皮肤真皮衰老相关microRNA表达谱分析及其在衰老成纤维细胞中的验证研究

发布时间：2017-12-26 16:25

本文关键词：皮肤真皮衰老相关microRNA表达谱分析及其在衰老成纤维细胞中的验证研究　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：研究背景皮肤衰老是机体衰老的重要外在表现。皮肤衰老不仅有损于美容,也可导致许多年龄相关性疾病,而给人带来身心损害。由于皮肤真皮包含多种类型的细胞及纵横交错的细胞外基质成分,因而在皮肤衰老过程中发挥着主导作用。目前有关真皮衰老的机制研究涉及了表观遗传学、基因组学、蛋白组学等多种分子生物学层面,然而nicroRNA (miRNA)与真皮衰老之间的关系尚不明确。miRNA是一类短链单链RNA,通过与靶基因3’端的非编码区序列特异性相结合从而发挥负性调节作用。miRNA可在多个层面影响机体的生理病理过程。了解真皮衰老相关miRNA的表达特点及调控网络有助于进一步加深对真皮衰老的认识,为后续抗衰老研究提供新的思路。研究目的1.初步了解真皮衰老进程中的miRNA差异表达谱,探讨该差异表达谱对应靶基因可能涉及的基因功能和调控通路,建立miRNA与部分衰老相关靶基因之间的网络关系。2.探索该差异表达的miRNA在年轻和衰老成纤维细胞中的变化情况,比较其与组织结果之间的一致性和差异性,并探讨这些miRNA可能参与的细胞衰老调控通路。研究方法在第一部分中,本研究选取12例源于非曝光部位的真皮样本,包括6例年轻真皮组织和6例衰老真皮组织,利用miRNA芯片初步筛选出衰老和年轻真皮差异表达的miRNA,运用实时定量RT-PCR验证部分miRNA表达情况。继而利用niRNA对应靶基因进行功能聚类分析和信号通路聚类分析,构建miRNA与可能涉及衰老调控的靶基因之间的调控网络,并得到网络中起核心调控作用的miRNA。在第二部分中,本研究将原代真皮成纤维细胞分别培养于正常营养和低营养培养基中并进行连续细胞传代,对各自营养状态下传代早期细胞与传代后期细胞进行多种衰老标记的比较,包括利用细胞计数法比较了细胞增殖曲线,利用SA-β-GA染色法比较了衰老细胞比例,运用实时定量RT-PCR方法比较了衰老相关标记基因的mRNA表达量。最后通过实时定量RT-PCR方法比较了部分真皮衰老相关miRNA在衰老代和年轻代成纤维细胞之间的变化情况。研究结果1. miRNA芯片结果分析显示,随着皮肤真皮衰老,真皮组织中共有40个miRNA差异性表达。2.实时定量RT-PCR显示衰老组织内miR-34家族及miR-29家族表达显著增加,与芯片结果具有一致性。3.靶基因功能聚类和通路聚类分析发现,miRNA对应靶基因的主要功能聚类包括细胞间粘附作用、胶原合成作用、基因转录的正性及负性调控作用等,主要通路聚类包括代谢调节通路、生长因子通路和细胞外基质与受体调控通路、DNA损伤反应通路等。4. miRNA-Gene-Network分析显示miR-34家族、miR-29家族和miR-424在调控网络中占有较大权重5.无论在正常营养培养条件抑或低营养培养条件下,相较于早期传代细胞而言,传代至后期时细胞的增殖能力均明显下降,SA-β-GA阳性细胞比例显著上升,p16基因的mRNA表达水平显著上调,而p53及p21基因的mRNA水平未见明显变化。6. miRNA实时定量结果显示,无论何种营养培养条件下,筛选出的11个miRNA(即miR-548q、miR-1226*、miR-601、miR-1207-5p、miR-134、miR-34b*、 miR-34a、miR-29c*、miR-181a, miR-154、miR-424)在衰老代细胞中均呈现出上调表达的趋势,其中绝大多数变化趋势与组织miRNA芯片结果相一致,但miR-181a、miR-154和miR-424在衰老细胞内的变化趋势与芯片结果之间存在矛盾。7. miRNA上调变化程度在各自营养培养条件下传代的衰老细胞中有所不同,正常营养条件下衰老代细胞以miR-34家族和miR-29家族上调最为显著,而低营养培养条件下衰老代细胞以miR-1226*、miR-601及miR-1207-5p上调为剧,miR-29家族未见明显上调。8.实时定量RT-PCR结果发现,低营养培养条件下的衰老代细胞内,miRNA部分靶基因如E2F3、c-Myc、CREB在mRNA水平上尚存在表达下降。研究结论真皮衰老进程中存在miRNA的差异性表达,差异表达miRNA的靶基因主要参与细胞粘附、胶原合成、基因转录调控中,主要涉及代谢通路、生长因子通路、细胞外基质与受体相互作用及DNA损伤反应通路等,且miR-34家族、miR-29家族及miR-424可能在真皮衰老过程中有较为重要的调控作用。在体外成纤维细胞的衰老模型中可观察到与真皮衰老进程大致相似的niRNA变化情况,但个别miRNA显示出的矛盾结果提示miRNA可能参与到除成纤维细胞衰老以外的真皮衰老的机制中,如炎症反应、血管新生异常等。成纤维细胞所处营养环境差异对调控衰老的miRNA表达情况存在影响。另外,本次研究建立的细胞衰老模型中,真皮衰老相关miRNA可能主要通过与p16通路发生联系从而对成纤维细胞衰老进程进行调控。
[Abstract]:Background the aging of the skin is an important external manifestation of the aging of the body. Skin aging is not only damaging to beauty, but also causes many age-related diseases, which can cause physical and mental damage to people. Skin dermis, which contains many types of cells and interlaced extracellular matrix, plays a leading role in the aging process of the skin. At present, the mechanism of dermal aging involves many molecular biological aspects, such as epigenetics, genomics and proteomics. However, the relationship between nicroRNA (miRNA) and dermal aging is not clear. MiRNA is a class of short chain single strand RNA, which plays a negative regulatory role by combining with the sequence specificity of the non coding region of the 3 'end of the target gene. MiRNA can affect the physiological and pathological processes of the body at many levels. Understanding the expression characteristics and regulatory network of dermis senescence related miRNA is helpful to further understand the senescence of dermis, and provide new ideas for subsequent anti-aging research. Research purposes 1.. We first understand the differential expression profiles of miRNA in the process of dermal senescence, and explore the differential expression profiles corresponding to the gene functions and regulatory pathways that target genes may be involved in, and establish the network relationship between miRNA and some aging related target genes. 2., we explored the change of miRNA expression in young and senescent fibroblasts, compared the consistency and difference between them, and discussed the possible regulatory pathways of miRNA in cell senescence. Research methods in the first part, this study selected 12 cases in non exposed parts of the leather samples, including 6 cases of young dermal tissues and 6 cases of aging dermis, screened the expression of aging and young dermal differences of miRNA using miRNA chip, using real-time RT-PCR to validate the miRNA expression. Then we use niRNA to analyze target genes by functional clustering analysis and signal pathway clustering analysis, and