本文關(guān)鍵詞：皮膚真皮衰老相關(guān)microRNA表達譜分析及其在衰老成纖維細胞中的驗證研究　出處：《復旦大學》2014年博士論文　論文類型：學位論文

  更多相關(guān)文章： 真皮 衰老 microRNA 差異性表達

【摘要】：研究背景皮膚衰老是機體衰老的重要外在表現(xiàn)。皮膚衰老不僅有損于美容,也可導致許多年齡相關(guān)性疾病,而給人帶來身心損害。由于皮膚真皮包含多種類型的細胞及縱橫交錯的細胞外基質(zhì)成分,因而在皮膚衰老過程中發(fā)揮著主導作用。目前有關(guān)真皮衰老的機制研究涉及了表觀遺傳學、基因組學、蛋白組學等多種分子生物學層面,然而nicroRNA (miRNA)與真皮衰老之間的關(guān)系尚不明確。miRNA是一類短鏈單鏈RNA,通過與靶基因3’端的非編碼區(qū)序列特異性相結(jié)合從而發(fā)揮負性調(diào)節(jié)作用。miRNA可在多個層面影響機體的生理病理過程。了解真皮衰老相關(guān)miRNA的表達特點及調(diào)控網(wǎng)絡有助于進一步加深對真皮衰老的認識,為后續(xù)抗衰老研究提供新的思路。研究目的1.初步了解真皮衰老進程中的miRNA差異表達譜,探討該差異表達譜對應靶基因可能涉及的基因功能和調(diào)控通路,建立miRNA與部分衰老相關(guān)靶基因之間的網(wǎng)絡關(guān)系。2.探索該差異表達的miRNA在年輕和衰老成纖維細胞中的變化情況,比較其與組織結(jié)果之間的一致性和差異性,并探討這些miRNA可能參與的細胞衰老調(diào)控通路。研究方法在第一部分中,本研究選取12例源于非曝光部位的真皮樣本,包括6例年輕真皮組織和6例衰老真皮組織,利用miRNA芯片初步篩選出衰老和年輕真皮差異表達的miRNA,運用實時定量RT-PCR驗證部分miRNA表達情況。繼而利用niRNA對應靶基因進行功能聚類分析和信號通路聚類分析,構(gòu)建miRNA與可能涉及衰老調(diào)控的靶基因之間的調(diào)控網(wǎng)絡,并得到網(wǎng)絡中起核心調(diào)控作用的miRNA。在第二部分中,本研究將原代真皮成纖維細胞分別培養(yǎng)于正常營養(yǎng)和低營養(yǎng)培養(yǎng)基中并進行連續(xù)細胞傳代,對各自營養(yǎng)狀態(tài)下傳代早期細胞與傳代后期細胞進行多種衰老標記的比較,包括利用細胞計數(shù)法比較了細胞增殖曲線,利用SA-β-GA染色法比較了衰老細胞比例,運用實時定量RT-PCR方法比較了衰老相關(guān)標記基因的mRNA表達量。最后通過實時定量RT-PCR方法比較了部分真皮衰老相關(guān)miRNA在衰老代和年輕代成纖維細胞之間的變化情況。研究結(jié)果1. miRNA芯片結(jié)果分析顯示,隨著皮膚真皮衰老,真皮組織中共有40個miRNA差異性表達。2.實時定量RT-PCR顯示衰老組織內(nèi)miR-34家族及miR-29家族表達顯著增加,與芯片結(jié)果具有一致性。3.靶基因功能聚類和通路聚類分析發(fā)現(xiàn),miRNA對應靶基因的主要功能聚類包括細胞間粘附作用、膠原合成作用、基因轉(zhuǎn)錄的正性及負性調(diào)控作用等,主要通路聚類包括代謝調(diào)節(jié)通路、生長因子通路和細胞外基質(zhì)與受體調(diào)控通路、DNA損傷反應通路等。4. miRNA-Gene-Network分析顯示miR-34家族、miR-29家族和miR-424在調(diào)控網(wǎng)絡中占有較大權(quán)重5.無論在正常營養(yǎng)培養(yǎng)條件抑或低營養(yǎng)培養(yǎng)條件下,相較于早期傳代細胞而言,傳代至后期時細胞的增殖能力均明顯下降,SA-β-GA陽性細胞比例顯著上升,p16基因的mRNA表達水平顯著上調(diào),而p53及p21基因的mRNA水平未見明顯變化。6. miRNA實時定量結(jié)果顯示,無論何種營養(yǎng)培養(yǎng)條件下,篩選出的11個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)在衰老代細胞中均呈現(xiàn)出上調(diào)表達的趨勢,其中絕大多數(shù)變化趨勢與組織miRNA芯片結(jié)果相一致,但miR-181a、miR-154和miR-424在衰老細胞內(nèi)的變化趨勢與芯片結(jié)果之間存在矛盾。7. miRNA上調(diào)變化程度在各自營養(yǎng)培養(yǎng)條件下傳代的衰老細胞中有所不同,正常營養(yǎng)條件下衰老代細胞以miR-34家族和miR-29家族上調(diào)最為顯著,而低營養(yǎng)培養(yǎng)條件下衰老代細胞以miR-1226*、miR-601及miR-1207-5p上調(diào)為劇,miR-29家族未見明顯上調(diào)。8.實時定量RT-PCR結(jié)果發(fā)現(xiàn),低營養(yǎng)培養(yǎng)條件下的衰老代細胞內(nèi),miRNA部分靶基因如E2F3、c-Myc、CREB在mRNA水平上尚存在表達下降。研究結(jié)論真皮衰老進程中存在miRNA的差異性表達,差異表達miRNA的靶基因主要參與細胞粘附、膠原合成、基因轉(zhuǎn)錄調(diào)控中,主要涉及代謝通路、生長因子通路、細胞外基質(zhì)與受體相互作用及DNA損傷反應通路等,且miR-34家族、miR-29家族及miR-424可能在真皮衰老過程中有較為重要的調(diào)控作用。在體外成纖維細胞的衰老模型中可觀察到與真皮衰老進程大致相似的niRNA變化情況,但個別miRNA顯示出的矛盾結(jié)果提示miRNA可能參與到除成纖維細胞衰老以外的真皮衰老的機制中,如炎癥反應、血管新生異常等。成纖維細胞所處營養(yǎng)環(huán)境差異對調(diào)控衰老的miRNA表達情況存在影響。另外,本次研究建立的細胞衰老模型中,真皮衰老相關(guān)miRNA可能主要通過與p16通路發(fā)生聯(lián)系從而對成纖維細胞衰老進程進行調(diào)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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R751

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