兔增殖性玻璃體視網(wǎng)膜病變視網(wǎng)膜蛋白質(zhì)組學(xué)的初步研究
發(fā)布時間：2018-08-10 21:44
【摘要】：目的:( 1 )建立兔增殖性玻璃體視網(wǎng)膜病變(proliferative vitreoretinopathy , PVR)動物模型;( 2 )采用雙向凝膠電泳(two-dimensional gel electrophoresis,2-DE)/高效液相色譜芯片串聯(lián)質(zhì)譜(high-performance liquid chromatography and tandem mass spectrometry using a chip cube nano-flow system ,HPLC-Chip MS/MS system)和Western blot方法篩選、鑒定PVR兔視網(wǎng)膜與正常兔表達有差異的蛋白,為探討PVR的發(fā)病機制尋找新的突破口。 方法:(1)采用部分玻璃體切除+造成裂孔和視網(wǎng)膜脫離后飼養(yǎng)8周的方法建立PVR動物模型;(2)獲取實驗組和對照組視網(wǎng)膜標本并用clean-up kit處理視網(wǎng)膜標本;(3)運用2-DE對視網(wǎng)膜蛋白進行二維分離;(4)采集凝膠圖像,以PDQuest軟件進行圖像分析,尋找有意義的差異蛋白點;(5)將差異蛋白質(zhì)點挖出、酶解,進行HPLC-Chip MS/MS鑒定;( 6 )使用Mascot軟件在UniProtKB/SWISS-PORT數(shù)據(jù)庫中搜索鑒定差異表達蛋白,生物信息學(xué)分析,得到差異蛋白的相關(guān)肽段的MS/MS質(zhì)譜圖;(7)對鑒定出的、有意義的差異蛋白aB-晶狀體蛋白運用Western blot方法進行一步驗證。 結(jié)果:(1)八周后,行部分玻璃體切除+造成裂孔和視網(wǎng)膜脫離的18只眼中,有3只眼誘導(dǎo)出了PVR B級, 3只眼誘導(dǎo)出了PVR C級。對照組中6只眼無一只眼誘導(dǎo)出PVR;(2)運用2-DE分離出PVR與正常兔視網(wǎng)膜的差異蛋白點,經(jīng)PDQuest8.0軟件進行分析后,篩選10個表達有差異的蛋白點,運用HPLC-Chip MS/MS進行分析鑒定,發(fā)現(xiàn)有8個蛋白在PVR組表達上調(diào)。其中滿足①肽評分8;②SPI(Structure predictability index for protein sequences) (%)70%;③分子量及等電點與雙向電泳的結(jié)果相符的蛋白有2個,即aB-晶狀體蛋白(Alpha-crystallin B chain - Oryctolagus cuniculus)和aA-晶狀體蛋白(Alpha-crystallin A chain - Oryctolagus cuniculus)。(3)Western blot顯示,aB-晶狀體蛋白在PVR B、C級的視網(wǎng)膜中檢測到,而在正常對照視網(wǎng)膜中未測到。 結(jié)論:aB-晶狀體蛋白為PVR的潛在生物標志物,可能在PVR的發(fā)生、發(fā)展中起著重要作用。
[Abstract]:Objective: (1) to establish (proliferative vitreoretinopathy, PVR) animal model of proliferative vitreoretinopathy in rabbits; (2) to screen by two-dimensional gel electrophoresis2-DE / high-performance liquid chromatography and tandem mass spectrometry using a chip cube nano-flow system HPLC-Chip MS/MS system and Western blot method. To identify the differentially expressed proteins in the retina of PVR rabbits and normal rabbits, and to find a new breakthrough in the pathogenesis of PVR. Methods: (1) PVR animal model was established by partial vitrectomy and retinal detachment for 8 weeks; (2) retinal specimens of experimental group and control group were obtained and treated with clean-up kit; (3) Retinal specimens were treated with 2-DE. Omentum protein was separated by two-dimensional method. (4) Gel images were collected. PDQuest software was used for image analysis to find meaningful differential protein spots; (5) the differential protein spots were extracted, hydrolyzed and identified by HPLC-Chip MS/MS; (6) Mascot software was used to search and identify differentially expressed proteins in UniProtKB/SWISS-PORT database, and bioinformatics analysis was used to identify the differentially expressed proteins. The MS/MS mass spectra of the related peptides of differentially expressed proteins were obtained. (7) A Western blot method was used to verify the identified and meaningful differentially expressed proteins. Results: (1) in 18 eyes with partial vitrectomy, PVR B grade was induced in 3 eyes and PVR C grade was induced in 3 eyes after partial vitrectomy. In 6 eyes of the control group, none of the eyes induced PVR. (2) the differential protein spots of PVR and normal rabbit retina were isolated by 2-DE. Ten differentially expressed protein spots were screened by PDQuest8.0 software and identified by HPLC-Chip MS/MS. Eight proteins were found to be up-regulated