miR-29及其靶基因COLI在增生性瘢痕形成中的作用及机制研究

发布时间：2018-07-27 15:52

【摘要】：背景:增生性瘢痕(HS)是皮肤创伤愈合过程中常见的并发症,发生率高达4~16%,严重影响组织功能恢复和外观。胶原的合成与降解代谢失衡是HS形成的根本原因,但导致胶原持续过度合成的病因学与分子机制尚不十分明确,临床上对HS的治疗是一大临床挑战。尽管采用了许多治疗方法,但其治疗效果却不尽相同。因此,需要探寻新的治疗靶点。在我们前期的研究中,基因芯片检测显示,正常皮肤(NS)组织和HS组织中micro RNA(miRNA)分子的表达存在明显差异,这些miRNA分子所调节的靶基因涵盖了细胞生命活动的多个方面,如细胞凋亡与增殖、周期与运动、合成与代谢,其中miR-29的差异最明显。而目前有研究认为,miR-29可以在转录后水平抑制胶原的合成,从而在多种纤维化疾病的发生中起重要的调控作用。但miR-29与HS形成关系的研究尚未见文献报道。因此,我们推测,持续低水平表达的miR-29丧失了对COLI基因表达的有效调控,进而导致胶原为主的细胞外基质(ECM)的过度沉积,最终形成HS。如果施用正义miR-29提高HS局部组织中miR-29表达水平,将有可能成功抑制组织的过度纤维化过程,为HS的临床防治提供新的靶点。目的:通过miRNA表达谱芯片筛选出HS组织及NS组织中显著差异表达的miRNA分子,探讨miR-29通过在转录后水平调控其靶基因COLI表达从而影响HS形成的作用机制,以期为HS的生物学治疗提供新的靶点。方法:1.收集HS组织和NS组织标本,经过HE染色后证明所取临床标本符合实验需要。分离并培养人HS及NS原代成纤维细胞。通过miRNA基因芯片筛选出HS及NS中差异表达的miRNA分子,并采用生物信息学方法预测miR-29下游靶基因。2.进一步使用荧光定量PCR方法及western blot法明确人NS与HS中miR-29c及COLI基因的差异性表达,并采用卡方检验或Fisher精确检验进行分析,探讨miR-29与COLI基因表达水平的相关性;3.构建COLI A1-3’UTR及其3种突变体的重组荧光素酶报告基因载体,分析正常成纤维细胞和瘢痕成纤维细胞中COLI A1-3’UTR荧光素酶报告载体活性是否存在显著差异,miR-29c模拟剂是否能显著降低报告载体的活性,从而验证COLI是否为miR-29c下游的有效靶基因。4.构建和包装人miR-29c过表达腺病毒,分别转染培养的NS成纤维细胞及HS成纤维细胞,采用流式细胞分析法及CCK-8法检测HS成纤维细胞在感染腺病毒过表达miR-29c后,细胞的增殖与凋亡是否受到显著影响。结果:1.收集的HS和NS组织标本经过HE染色后均证明符合实验需要。免疫荧光染色鉴定HS组织及NS组织原代培养所得的细胞确实为成纤维细胞;2.基因芯片技术筛选发现,HS组织中miR-143、miR-32、miR-92b及miR-25等45个miRNA的表达水平与NS组织相比上调,同时miR-29a、miR-29b、miR-29c、miR-713、miR-195等68个miRNA显著表达下调,提示它们可能在HS的发生发展过程中发挥作用。其中,miR-29的改变最为显著,在12个HS样本中miR-29分子表达均显著低于NS组织(平均降低15.3倍)。3.荧光定量PCR方法对筛选出的miRNA-29在HS及NS组织中的表达情况进行检测,结果发现,无论是在组织中还是原代成纤维细胞中,miR-29c在HS中的表达显著低于NS组织。而miRNA-29家族的其他成员在HS/NS中的表达量均无显著差异。4.应用生物信息学数据库及软件对miR-29靶基因进行预测,初步推测COLI是miR-29可能的靶基因。荧光定量PCR及Western Blot检测表明COLI基因在NS组织中的表达量显著低于HS组织,COLI在HS组织及NS组织原代培养所得的成纤维细胞中的表达量与组织中表达趋势相符,卡方检验分析发现,无论在HS及NS组织中还是在原代培养的成纤维细胞中,miR-29与COLI基因表达水平均具有显著负相关性。5.构建了COLI基因及miR-29c相应结合区的突变体荧光素酶报告基因载体,酶切及测序证实构建成功。荧光素酶活性的检测结果显示:瘢痕成纤维细胞中,COLI A1-3’UTR荧光素酶报告载体活性显著高于正常成纤维细胞。在正常成纤维细胞及增生性瘢痕成纤维细胞中,miR-29c模拟剂均能有效降低COLI A1-3’UTR荧光素酶报告载体活性,却不能有效降低其突变体的荧光素酶报告载体活性。进一步说明miR-29c可直接作用于COLI A1-3’UTR从而发挥作用。6.成功构建和包装人miR-29c过表达腺病毒后,分别转染培养的NS成纤维细胞及HS成纤维细胞,采用定量PCR结合WB检测发现miR-29过表达后COLI基因表达显著受到抑制,细胞增殖实验及流式细胞分析发现miR-29过表达后成纤维细胞的增殖率显著提高而凋亡率显著下降。结论:1.基因芯片技术筛选发现,HS组织中miR-143、miR-32、miR-92b及miR-25等45个miRNA的表达水平与NS组织相比上调,同时miR-29a、miR-29b、miR-29c、miR-713、miR-195等68个miRNA显著表达下调,提示它们可能在HS的发生发展过程中发挥作用。其中,miR-29的表达差异最为显著。2.在无论是组织还是细胞,miR-29c在HS中的表达均显著低于NS,降低HS成纤维细胞中miR-29c的表达可以促进其增殖并抑制其凋亡,提示miR-29在HS的发生发展中发挥负调节作用。3.COLI基因是miR-29c直接作用的下游靶基因,miR-29对COLI基因表达发挥负调控作用,miR-29在增生性瘢痕组织中的持续性低水平表达使其对靶基因COL1表达丧失了有效的调控,造成COL1基因过表达,导致胶原为主的ECM的过度沉积,最终促进了增生性瘢痕组织的形成。miR-29c有可能成为增生性瘢痕治疗的新靶点。
[Abstract]:Background: hypertrophic scar (HS) is a common complication in the process of skin wound healing. The occurrence rate is up to 4~16%, which seriously affects the recovery and appearance of tissue function. The synthesis of collagen and the imbalance of degradation metabolism are the fundamental causes of the formation of HS. However, the etiology and molecular mechanism of the continuous hypersynthesis of collagen are not very clear. The treatment of HS is clinically. Therapy is a major clinical challenge. Although many treatments are used, the therapeutic effect is not the same. Therefore, new therapeutic targets need to be explored. In our previous study, gene chip detection showed that the expression of micro RNA (miRNA) subdivision in normal skin (NS) tissues and HS tissues was significantly different from those of miRNA molecules. The target gene covers many