本文選題：miR-29b + 沉默��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：研究背景 青光眼是全球第一位不可逆性致盲眼病。在藥物和激光不能控制眼壓時(shí),濾過性手術(shù)(Glaucoma Filtration Surgery, GFS)仍是目前治療青光眼的主要手段。但是,由于Tenon's囊成纖維細(xì)胞過度增生、瘢痕形成,導(dǎo)致濾過性手術(shù)后1年的失敗率高達(dá)15%。濾過道瘢痕形成是導(dǎo)致手術(shù)失敗的主要原因。其中TGF-β誘導(dǎo)Tenon's囊成纖維細(xì)胞(HTFs)向肌成纖維細(xì)胞轉(zhuǎn)化,其持續(xù)存在并合成膠原等細(xì)胞外基質(zhì)是濾過道瘢痕化的中心環(huán)節(jié)。 多年來,許多國內(nèi)外專家致力于GFS術(shù)后抗瘢痕形成的研究。但是由于目前抗瘢痕形成藥物的應(yīng)用時(shí)間難以個(gè)體化,往往出現(xiàn)作用不明顯或由于作用時(shí)間過長及對(duì)眼部組織的廣泛作用等而導(dǎo)致系列嚴(yán)重的并發(fā)癥的發(fā)生。因此,闡明GFS術(shù)后濾過道瘢痕化的發(fā)病機(jī)制并積極探索效果更好、更特異性的治療手段很有必要。 miRNA是一類長度約21-25個(gè)核苷酸的小分子RNA,通過與目的mRNA3'端非編碼區(qū)結(jié)合促使靶基因抑制或降解,在細(xì)胞的增殖、凋亡、分化、個(gè)體發(fā)育以及機(jī)體代謝等一系列生命過程中扮演重要角色。miRNA在心、肝、腎等全身器官纖維化疾病的治療中顯示出了巨大的潛力,已經(jīng)成為進(jìn)一步開發(fā)抗纖維增生和抑制新生血管形成藥物的新靶點(diǎn)。 GFS術(shù)后瘢痕形成作為一種常見的不可逆性結(jié)局,面臨著與全身性器官纖維化相似的治療挑戰(zhàn)。在眼部很多組織中檢測(cè)出與其它器官相同的miRNA表達(dá),意味著可能存在共同的抗纖維增生策略。已經(jīng)用于其它器官的成功的miRNA治療可以用于GFS術(shù)后抗瘢痕形成。 目的 篩選人Tenon's囊成纖維細(xì)胞(HTFs)的miRNA表達(dá)情況。研究miRNA差異表達(dá)與青光眼手術(shù)后瘢痕形成的相關(guān)性,闡明miR-29b過表達(dá)對(duì)HTFs增殖的影響及可能的作用機(jī)制,探索以miRNA為基礎(chǔ)的針對(duì)青光眼手術(shù)后減輕或防止瘢痕形成的可能性。 方法 手術(shù)中取斜視患者的Tenon's囊組織,組織貼塊法培養(yǎng)原代HTFs,并利用TGFβ1活化誘導(dǎo)HTFs,分別行免疫組織化學(xué)鑒定。通過高通量miRNA芯片篩選經(jīng)TGFβ1誘導(dǎo)前后HTFs miRNA表達(dá)譜。差異表達(dá)的miRNA利用實(shí)時(shí)定量Real Time-PCR鑒定。生物信息學(xué)方法(TargetScan; Envisioneering Medical Technologies, St. Louis, MO)預(yù)測(cè)特異性候選miRNA。在TGF-β1誘導(dǎo)前后的HTFs中瞬時(shí)轉(zhuǎn)染miR-29b,結(jié)合運(yùn)用實(shí)時(shí)定量Real Time-PCR、Western blot蛋白質(zhì)印跡技術(shù),系統(tǒng)研究miR-29b與促纖維增生PI3K/Akt/Sβ1信號(hào)通路之間的作用機(jī)制,探討miR-29b差異表達(dá)與PI3K/Akt/Sβ1信號(hào)通路磷酸化水平之間的關(guān)系。結(jié)果 TGF-β1誘導(dǎo)前后的HTFs存在miRNA表達(dá)差異,其中表達(dá)上調(diào)2倍的miRNA有38個(gè),表達(dá)下調(diào)2倍的miRNA有31個(gè)。miR-29b在TGFβ1誘導(dǎo)后的HTFs中較正常HTFs表達(dá)量顯著降低。miR-29b靶向調(diào)控PI3Kp85α、Sp1、COL1A1等促纖維化基因。TGFβ1刺激原代HTFs后,COL1A1蛋白表達(dá)量明顯增加。miR-29b通過調(diào)控PI3Kp85α、Sp1、COL1A1靶基因,抑制PI3K/Akt/Sp1信號(hào)通路的磷酸化水平。過表達(dá)miR-29b能夠抑制促纖維增生PI3K/Akt/Sp1信號(hào)通路的活化,有效減少Ⅰ型膠原表達(dá)。 結(jié)論 采用TGF-β1誘導(dǎo)HTFs活化的方法在體外成功地建立了GFS術(shù)后濾過道瘢痕形成的細(xì)胞模型。TGF-β1誘導(dǎo)前后的HTFs存在miRNA表達(dá)差異。其中,miR-29b靶向調(diào)控COL1A1、PI3Kp85α、 Sp1基因,通過抑制PI3K/Akt/Sp1信號(hào)通路的磷酸化水平來調(diào)控HTFs的膠原沉積。過表達(dá)miR-29b能夠有效降低HTFs膠原表達(dá),這或許能為抑制GFS術(shù)后濾過道的瘢痕形成提供新的治療手段。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R779.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 卿國平,段宣初;濾過泡相關(guān)性眼部感染[J];中國實(shí)用眼科雜志;2002年09期

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本文編號(hào)：1962440