miR-29b沉默Ⅰ型胶原基因抑制青光眼手术后滤过道瘢痕形成的实验研究

发布时间：2018-06-01 02:05

  本文选题：miR-29b + 沉默　； 参考：《中南大学》2012年博士论文

【摘要】：研究背景 青光眼是全球第一位不可逆性致盲眼病。在药物和激光不能控制眼压时,滤过性手术(Glaucoma Filtration Surgery, GFS)仍是目前治疗青光眼的主要手段。但是,由于Tenon's囊成纤维细胞过度增生、瘢痕形成,导致滤过性手术后1年的失败率高达15%。滤过道瘢痕形成是导致手术失败的主要原因。其中TGF-β诱导Tenon's囊成纤维细胞(HTFs)向肌成纤维细胞转化,其持续存在并合成胶原等细胞外基质是滤过道瘢痕化的中心环节。 多年来,许多国内外专家致力于GFS术后抗瘢痕形成的研究。但是由于目前抗瘢痕形成药物的应用时间难以个体化,往往出现作用不明显或由于作用时间过长及对眼部组织的广泛作用等而导致系列严重的并发症的发生。因此,阐明GFS术后滤过道瘢痕化的发病机制并积极探索效果更好、更特异性的治疗手段很有必要。 miRNA是一类长度约21-25个核苷酸的小分子RNA,通过与目的mRNA3'端非编码区结合促使靶基因抑制或降解,在细胞的增殖、凋亡、分化、个体发育以及机体代谢等一系列生命过程中扮演重要角色。miRNA在心、肝、肾等全身器官纤维化疾病的治疗中显示出了巨大的潜力,已经成为进一步开发抗纤维增生和抑制新生血管形成药物的新靶点。 GFS术后瘢痕形成作为一种常见的不可逆性结局,面临着与全身性器官纤维化相似的治疗挑战。在眼部很多组织中检测出与其它器官相同的miRNA表达,意味着可能存在共同的抗纤维增生策略。已经用于其它器官的成功的miRNA治疗可以用于GFS术后抗瘢痕形成。 目的 筛选人Tenon's囊成纤维细胞(HTFs)的miRNA表达情况。研究miRNA差异表达与青光眼手术后瘢痕形成的相关性,阐明miR-29b过表达对HTFs增殖的影响及可能的作用机制,探索以miRNA为基础的针对青光眼手术后减轻或防止瘢痕形成的可能性。 方法 手术中取斜视患者的Tenon's囊组织,组织贴块法培养原代HTFs,并利用TGFβ1活化诱导HTFs,分别行免疫组织化学鉴定。通过高通量miRNA芯片筛选经TGFβ1诱导前后HTFs miRNA表达谱。差异表达的miRNA利用实时定量Real Time-PCR鉴定。生物信息学方法(TargetScan; Envisioneering Medical Technologies, St. Louis, MO)预测特异性候选miRNA。在TGF-β1诱导前后的HTFs中瞬时转染miR-29b,结合运用实时定量Real Time-PCR、Western blot蛋白质印迹技术,系统研究miR-29b与促纤维增生PI3K/Akt/Sβ1信号通路之间的作用机制,探讨miR-29b差异表达与PI3K/Akt/Sβ1信号通路磷酸化水平之间的关系。结果 TGF-β1诱导前后的HTFs存在miRNA表达差异,其中表达上调2倍的miRNA有38个,表达下调2倍的miRNA有31个。miR-29b在TGFβ1诱导后的HTFs中较正常HTFs表达量显著降低。miR-29b靶向调控PI3Kp85α、Sp1、COL1A1等促纤维化基因。TGFβ1刺激原代HTFs后,COL1A1蛋白表达量明显增加。miR-29b通过调控PI3Kp85α、Sp1、COL1A1靶基因,抑制PI3K/Akt/Sp1信号通路的磷酸化水平。过表达miR-29b能够抑制促纤维增生PI3K/Akt/Sp1信号通路的活化,有效减少Ⅰ型胶原表达。 结论 采用TGF-β1诱导HTFs活化的方法在体外成功地建立了GFS术后滤过道瘢痕形成的细胞模型。TGF-β1诱导前后的HTFs存在miRNA表达差异。其中,miR-29b靶向调控COL1A1、PI3Kp85α、 Sp1基因,通过抑制PI3K/Akt/Sp1信号通路的磷酸化水平来调控HTFs的胶原沉积。过表达miR-29b能够有效降低HTFs胶原表达,这或许能为抑制GFS术后滤过道的瘢痕形成提供新的治疗手段。
[Abstract]:Research background
Glaucoma is the world's first irreversible blinding eye disease. Glaucoma Filtration Surgery (GFS) is still the main treatment for glaucoma when drug and laser can not control IOP. However, the failure rate of 1 years after filtration surgery is as high as 15%. due to the excessive proliferation of Tenon's fibroblasts and the formation of scar. The formation of filter scar formation is the main cause of the failure of the operation. In which TGF- beta induces the transformation of Tenon's cystic fibroblast (HTFs) into myofibroblast, and its continuous existence and synthesis of extracellular matrix, such as collagen, is the central link in the scar formation of the filter.
Many domestic and foreign experts have been working on the research of anti scar formation after GFS. However, due to the difficult time to individualization of the application time of anti scar formation drugs, a series of serious complications are often caused by the lack of effect or the long action time and extensive effect on the eye tissue. Therefore, it is clear that after the operation of GFS The pathogenetic mechanism of filtering scar is better and the effect of exploration is better. More specific treatment is necessary.
