本文關鍵詞： 增生性瘢痕 miR-31 缺氧誘導因子 缺氧誘導因子抑制因子 細胞外基質　出處：《蚌埠醫(yī)學院》2015年碩士論文　論文類型：學位論文

【摘要】：目的創(chuàng)傷后的瘢痕增生,是由于細胞外基質的過度沉積或者降解減少而產生,給患者帶來疼痛,瘙癢,畸形和甚至毀容等問題。但其產生的生物學調控機制仍然不詳,目前臨床上并沒有較有效的治療手段。Micro RNA(微小RNA)是一種可在轉錄后水平調控基因表達的內源性非編碼小分子RNA,在前期研究中,我們發(fā)現HS(Hypertrophic scar增生性疤痕)組織中mi R-31(Micro RNA-31)表達量最高,它的出現可能會給我們提供新的思路和治療靶點。HIF1α(hypoxia inhibit factor 1α缺氧誘導因子1α)在近年來的研究中被發(fā)現對疤痕的產生有促進作用,但具體機制仍不清楚。本文我們將探索mi R-31與HIF1α是如何促進疤痕的增生,二者間是否存在影響,并通過裸鼠體內試驗來驗證mi R-31作用的有效性。本實驗深入的研究了mi R-31在增生性瘢痕形成中的作用及生物學機制,并為增生性疤痕的預防和臨床治療提供了理論依據和新的切入點。方法1、正常皮膚真皮組織及增生性瘢痕真皮組織的樣本采集;福爾馬林溶液浸泡后用石蠟進行包埋固定,切片給予HE染色、Masson染色和免疫組化染色;RT-PCR檢測正常皮膚真皮組織和增生性瘢痕組織中mi R-31的相對表達差異;WB(western blot)檢測NS(Normal skin正常皮膚組織)與HS組織中HIF1α的蛋白表達差異。2、進行人NSFBs(Normal skin fibroblasts正常皮膚成纖維細胞)和HSFBs(Hypertrophic scar fibroblats增生性疤痕成纖維細胞)的培養(yǎng),RT-PCR對不同代次細胞檢測其mi R-31;Fn(Fibronectin纖維鏈接蛋白);Col1a1(collage1a1膠原1 a1);Col3a1(collage3a1膠原3 a1);TGF-β(Transforming growth factor beta轉化生長因子-β)的表達量,驗證細胞模型構建的可用性。構建mi R-31過表達干擾載體,并轉染入NSFBs;反義干擾載體,并轉染入HSFBs。RT-PCR、WB檢測轉染后細胞的mi R-31;Fn;Col1a1;Col3a1的表達量,從而觀察mi R-31對成纖維細胞生物學特性的影響。3、檢索生物信息數據庫,包括:Targetscan,mi Randa,Pic Tar等,通過計算機運算非翻譯區(qū)的核苷酸結合度來預測mi R-31的靶基因,篩選與缺氧相關的特定靶基因FIH(Factor inhibit HIF缺氧誘導因子抑制因子)。螢火素酶報告及RT-PCR、WB檢測轉染后細胞的FIH的表達量,驗證mi R-31靶向抑制FIH。4、RT-PCR、WB檢測細胞過表達和反義表達mi R-31后,HIF1α;TIMP-1(Tissue inhibitor of metalloproteinase基質金屬蛋白抑制因子1);MMP1(matrix metalloproteinases-1基質金屬蛋白酶-1)的表達量,從而觀察mi R-31對HIF1α及其下游調控蛋白的影響。5、將HSFBs培養(yǎng)于缺氧的環(huán)境中,并于24h;48h;72h三個時間節(jié)點檢測Fn;Col1a1;Col3a1;TIMP-1;MMP1的蛋白表達量,從而闡明HIF1α促進疤痕增生的原因。6、建立體外裸鼠疤痕模型,觀察mi R-31抑制劑局部干預后Fn;Col1a1;Col3a1的表達量變化。結果HS組織及細胞中mi R-31及HIF1α表達量都顯著升高,體外實檢測到Fn;Col1a1;Col3a1三個纖維化指標的表達量與mi R-31呈正相關,從而得出mi R-31可以促進HS的發(fā)生。此外HIF1α與mi R-31在成纖維細胞中有類似的作用,也可以促進纖維化指標的表達,成纖維細胞梯度低氧實驗驗證了TIMP-1是HIF1α的下游作用基因,與HIF1α的表達正相關。MMP1已多次被驗證有促進Fn;Col1a1;Col3a1降解的作用,而TIMP-1是MMP1的抑制因子,所以隨著缺氧的加深,纖維化指標的過度表達是通過增強TIMP-1對MMP1的抑制作用而實現的。FIH是缺氧誘導因子的抑制因子,它可以抑制HIF1α對下游基因的轉錄誘導作用。螢火素酶報告,RT-PCR及WB實驗得出FIH是mi R-31的靶基因,并且HIF1α對疤痕的這一促進作用可以被mi R-31所增強。綜上我們得出mi R-31增強HIF1α-TIMP-1-MMP1通路促進HS發(fā)生。為了進一步驗證mi R-31在HS中作用的確定性,設計動物實驗。mi R-31抑制劑局部干預裸鼠疤痕后,HE,MASSON及免疫組化染色結果提示Fn;Col1a1;Col3a1的表達量呈下降趨勢。結論1、與正常皮膚組織相較,增生性瘢痕中mi R-31的相對表達量增高;2、mi R-31在轉錄后水平靶向抑制FIH的表達,從而增強HIF1α通路的對增生性瘢痕促進的作用。3、mi R-31抑制劑對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.

【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R622

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