發(fā)布時(shí)間：2017-12-26 16:25

  本文關(guān)鍵詞：皮膚真皮衰老相關(guān)microRNA表達(dá)譜分析及其在衰老成纖維細(xì)胞中的驗(yàn)證研究　出處：《復(fù)旦大學(xué)》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 真皮 衰老 microRNA 差異性表達(dá)

【摘要】：研究背景皮膚衰老是機(jī)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(dǎo)致許多年齡相關(guān)性疾病,而給人帶來(lái)身心損害。由于皮膚真皮包含多種類型的細(xì)胞及縱橫交錯(cuò)的細(xì)胞外基質(zhì)成分,因而在皮膚衰老過(guò)程中發(fā)揮著主導(dǎo)作用。目前有關(guān)真皮衰老的機(jī)制研究涉及了表觀遺傳學(xué)、基因組學(xué)、蛋白組學(xué)等多種分子生物學(xué)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(guò)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(fù)性調(diào)節(jié)作用。miRNA可在多個(gè)層面影響機(jī)體的生理病理過(guò)程。了解真皮衰老相關(guān)miRNA的表達(dá)特點(diǎn)及調(diào)控網(wǎng)絡(luò)有助于進(jìn)一步加深對(duì)真皮衰老的認(rèn)識(shí),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(jìn)程中的miRNA差異表達(dá)譜,探討該差異表達(dá)譜對(duì)應(yīng)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(luò)關(guān)系。2.探索該差異表達(dá)的miRNA在年輕和衰老成纖維細(xì)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(xì)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(dá)的miRNA,運(yùn)用實(shí)時(shí)定量RT-PCR驗(yàn)證部分miRNA表達(dá)情況。繼而利用niRNA對(duì)應(yīng)靶基因進(jìn)行功能聚類分析和信號(hào)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(luò),并得到網(wǎng)絡(luò)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(xì)胞分別培養(yǎng)于正常營(yíng)養(yǎng)和低營(yíng)養(yǎng)培養(yǎng)基中并進(jìn)行連續(xù)細(xì)胞傳代,對(duì)各自營(yíng)養(yǎng)狀態(tài)下傳代早期細(xì)胞與傳代后期細(xì)胞進(jìn)行多種衰老標(biāo)記的比較,包括利用細(xì)胞計(jì)數(shù)法比較了細(xì)胞增殖曲線,利用SA-β-GA染色法比較了衰老細(xì)胞比例,運(yùn)用實(shí)時(shí)定量RT-PCR方法比較了衰老相關(guān)標(biāo)記基因的mRNA表達(dá)量。最后通過(guò)實(shí)時(shí)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(xì)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(gè)miRNA差異性表達(dá)。2.實(shí)時(shí)定量RT-PCR顯示衰老組織內(nèi)miR-34家族及miR-29家族表達(dá)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(duì)應(yīng)靶基因的主要功能聚類包括細(xì)胞間粘附作用、膠原合成作用、基因轉(zhuǎn)錄的正性及負(fù)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(zhǎng)因子通路和細(xì)胞外基質(zhì)與受體調(diào)控通路、DNA損傷反應(yīng)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(luò)中占有較大權(quán)重5.無(wú)論在正常營(yíng)養(yǎng)培養(yǎng)條件抑或低營(yíng)養(yǎng)培養(yǎng)條件下,相較于早期傳代細(xì)胞而言,傳代至后期時(shí)細(xì)胞的增殖能力均明顯下降,SA-β-GA陽(yáng)性細(xì)胞比例顯著上升,p16基因的mRNA表達(dá)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(shí)時(shí)定量結(jié)果顯示,無(wú)論何種營(yíng)養(yǎng)培養(yǎng)條件下,篩選出的11個(gè)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(xì)胞中均呈現(xiàn)出上調(diào)表達(dá)的趨勢(shì),其中絕大多數(shù)變化趨勢(shì)與組織miRNA芯片結(jié)果相一致,但miR-181a、miR-154和miR-424在衰老細(xì)胞內(nèi)的變化趨勢(shì)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(yíng)養(yǎng)培養(yǎng)條件下傳代的衰老細(xì)胞中有所不同,正常營(yíng)養(yǎng)條件下衰老代細(xì)胞以miR-34家族和miR-29家族上調(diào)最為顯著,而低營(yíng)養(yǎng)培養(yǎng)條件下衰老代細(xì)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(shí)時(shí)定量RT-PCR結(jié)果發(fā)現(xiàn),低營(yíng)養(yǎng)培養(yǎng)條件下的衰老代細(xì)胞內(nèi),miRNA部分靶基因如E2F3、c-Myc、CREB在mRNA水平上尚存在表達(dá)下降。研究結(jié)論真皮衰老進(jìn)程中存在miRNA的差異性表達(dá),差異表達(dá)miRNA的靶基因主要參與細(xì)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(zhǎng)因子通路、細(xì)胞外基質(zhì)與受體相互作用及DNA損傷反應(yīng)通路等,且miR-34家族、miR-29家族及miR-424可能在真皮衰老過(guò)程中有較為重要的調(diào)控作用。在體外成纖維細(xì)胞的衰老模型中可觀察到與真皮衰老進(jìn)程大致相似的niRNA變化情況,但個(gè)別miRNA顯示出的矛盾結(jié)果提示miRNA可能參與到除成纖維細(xì)胞衰老以外的真皮衰老的機(jī)制中,如炎癥反應(yīng)、血管新生異常等。成纖維細(xì)胞所處營(yíng)養(yǎng)環(huán)境差異對(duì)調(diào)控衰老的miRNA表達(dá)情況存在影響。另外,本次研究建立的細(xì)胞衰老模型中,真皮衰老相關(guān)miRNA可能主要通過(guò)與p16通路發(fā)生聯(lián)系從而對(duì)成纖維細(xì)胞衰老進(jìn)程進(jìn)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(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R751

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