aspects of cell life activities, such as cell apoptosis and proliferation, cycle and movement, synthesis and metabolism, in which the difference of miR-29 is most obvious. At present, studies have suggested that miR-29 can inhibit the synthesis of collagen at post transcriptional level and thus play an important role in the development of various fibrotic diseases. But miR-29 and HS The study of the formation of the relationship has not yet been reported. Therefore, we speculate that the continuous low level expression of miR-29 has lost the effective regulation of the expression of COLI gene, leading to the excessive deposition of collagen based extracellular matrix (ECM), and eventually the formation of HS. may be successful if the miR-29 expression level in the HS local tissue is developed by the use of justice miR-29. The process of inhibiting the excessive fibrosis of the tissues provides new targets for the clinical prevention and control of HS. Objective: to screen out the significant differential miRNA molecules expressed in HS tissues and NS tissues by miRNA expression chip, and to explore the mechanism of miR-29 by regulating the expression of the target gene COLI in the post transcriptional level and affecting the formation of HS, so as to be a biological treatment for HS. New therapeutic targets were provided. Methods: 1. the specimens of HS and NS tissues were collected. After HE staining, it was proved that the clinical specimens were in conformity with the experimental needs. The human HS and NS primary fibroblasts were isolated and cultured. The miRNA molecules expressed differently in HS and NS were screened by miRNA gene chip, and the target gene.2. in the downstream of miR-29 was predicted by bioinformatics. Further using the fluorescence quantitative PCR method and the Western blot method to clarify the differential expression of miR-29c and COLI genes in human NS and HS, and using chi square test or Fisher accurate test to analyze the correlation between miR-29 and COLI gene expression level. 3. the recombinant luciferase reporter gene vector of COLI A1-3 'and its 3 mutants was constructed. Whether there are significant differences in the activity of COLI A1-3 'UTR luciferase reporter vector in normal fibroblasts and scar fibroblasts, whether miR-29c simulant can significantly reduce the activity of the reported carrier, thus verifying whether COLI is an effective target gene for the downstream of miR-29c,.4. construction and packaging of human miR-29c overexpressed adenovirus, respectively. NS fibroblasts and HS fibroblasts were cultured. The proliferation and apoptosis of HS fibroblasts were detected by flow cytometry and CCK-8 method. Results: 1. the specimens of HS and NS collected from HS and NS proved to be in accordance with the experimental needs. Immunofluorescence staining was used. The cells in the primary culture of HS tissue and NS tissue were fibroblasts. 