MiRNA is a small molecule RNA with a length of about 21-25 nucleotides. It plays an important role in cell proliferation, apoptosis, differentiation, ontogenesis, and body metabolism by combining with the non coding region of the target mRNA3'terminal to inhibit or degrade the target gene..miRNA is used in the treatment of heart, liver, kidney and other systemic fibrosis diseases. It has shown great potential and has become a new target for further developing anti fibrotic proliferation and inhibiting angiogenesis.
Cicatricial formation after GFS is a common and irreversible outcome that faces a similar treatment challenge to systemic organ fibrosis. Detection of the same miRNA expression as other organs in many tissues means there may be a common anti fibrous proliferation strategy. The successful miRNA therapy used in other organs can be used for the treatment of other organs. Anti scar formation after GFS.
objective
The expression of miRNA in human Tenon's fibroblast (HTFs) was screened. The correlation between the differential expression of miRNA and the formation of scar after glaucoma surgery was studied. The effect of miR-29b overexpression on the proliferation of HTFs and the possible mechanism of action were elucidated, and the possibility of reducing or preventing scar formation after glaucoma surgery was explored by miRNA.
Method
The Tenon's capsule tissue of the patients with strabismus was taken during the operation, the original HTFs was cultured with tissue patch method and the activation of HTFs was induced by TGF beta 1 activation. The expression of HTFs miRNA before and after TGF beta 1 was screened through high throughput miRNA chip. The differential expression of miRNA used real-time quantitative Real Time-PCR identification. Bioinformatics Method (Targ) EtScan, Envisioneering Medical Technologies, St. Louis, MO) predicted the transient transfection of the specific candidate miRNA. in HTFs before and after TGF- beta 1. To explore the relationship between miR-29b differential expression and phosphorylation level of PI3K/Akt/S beta 1 signaling pathway.
There was a difference in the expression of miRNA in HTFs before and after TGF- beta 1, of which 38 of the expression was up to 2 times of miRNA, and 31.MiR-29b in TGF beta 1 induced by TGF beta 1 significantly decreased the.MiR-29b target regulation PI3Kp85 alpha in HTFs, and Sp1. The amount of.MiR-29b can inhibit the phosphorylation level of PI3K/Akt/Sp1 signaling pathway by regulating PI3Kp85 alpha, Sp1, COL1A1 target gene. Overexpression of miR-29b can inhibit the activation of PI3K/Akt/Sp1 signaling pathway and effectively reduce the expression of type I collagen.
conclusion
TGF- beta 1 induced HTFs activation in vitro successfully established the miRNA expression difference of HTFs before and after.TGF- beta 1 induced by GFS. Among them, miR-29b targeted COL1A1, PI3Kp85 alpha, Sp1 genes, and regulated the deposition of HTFs collagen by inhibiting the phosphorylation level of PI3K/Akt/Sp1 signaling pathway. Overexpression of miR-29b can effectively reduce the expression of HTFs collagen, which may provide a new treatment for inhibiting scar formation after filtering operation in GFS.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R779.6

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