zhuǎn)導(dǎo)及MicroRNA表達(dá)譜的研究 用Western blot方法研究小鼠毛發(fā)周期中細(xì)胞周期及凋亡相關(guān)的信號(hào)通路,如細(xì)胞周期蛋白A、B、D、E,細(xì)胞周期調(diào)節(jié)、凋亡相關(guān)的P53、P21、P38、BCL2、Bax、Rb,信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的TGF-β1等的表達(dá)。提取小鼠皮膚組織的RNA,進(jìn)行miRNA微陣列芯片檢測(cè)并行數(shù)據(jù)分析,結(jié)果發(fā)現(xiàn)miR-690、miR-1308、miR-291a-5p、miR-212、miR-31有2倍以上的差異表達(dá)。 三、針對(duì)雄激素受體的RNA干擾對(duì)雄激素脫發(fā)基因治療的基礎(chǔ)研究 構(gòu)建針對(duì)雄激素受體(androgen receptor,AR)RNA干擾的真核表達(dá)質(zhì)粒,將其轉(zhuǎn)染人毛乳頭細(xì)胞(dermal papilla cell,DPC)后發(fā)現(xiàn)其能抑制AR的表達(dá)。MTT法及3H-TdR摻入法證明RNAi質(zhì)粒轉(zhuǎn)染對(duì)人DPC增殖無(wú)促進(jìn)作用,流式細(xì)胞儀檢測(cè)結(jié)果顯示轉(zhuǎn)染不會(huì)引起細(xì)胞的凋亡及細(xì)胞周期的改變。RNAi質(zhì)粒能在轉(zhuǎn)染體外培養(yǎng)的毛囊,但對(duì)毛發(fā)的生長(zhǎng)無(wú)明顯的促進(jìn)作用。脂質(zhì)體轉(zhuǎn)染的方法能成功將RNAi質(zhì)粒轉(zhuǎn)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(zhì)表達(dá)譜與miRNA表達(dá)譜,發(fā)現(xiàn)了差異表達(dá)的多個(gè)蛋白及miRNA,并深入研究了小鼠毛發(fā)周期中相關(guān)信號(hào)傳導(dǎo)的分子機(jī)制。選擇雄激素受體為靶標(biāo),采用RNA干擾的方法研究其轉(zhuǎn)染人毛乳頭細(xì)胞、人毛囊和小鼠皮膚的生物學(xué)效應(yīng)。本實(shí)驗(yàn)為今后研究毛發(fā)生長(zhǎng)的宏觀調(diào)控網(wǎng)絡(luò)與毛發(fā)疾病的診斷和基因治療提供了實(shí)驗(yàn)依據(jù),從而為進(jìn)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R758.7

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本文編號(hào)：2010817