本文選題：濾過手術(shù)　切入點：濾過泡　出處：《北京協(xié)和醫(yī)學院》2011年博士論文　論文類型：學位論文

【摘要】：背景 青光眼濾過術(shù)后濾過泡瘢痕化一直是眼科研究的熱點和難點。雖然對其進行了大量的研究,但僅有絲裂霉素C (mitmycin C, MMC)和5-氟尿嘧啶(5-fluorouracil, 5-FU)應(yīng)用于臨床,然而由于這兩種藥物的嚴重并發(fā)癥而使其臨床應(yīng)用受到限制。膠原蛋白的異常合成與沉積是纖維化疾病的病理基礎(chǔ),亦是瘢痕形成的重要原因。熱休克蛋白47(heat shock protein, HSP47)是一種相對分子質(zhì)量為47000的熱休克蛋白,存在于膠原蛋白分泌細胞的內(nèi)質(zhì)網(wǎng)中,與前膠原特異性結(jié)合,參與前膠原的折疊、裝配、修飾、轉(zhuǎn)運等過程,被認為是膠原特異性分子伴侶,為膠原合成所必需。多種纖維化疾病模型和人類纖維化疾病的研究均證實,HSP47的表達上調(diào)與膠原合成增加以及纖維化的發(fā)生、發(fā)展密切相關(guān)。抑制HSP47的表達可以抑制膠原合成,減弱纖維化程度。因此我們假設(shè)HSP47有可能成為治療纖維化疾病的有效靶點。 目的 探討HSP47在大鼠濾過術(shù)后濾過泡瘢痕化過程中的作用及其作用機制,以及通過此途徑可能減輕濾過泡瘢痕化的方法。 方法 1.建立濾過術(shù)后結(jié)膜濾過泡瘢痕化大鼠模型。 2.應(yīng)用免疫組織化學染色方法,了解HSP47在大鼠濾過術(shù)后濾過泡中的表達情況。 3.應(yīng)用天狼星紅-苫味酸染色方法,了解Ⅰ型膠原和Ⅲ型膠原在大鼠濾過術(shù)后濾過泡中的表達情況。 4.應(yīng)用實時熒光定量PCR和Western blot方法,分別檢測HSP47、I型膠原和Ⅲ型膠原在大鼠濾過術(shù)后不同時間點濾過泡中基因轉(zhuǎn)錄水平和蛋白質(zhì)水平表達變化的情況。 5.采用抗HSP47單克隆抗體結(jié)膜下注射的方法,檢測抑制HSP47蛋白的表達情況,應(yīng)用實時熒光定量PCR和Western blot方法,分別檢測Ⅰ型膠原和Ⅲ型膠原表達的變化。 結(jié)果 1.選用大鼠作為實驗動物,于濾過術(shù)中切除全層角鞏膜緣組織,成功建立了濾過術(shù)后結(jié)膜濾過泡瘢痕化動物模型。 2.HSP47在大鼠濾過術(shù)后濾過泡和正常結(jié)膜組織均有表達。表達染色的細胞絕大多數(shù)為梭型的成纖維細胞,少數(shù)為血管內(nèi)皮細胞,定位于細胞漿。濾過術(shù)后1w,HSP47蛋白較正常結(jié)膜組織明顯增多；濾過術(shù)后2w, HSP47蛋白較術(shù)后1w有所減少,但是仍然比正常結(jié)膜組織表達水平高。 3.應(yīng)用天狼星紅-苦味酸染色方法可以清晰地顯示Ⅰ型膠原和Ⅲ型膠原在大鼠濾過術(shù)后濾過泡的表達情況。濾過術(shù)后1w,大鼠濾過泡膠原蛋白較正常結(jié)膜組織增多,以Ⅲ型膠原增加為主；濾過術(shù)后2w,大鼠濾過泡膠原蛋白較術(shù)后1w明顯增加,以Ⅰ型膠原增加為主。 4.大鼠濾過術(shù)后濾過泡中HSP47和Ⅲ型膠原在mRNA水平與蛋白質(zhì)水平的表達均在術(shù)后2d就有增加,術(shù)后5d、8d、11d時明顯增加,與正常對照組相比均有顯著的統(tǒng)計學差異(P0.05),術(shù)后8d均達到峰值,術(shù)后11d均下降,但仍高于術(shù)后5d的表達水平。在濾過術(shù)后2d、5d時,Ⅰ型膠原在mRNA水平與蛋白質(zhì)水平的表達增加,術(shù)后8d、11d時明顯增加,與正常對照組相比有顯著的統(tǒng)計學差異(P0.05)。 5.抗HSP47單克隆抗體結(jié)膜下注射1ug后,在蛋白質(zhì)水平抑制了HSP47的表達,Ⅰ型膠原和Ⅲ型膠原在mRNA水平與蛋白質(zhì)水平的表達均下降,與生理鹽水組相比差異有顯著的統(tǒng)計學意義(P0.05),與MMC組相比差異無統(tǒng)計學意義(P0.05)。 結(jié)論 1.HSP47在大鼠濾過術(shù)后濾過泡成纖維細胞和血管內(nèi)皮細胞的表達較正常結(jié)膜組織明顯增高。Ⅰ型膠原和Ⅲ型膠原在大鼠濾過術(shù)后濾過泡的表達較正常結(jié)膜組織明顯增高。手術(shù)創(chuàng)傷可以促使HSP47、Ⅰ型膠原和Ⅲ型膠原的mRNA和蛋白質(zhì)的表達上調(diào),表明HSP47、Ⅰ型膠原和Ⅲ型膠原在大鼠濾過泡瘢痕化過程中可能會發(fā)揮重要作用。 2.抑制濾過術(shù)后濾過泡中HSP47的表達,可以減少Ⅰ型膠原和Ⅲ型膠原在濾過泡的沉積,從而減輕濾過泡的瘢痕化反應(yīng)。表明HSP47在大鼠濾過泡瘢痕化過程中通過調(diào)控Ⅰ型膠原和Ⅲ型膠原的表達而發(fā)揮作用。抗HSP47單克隆抗體有可能成為未來青光眼抗濾過泡瘢痕化的藥物。
[Abstract]:background
Glaucoma filtering bleb scarring has been a hotspot and difficulty in ophthalmology. Although a lot of research, but only mitomycin C (mitmycin C MMC) and 5- fluorouracil (5-fluorouracil, 5-FU) in clinical application, however, because of the serious complications of the two drugs and its clinical application is limited. Abnormal synthesis and deposition of collagen is the pathological basis of fibrotic disease, is also an important reason for scar formation. Heat shock protein 47 (heat shock, protein, HSP47) is a relative molecular mass of 47000 heat shock proteins exist in the collagen secreting cells in the endoplasmic reticulum, combined with collagen specific participation procollagen folding, assembly, modification, transport process, is considered as a collagen specific molecular chaperone, essential for collagen synthesis. Research on a variety of fibrotic diseases model and human fibrotic diseases It has been confirmed that the up regulation of HSP47 is closely related to the increase of collagen synthesis and the development of fibrosis. Inhibition of HSP47 expression can inhibit collagen synthesis and weaken fibrosis. Therefore, we hypothesized that HSP47 may become an effective target for treatment of fibrotic diseases.
objective
To explore the role and mechanism of HSP47 in the process of filtering bleb scar after filtering operation in rats, and the method of reducing the scarring of filter bubbles by this way.
Method
1. the rat model of cicatricial vesicles of conjunctival filtration bleb after filtration was established.
2. the expression of HSP47 in filter bleb after filtration was studied by immunohistochemical staining.