2. gene chip technology screening showed that the expression levels of 45 miRNA in HS tissues were up regulated compared with NS tissues, while miR-29a, miR-29b, miR-29c, etc. In the process of the development of HS, the change of miR-29 is the most significant. In 12 HS samples, the expression of miR-29 molecules is significantly lower than that of NS tissue (averaging 15.3 times). The.3. fluorescence quantitative PCR method is used to detect the expression of the selected miRNA-29 in HS and NS tissues. The results have been found, both in the tissue and in the original generation. In the fibrous cells, the expression of miR-29c in HS was significantly lower than that of NS tissue. There was no significant difference in the expression of other members of the miRNA-29 family in HS/NS..4. applied bioinformatics database and software to predict the miR-29 target gene. It was preliminarily presumed that COLI was a possible target gene for miR-29. Fluorescent quantitative PCR and Western Blot test showed that COLI The expression of gene in NS tissues was significantly lower than that in HS tissue. The expression of COLI in the fibroblasts derived from HS and NS tissues was consistent with the expression trend in the tissue. The chi square test found that both miR-29 and COLI gene expression levels were significant in both HS and NS tissues and in the primary cultured fibroblasts. The negative correlation.5. was used to construct the COLI gene and the mutant luciferase reporter gene vector of the corresponding binding region of miR-29c. The enzyme activity was confirmed by enzyme digestion and sequencing. The results of luciferase activity showed that the activity of COLI A1-3 'UTR luciferase reporter vector was significantly higher than normal fibroblast in scar fibroblasts. In vascular and hypertrophic scar fibroblasts, miR-29c simulants can effectively reduce the activity of the COLI A1-3 'UTR luciferase reporter carrier, but it can not effectively reduce the activity of the luciferase reporter vector of the mutant, and further indicates that miR-29c can directly act on COLI A1-3' UTR to play a role of.6. successfully construct and package the miR-29 of the COLI A1-3 'UTR. After C overexpressed the adenovirus, the cultured NS fibroblasts and HS fibroblasts were transfected respectively. The quantitative PCR combined with WB detection showed that the expression of COLI gene was significantly inhibited after miR-29 overexpression. The proliferation test and flow cytometry showed that the proliferation rate of fibroblasts increased significantly and the apoptosis rate decreased significantly after miR-29 overexpression. Conclusion: 1. gene chip technology screening showed that the expression levels of 45 miRNA, such as miR-143, miR-32, miR-92b and miR-25 in HS tissues were up regulated compared with those of NS tissues, while miR-29a, miR-29b, miR-29c, miR-713, miR-195 and so on were down regulated, suggesting that they might play a role in the development of the miRNA. The most significant.2. in tissues and cells, the expression of miR-29c in HS is significantly lower than that of NS, and the reduction of miR-29c in HS fibroblasts can promote its proliferation and inhibit its apoptosis, suggesting that miR-29 plays a negative regulatory role in the development of HS, the.3.COLI gene is the downstream target gene directly acting on miR-29c, miR-29 on COLI genes. The expression of negative regulation in the expression of miR-29 in hypertrophic scar tissue makes it lose effective regulation on the expression of target gene COL1, resulting in overexpression of COL1 gene, resulting in the overdeposition of collagen based ECM, which ultimately promotes the formation of hypertrophic scar tissue that may become a hypertrophic scar treatment. New target.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R622

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