miR-31靶向抑制FIH并调控其下游HIF1α-TIMP1-MMP1通路促进增生性瘢痕形成

发布时间：2018-02-25 02:26

本文关键词： 增生性瘢痕 miR-31 缺氧诱导因子缺氧诱导因子抑制因子细胞外基质　出处：《蚌埠医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的创伤后的瘢痕增生,是由于细胞外基质的过度沉积或者降解减少而产生,给患者带来疼痛,瘙痒,畸形和甚至毁容等问题。但其产生的生物学调控机制仍然不详,目前临床上并没有较有效的治疗手段。Micro RNA(微小RNA)是一种可在转录后水平调控基因表达的内源性非编码小分子RNA,在前期研究中,我们发现HS(Hypertrophic scar增生性疤痕)组织中mi R-31(Micro RNA-31)表达量最高,它的出现可能会给我们提供新的思路和治疗靶点。HIF1α(hypoxia inhibit factor 1α缺氧诱导因子1α)在近年来的研究中被发现对疤痕的产生有促进作用,但具体机制仍不清楚。本文我们将探索mi R-31与HIF1α是如何促进疤痕的增生,二者间是否存在影响,并通过裸鼠体内试验来验证mi R-31作用的有效性。本实验深入的研究了mi R-31在增生性瘢痕形成中的作用及生物学机制,并为增生性疤痕的预防和临床治疗提供了理论依据和新的切入点。方法1、正常皮肤真皮组织及增生性瘢痕真皮组织的样本采集;福尔马林溶液浸泡后用石蜡进行包埋固定,切片给予HE染色、Masson染色和免疫组化染色;RT-PCR检测正常皮肤真皮组织和增生性瘢痕组织中mi R-31的相对表达差异;WB(western blot)检测NS(Normal skin正常皮肤组织)与HS组织中HIF1α的蛋白表达差异。2、进行人NSFBs(Normal skin fibroblasts正常皮肤成纤维细胞)和HSFBs(Hypertrophic scar fibroblats增生性疤痕成纤维细胞)的培养,RT-PCR对不同代次细胞检测其mi R-31;Fn(Fibronectin纤维链接蛋白);Col1a1(collage1a1胶原1 a1);Col3a1(collage3a1胶原3 a1);TGF-β(Transforming growth factor beta转化生长因子-β)的表达量,验证细胞模型构建的可用性。构建mi R-31过表达干扰载体,并转染入NSFBs;反义干扰载体,并转染入HSFBs。RT-PCR、WB检测转染后细胞的mi R-31;Fn;Col1a1;Col3a1的表达量,从而观察mi R-31对成纤维细胞生物学特性的影响。3、检索生物信息数据库,包括:Targetscan,mi Randa,Pic Tar等,通过计算机运算非翻译区的核苷酸结合度来预测mi R-31的靶基因,筛选与缺氧相关的特定靶基因FIH(Factor inhibit HIF缺氧诱导因子抑制因子)。萤火素酶报告及RT-PCR、WB检测转染后细胞的FIH的表达量,验证mi R-31靶向抑制FIH。4、RT-PCR、WB检测细胞过表达和反义表达mi R-31后,HIF1α;TIMP-1(Tissue inhibitor of metalloproteinase基质金属蛋白抑制因子1);MMP1(matrix metalloproteinases-1基质金属蛋白酶-1)的表达量,从而观察mi R-31对HIF1α及其下游调控蛋白的影响。5、将HSFBs培养于缺氧的环境中,并于24h;48h;72h三个时间节点检测Fn;Col1a1;Col3a1;TIMP-1;MMP1的蛋白表达量,从而阐明HIF1α促进疤痕增生的原因。6、建立体外裸鼠疤痕模型,观察mi R-31抑制剂局部干预后Fn;Col1a1;Col3a1的表达量变化。结果HS组织及细胞中mi R-31及HIF1α表达量都显著升高,体外实检测到Fn;Col1a1;Col3a1三个纤维化指标的表达量与mi R-31呈正相关,从而得出mi R-31可以促进HS的发生。此外HIF1α与mi R-31在成纤维细胞中有类似的作用,也可以促进纤维化指标的表达,成纤维细胞梯度低氧实验验证了TIMP-1是HIF1α的下游作用基因,与HIF1α的表达正相关。MMP1已多次被验证有促进Fn;Col1a1;Col3a1降解的作用,而TIMP-1是MMP1的抑制因子,所以随着缺氧的加深,纤维化指标的过度表达是通过增强TIMP-1对MMP1的抑制作用而实现的。FIH是缺氧诱导因子的抑制因子,它可以抑制HIF1α对下游基因的转录诱导作用。萤火素酶报告,RT-PCR及WB实验得出FIH是mi R-31的靶基因,并且HIF1α对疤痕的这一促进作用可以被mi R-31所增强。综上我们得出mi R-31增强HIF1α-TIMP-1-MMP1通路促进HS发生。为了进一步验证mi R-31在HS中作用的确定性,设计动物实验。mi R-31抑制剂局部干预裸鼠疤痕后,HE,MASSON及免疫组化染色结果提示Fn;Col1a1;Col3a1的表达量呈下降趋势。结论1、与正常皮肤组织相较,增生性瘢痕中mi R-31的相对表达量增高;2、mi R-31在转录后水平靶向抑制FIH的表达,从而增强HIF1α通路的对增生性瘢痕促进的作用。3、mi R-31抑制剂对裸鼠疤痕有抑制作用。
[Abstract]:The purpose of post-traumatic hypertrophic scar, is due to the excessive deposition of extracellular matrix degradation or reduced to patients with pain, itching, deformity and even disfigured. But the biological regulation of its formation mechanism is still unknown, currently no clinical effective treatment for.Micro RNA (micro RNA) is a kind of in the endogenous regulation of gene expression in the post transcriptional level of non encoding small molecule RNA, in our previous study, we found that HS (Hypertrophic scar of hypertrophic scar tissue) mi R-31 (Micro RNA-31) the highest expression level, this may provide us with new ideas and therapeutic targets of.HIF1 alpha (hypoxia inhibit factor 1 alpha hypoxia inducible factor 1 alpha) in recent studies found on scar have stimulative effect, but the mechanism is not clear. In this paper, we will explore the MI R-31 and HIF1 alpha is how to promote the scars. Students, whether effects exist between the two, and to verify the effectiveness of the MI R-31 function through in vivo experiment. This experiment studied the MI R-31 in the formation of hypertrophic scar and the role of biological mechanisms, and provide a theoretical basis and a new starting point for the prevention of hypertrophic scars and clinical treatment. 