in PVR group. Among them, there are 2 proteins that meet the 1 peptide score of 8 ~ 2 SPI (Structure predictability index for protein sequences) (%) 70 ~ (-3) molecular weight and isoelectric point, which are consistent with the results of two-dimensional electrophoresis. Alpha-crystallin B chain-Oryctolagus cuniculus) and Alpha-crystallin A chain-Oryctolagus cuniculus). (3) Western blot were detected in the retina of PVR B C grade, but not in the normal control retina. Conclusion as a potential biomarker of PVR, the PVR may play an important role in the pathogenesis and development of PVR.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R776.4;R774.1
本文編號：2176299
[Abstract]:Objective: (1) to establish (proliferative vitreoretinopathy, PVR) animal model of proliferative vitreoretinopathy in rabbits; (2) to screen by two-dimensional gel electrophoresis2-DE / high-performance liquid chromatography and tandem mass spectrometry using a chip cube nano-flow system HPLC-Chip MS/MS system and Western blot method. To identify the differentially expressed proteins in the retina of PVR rabbits and normal rabbits, and to find a new breakthrough in the pathogenesis of PVR. Methods: (1) PVR animal model was established by partial vitrectomy and retinal detachment for 8 weeks; (2) retinal specimens of experimental group and control group were obtained and treated with clean-up kit; (3) Retinal specimens were treated with 2-DE. Omentum protein was separated by two-dimensional method. (4) Gel images were collected. PDQuest software was used for image analysis to find meaningful differential protein spots; (5) the differential protein spots were extracted, hydrolyzed and identified by HPLC-Chip MS/MS; (6) Mascot software was used to search and identify differentially expressed proteins in UniProtKB/SWISS-PORT database, and bioinformatics analysis was used to identify the differentially expressed proteins. The MS/MS mass spectra of the related peptides of differentially expressed proteins were obtained. (7) A Western blot method was used to verify the identified and meaningful differentially expressed proteins. Results: (1) in 18 eyes with partial vitrectomy, PVR B grade was induced in 3 eyes and PVR C grade was induced in 3 eyes after partial vitrectomy. In 6 eyes of the control group, none of the eyes induced PVR. (2) the differential protein spots of PVR and normal rabbit retina were isolated by 2-DE. Ten differentially expressed protein spots were screened by PDQuest8.0 software and identified by HPLC-Chip MS/MS. Eight proteins were found to be up-regulated in PVR group. Among them, there are 2 proteins that meet the 1 peptide score of 8 ~ 2 SPI (Structure predictability index for protein sequences) (%) 70 ~ (-3) molecular weight and isoelectric point, which are consistent with the results of two-dimensional electrophoresis. Alpha-crystallin B chain-Oryctolagus cuniculus) and Alpha-crystallin A chain-Oryctolagus cuniculus). (3) Western blot were detected in the retina of PVR B C grade, but not in the normal control retina. Conclusion as a potential biomarker of PVR, the PVR may play an important role in the pathogenesis and development of PVR.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R776.4;R774.1
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