3. application of Sirius red - Shan flavor acid staining method, understand the type I collagen and collagen expression of bleb in rats after trabeculectomy.
4. real-time fluorescence quantitative PCR and Western blot were used to detect the expression level of HSP47, collagen type I and type III collagen at different time points after filtration in rats.
5., the expression of HSP47 protein was inhibited by subconjunctival injection of anti HSP47 monoclonal antibody. The expression of collagen type I and type III collagen was detected by real-time fluorescence quantitative PCR and Western blot.
Result
1. rats were selected as experimental animals, and the whole layer of corneo sclera was removed during filtration, and the animal model of cicatricial membrane filtration bleb after filtration was successfully established.
Expression of 2.HSP47 in rat filtration bleb and normal conjunctival tissue. The expression of fibroblast cells stained for the vast majority of the shuttle, a small number of endothelial cells, localized in the cytoplasm. After filtration surgery 1W, HSP47 protein increased significantly compared with the normal conjunctiva; after filtration surgery 2W, HSP47 protein. After 1W has decreased, but still higher than the normal conjunctival tissue high expression level.
3. application of Sirius red picric acid staining method can clearly show the type I collagen and type III collagen in rats with filtration bleb after trabeculectomy. The expression of 1W in rats compared with normal conjunctival bleb collagen tissue increased, with the increase of type III collagen; after filtration surgery 2W rats bleb collagen compared with postoperative 1W increased significantly with the increase of collagen I.
The expression of bleb of HSP47 and collagen in mRNA level and protein level of filtration after 4. rats were in the postoperative 2D had increased after 5D, 8D, 11d increased significantly when compared with the control group there was statistical differences (P0.05), after 8D peak operation after 11d decreased, but still higher than the expression level of 5D in filtration surgery after operation. 2D, 5D, collagen type I expression in mRNA level and protein level increased after 8D, 11d increased significantly, there was statistically significant difference compared with the normal control group (P0.05).
5. monoclonal antibodies against HSP47 subconjunctival injection of 1ug after HSP47 expression inhibited at the protein level, the expression of type I collagen and type III collagen in mRNA and protein level were decreased, compared with saline group the difference was statistically significant (P0.05), and MMC group compared the difference was not statistically significant (P0.05).
conclusion
1.HSP47 in rat filtration bleb expression of fibroblasts and vascular endothelial cells than in normal conjunctival tissue increased significantly. The expression of type I collagen and type III collagen in rats with filtration bleb than normal conjunctival tissue increased significantly. Surgical trauma can promote the expression of HSP47, mRNA and protein of type I collagen and type III collagen showed HSP47, type I collagen and type III collagen in rats of bleb scarring may play an important role in the process.
The expression of HSP47 in inhibiting bleb 2. after filtration surgery, can reduce collagen and collagen deposition bleb, thereby reducing bleb scarring. It shows that the process of HSP47 in rats in filtering bleb scarring by regulating the expression of type I collagen and type III collagen and play a role in resistance. HSP47 monoclonal antibody has the potential to become the future of anti glaucoma bleb scarring drugs.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R779.6

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