1 samples of normal skin and hypertrophic scar dermal dermis; was embedded with paraffin soaked Faure Marin solution, were given HE staining, Masson staining and immunohistochemical staining; the relative expression of RT-PCR detected in normal dermis tissue and hypertrophic scar tissue in MI R-31 WB (Western blot); the detection of NS (Normal skin of normal skin tissue) between.2 and HIF1 alpha HS tissues the expression of NSFBs (Normal skin, fibroblasts of normal skin fibroblasts (Hypertrophic) and HSFBs scar fibroblat S of hypertrophic scar fibroblasts cultured on RT-PCR cells), different generations of the MI R-31 Fn (detection; Fibronectin fiber link protein (collage1a1); Col1a1 1 A1; Col3a1 (collagen) collage3a1 collagen 3 A1); TGF- (Transforming growth factor beta beta transforming growth factor beta) expression available verify the cell model was constructed. Construction of MI R-31 expression vector and transfected into NSFBs; Antisense Vector and transfected into HSFBs.RT-PCR, WB detection of MI R-31 of transfected cells; Fn; Col1a1; the expression of Col3a1 and MI to observe the effect of R-31 on the biological characteristics of fibroblasts.3, retrieval of biological the information database, including: Targetscan, MI Randa, Pic Tar, by computing the untranslated region of nucleotide binding to the predicted target gene of MI R-31, screening of specific target gene FIH and related Factor inhibit HIF (hypoxia hypoxia inducible factor inhibitor Sub). Luciferase reporter and RT-PCR, WB cells and the expression of FIH was detected after the verification of MI R-31, targeting FIH.4, RT-PCR, WB detection of cell overexpression and antisense expression of MI R-31, HIF1 TIMP-1 (Tissue inhibitor of alpha; metalloproteinase matrix metalloproteinase inhibitor 1 (matrix); MMP1 metalloproteinases-1 matrix metalloproteinase -1) expression, to observe the effect of R-31 on HIF1 alpha MI and its downstream regulatory protein.5, HSFBs were cultured in hypoxia environment, and 24h; 48h; 72h three time nodes detect Fn; Col1a1; Col3a1; TIMP-1; expression of MMP1 protein, so as to clarify the HIF1 alpha.6 cause of hypertrophic scar and scar in nude mice to establish in vitro model, observe the MI R-31 inhibitor Fn Col1a1; local intervention; expression of Col3a1. Results the expression of HS and R-31 in MI tissues and cells of HIF1 alpha amount was significantly increased, and in vitro experiments to detect Fn; Col1a1; Col3a1 three positive expression and fibrosis index of MI R-31, so that MI R-31 can promote the occurrence of HS. In addition HIF1 alpha and MI R-31 in a similar role in fiber cells, can promote the expression of fibrosis, fibroblast gradient hypoxia experiment proved that TIMP-1 is the downstream effects of HIF1 alpha gene the expression of HIF1, and a positive correlation of.MMP1 has been verified to promote Fn; Col1a1; Col3a1 degradation, TIMP-1 inhibitor MMP1, so as to deepen the over expression of hypoxia, fibrosis is realized by enhancing the inhibitory effect of TIMP-1 on the MMP1.FIH is the inhibitor of hypoxia inducible factor. It can induce transcription inhibition of HIF1 alpha on downstream genes. Luciferase reporter, RT-PCR and WB experiment showed that FIH was a target gene of MI R-31, and HIF1 alpha on scar this role can be mi R-31 These are enhanced. We conclude that MI enhanced R-31 HIF1 alpha -TIMP-1-MMP1 pathway to promote the occurrence of HS. In order to further verify the role of MI R-31 in HS to determine the design of animal experiment of.Mi R-31 inhibitor intervention in nude mice after local scar, HE, MASSON and immunohistochemical staining showed that Fn; Col1a1; the expression of Col3a1 was decreased. Conclusion 1, compared with the normal skin tissues, the relative expression in hypertrophic scar mi R-31 increased; 2, MI R-31 at the post transcriptional level inhibit the expression of FIH, thereby enhancing the HIF1 alpha pathway of hypertrophic scar and promote the role of.3, MI R-31 inhibitor has inhibitory effect on scar in nude mice.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622

【相似文献】