construct a regulatory network between miRNA and target genes that may be involved in aging, and get the miRNA that plays a core regulatory role in the network. In the second part, this research will be the primary dermal fibroblasts were cultured into normal nutrition and low nutrient medium and continuous cell passage, compared a variety of senescence marker on their respective nutritional status in early passage cells and late passage cells, including the cell count method compared the cell proliferation curve, using SA- beta -GA staining compared the proportion of senescent cells, using real-time quantitative RT-PCR method for comparison of senescence associated marker gene expression of mRNA. Finally, the changes of miRNA between senescent and young generation fibroblasts were compared by real time quantitative RT-PCR method. Results 1. miRNA chip results showed that there were 40 differential expressions of miRNA in dermis with skin aging. 2. real time quantitative RT-PCR showed that the expression of miR-34 family and miR-29 family increased significantly in senescent tissue, which was consistent with the results of chip. The 3. target gene function clustering and pathway cluster analysis showed that the main function of clustering miRNA corresponding target genes including cell adhesion, collagen synthesis, gene transcription positive and negative role, mainly including metabolic pathways and pathway cluster growth factor pathway and extracellular matrix receptor pathway, DNA damage the reaction pathway. 4. miRNA-Gene-Network analysis showed that the miR-34 family, miR-29 family and miR-424 have a large weight in the regulation of the network culture in the normal 5. regardless of nutrient conditions or low nutrient conditions, compared with early passage cells, the passage to the late cell proliferation was significantly decreased, significantly increased the proportion of positive cells of SA- beta -GA, p16 gene the expression level of mRNA was significantly increased, while the p53 and p21 gene mRNA levels showed no significant change. 6. real time quantitative miRNA results showed that regardless of nutrient conditions, 11 miRNA screened (i.e. miR-548q, miR-1226*, miR-601, miR-1207-5p, miR-134, miR-34b*, miR-34a, miR-29c*, miR-181a, miR-154, miR-424) in aging generation cells showed increased expression of the trend, most of which changes with the organization the miRNA chip is consistent, but there is a contradiction between trends in aging cells of miR-181a, miR-154 and miR-424 and the chip results. 7. miRNA raised change culture conditions passaged in senescent cells differ in their nutrient nutrition conditions, normal aging cells to the miR-34 family and the miR-29 family raised the most significant, and low nutrient conditions of aging generation cells by miR-1226*, miR-601 and miR-1207-5p increased to miR-29 family drama, no obvious increase. 8., real time quantitative RT-PCR results showed that miRNA partial target genes such as E2F3, c-Myc and CREB still had decreased expression at the mRNA level in the senescent cells under low nutrient condition. There is miRNA expression difference conclusion dermis in aging process, differential expression of miRNA target genes involved in cell adhesion, collagen synthesis, gene transcription regulation, mainly involved in metabolic pathways, growth factor pathway, extracellular matrix and receptor interaction and DNA damage response pathway, and the miR-34 family, miR-29 family and miR-424 there may be an important regulatory role in the dermal aging process. Observed changes of niRNA were similar and the aging process can be dermal fibroblasts in the aging model in vitro, but the contradiction between individual miRNA shows the results suggest that miRNA may be involved in the mechanisms of fiber cell aging except outside dermal aging, such as inflammation, blood vessel abnormalities in newborn. The differences in the nutritional environment of fibroblasts have an effect on the expression of miRNA in the regulation of aging. In addition, in the cell aging model established in this study, miRNA may be related mainly to the p16 pathway and thus the formation of the dermal senescence.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R751

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