热休克蛋白47在大鼠结膜滤过泡瘢痕化过程中的作用及其作用机制的研究

发布时间：2018-03-18 02:03

本文选题：滤过手术　切入点：滤过泡　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：背景青光眼滤过术后滤过泡瘢痕化一直是眼科研究的热点和难点。虽然对其进行了大量的研究,但仅有丝裂霉素C (mitmycin C, MMC)和5-氟尿嘧啶(5-fluorouracil, 5-FU)应用于临床,然而由于这两种药物的严重并发症而使其临床应用受到限制。胶原蛋白的异常合成与沉积是纤维化疾病的病理基础,亦是瘢痕形成的重要原因。热休克蛋白47(heat shock protein, HSP47)是一种相对分子质量为47000的热休克蛋白,存在于胶原蛋白分泌细胞的内质网中,与前胶原特异性结合,参与前胶原的折叠、装配、修饰、转运等过程,被认为是胶原特异性分子伴侣,为胶原合成所必需。多种纤维化疾病模型和人类纤维化疾病的研究均证实,HSP47的表达上调与胶原合成增加以及纤维化的发生、发展密切相关。抑制HSP47的表达可以抑制胶原合成,减弱纤维化程度。因此我们假设HSP47有可能成为治疗纤维化疾病的有效靶点。目的探讨HSP47在大鼠滤过术后滤过泡瘢痕化过程中的作用及其作用机制,以及通过此途径可能减轻滤过泡瘢痕化的方法。方法 1.建立滤过术后结膜滤过泡瘢痕化大鼠模型。 2.应用免疫组织化学染色方法,了解HSP47在大鼠滤过术后滤过泡中的表达情况。 3.应用天狼星红-苫味酸染色方法,了解Ⅰ型胶原和Ⅲ型胶原在大鼠滤过术后滤过泡中的表达情况。 4.应用实时荧光定量PCR和Western blot方法,分别检测HSP47、I型胶原和Ⅲ型胶原在大鼠滤过术后不同时间点滤过泡中基因转录水平和蛋白质水平表达变化的情况。 5.采用抗HSP47单克隆抗体结膜下注射的方法,检测抑制HSP47蛋白的表达情况,应用实时荧光定量PCR和Western blot方法,分别检测Ⅰ型胶原和Ⅲ型胶原表达的变化。结果 1.选用大鼠作为实验动物,于滤过术中切除全层角巩膜缘组织,成功建立了滤过术后结膜滤过泡瘢痕化动物模型。 2.HSP47在大鼠滤过术后滤过泡和正常结膜组织均有表达。表达染色的细胞绝大多数为梭型的成纤维细胞,少数为血管内皮细胞,定位于细胞浆。滤过术后1w,HSP47蛋白较正常结膜组织明显增多；滤过术后2w, HSP47蛋白较术后1w有所减少,但是仍然比正常结膜组织表达水平高。 3.应用天狼星红-苦味酸染色方法可以清晰地显示Ⅰ型胶原和Ⅲ型胶原在大鼠滤过术后滤过泡的表达情况。滤过术后1w,大鼠滤过泡胶原蛋白较正常结膜组织增多,以Ⅲ型胶原增加为主；滤过术后2w,大鼠滤过泡胶原蛋白较术后1w明显增加,以Ⅰ型胶原增加为主。 4.大鼠滤过术后滤过泡中HSP47和Ⅲ型胶原在mRNA水平与蛋白质水平的表达均在术后2d就有增加,术后5d、8d、11d时明显增加,与正常对照组相比均有显著的统计学差异(P0.05),术后8d均达到峰值,术后11d均下降,但仍高于术后5d的表达水平。在滤过术后2d、5d时,Ⅰ型胶原在mRNA水平与蛋白质水平的表达增加,术后8d、11d时明显增加,与正常对照组相比有显著的统计学差异(P0.05)。 5.抗HSP47单克隆抗体结膜下注射1ug后,在蛋白质水平抑制了HSP47的表达,Ⅰ型胶原和Ⅲ型胶原在mRNA水平与蛋白质水平的表达均下降,与生理盐水组相比差异有显著的统计学意义(P0.05),与MMC组相比差异无统计学意义(P0.05)。结论 1.HSP47在大鼠滤过术后滤过泡成纤维细胞和血管内皮细胞的表达较正常结膜组织明显增高。Ⅰ型胶原和Ⅲ型胶原在大鼠滤过术后滤过泡的表达较正常结膜组织明显增高。手术创伤可以促使HSP47、Ⅰ型胶原和Ⅲ型胶原的mRNA和蛋白质的表达上调,表明HSP47、Ⅰ型胶原和Ⅲ型胶原在大鼠滤过泡瘢痕化过程中可能会发挥重要作用。 2.抑制滤过术后滤过泡中HSP47的表达,可以减少Ⅰ型胶原和Ⅲ型胶原在滤过泡的沉积,从而减轻滤过泡的瘢痕化反应。表明HSP47在大鼠滤过泡瘢痕化过程中通过调控Ⅰ型胶原和Ⅲ型胶原的表达而发挥作用。抗HSP47单克隆抗体有可能成为未来青光眼抗滤过泡瘢痕化的药物。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R779.6

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