毛发周期的调控网络与雄激素性脱发基因治疗的基础研究

发布时间：2018-06-12 19:30

本文选题：毛发周期 + 蛋白质组学　；参考：《南京医科大学》2010年博士论文

【摘要】： 毛发疾病是临床的常见病和多发病,但在治疗上缺乏非常有效的方法。随着人们生活水平的提高,人们对健康毛发的要求也日益强烈。毛发的生长与脱落取决于毛囊周期性的生长。毛发的生长周期分为三期:即生长期(anagen)、退行期(catagen)及休止期(telogen)。雄激素性脱发是一种具有遗传倾向、渐进性头发脱落,病理表现为毛囊体积及密度逐渐缩小,生长期与休止期毛囊的比例逐渐降低。如果能够局部下调毛囊内雄激素受体的表达水平,将有望成功地治疗或逆转由雄激素介导的雄激素性脱发。本研究构建了小鼠毛发周期的动物模型,用蛋白组学的方法研究差异表达的因子,通过Western blot和免疫组化来验证;进一步研究毛发的生长周期中相关信号通路及MicroRNA的表达谱;选择雄激素受体为靶标,用RNA干扰的方法来研究其在雄激素脱发基因治疗中的作用。主要研究内容和结果如下: 一、小鼠毛发周期的蛋白质组学研究构建小鼠毛发周期的动物模型,共筛选出95个差异表达蛋白,其中45个通过质谱分析和SwissProt蛋白数据库检索得到鉴定,如肌球蛋白轻链、原肌球蛋白1、膜联蛋白A、核纤层蛋白A/C、延伸因子1、细胞色素b、磷酸甘油醛脱氢酶等,这些蛋白主要与细胞凋亡、细胞增殖与分化和信号转导等有关。其中5个通过Western blot和免疫组化证实它们在毛发周期中有差异表达。二、小鼠毛发周期相关信号转导及MicroRNA表达谱的研究用Western blot方法研究小鼠毛发周期中细胞周期及凋亡相关的信号通路,如细胞周期蛋白A、B、D、E,细胞周期调节、凋亡相关的P53、P21、P38、BCL2、Bax、Rb,信号转导相关的TGF-β1等的表达。提取小鼠皮肤组织的RNA,进行miRNA微阵列芯片检测并行数据分析,结果发现miR-690、miR-1308、miR-291a-5p、miR-212、miR-31有2倍以上的差异表达。三、针对雄激素受体的RNA干扰对雄激素脱发基因治疗的基础研究构建针对雄激素受体(androgen receptor,AR)RNA干扰的真核表达质粒,将其转染人毛乳头细胞(dermal papilla cell,DPC)后发现其能抑制AR的表达。MTT法及3H-TdR掺入法证明RNAi质粒转染对人DPC增殖无促进作用,流式细胞仪检测结果显示转染不会引起细胞的凋亡及细胞周期的改变。RNAi质粒能在转染体外培养的毛囊,但对毛发的生长无明显的促进作用。脂质体转染的方法能成功将RNAi质粒转染小鼠的皮肤。综上所述,本研究成功建立了小鼠毛发周期的蛋白质表达谱与miRNA表达谱,发现了差异表达的多个蛋白及miRNA,并深入研究了小鼠毛发周期中相关信号传导的分子机制。选择雄激素受体为靶标,采用RNA干扰的方法研究其转染人毛乳头细胞、人毛囊和小鼠皮肤的生物学效应。本实验为今后研究毛发生长的宏观调控网络与毛发疾病的诊断和基因治疗提供了实验依据,从而为进一步开发防治脱发的新型药物提供可能的方向。
[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.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R758.7

【参考文献】