血管抑素抑制兔角膜碱烧伤后新生血管的蛋白组研究
发布时间：2018-09-17 09:37
【摘要】：1.研究目的及意义 角膜组织没有血管的透明组织。维持其透明性的一个很重要的原因是它没有血管。在感染、外伤、免疫反应、排斥反应、佩戴角膜接触镜、碱烧伤、基质溃疡、角膜缘干细胞病变等病理情况下,从角膜缘血管网形成的新生血管逐渐侵入角膜,从而形成角膜新生血管(corneal neovascularization, CNV)。诚然,CNV在感染清除、伤口愈合等方面具有一定的积极作用。但是,CNV导致角膜的透明性降低,从而严重的影响视力,另外,CNV可破坏角膜正常微环境,使眼前节生理性的免疫豁免消失,因而成为角膜移植排斥反应的高危因素。因此,针对角膜新生血管的治疗业已成为眼科的研究热点。近年来的研究证实,血管抑素(angiostatin, AS)存在着明确的抗角膜新生血管形成的作用,角膜上皮细胞被证明可以分泌血管抑素,但在角膜受到损伤后分泌减少。另外,血管抑素被证实通过病毒载体或者局部运用可以抑制角膜新生血管形成。但相关作用机制研究的不足,影响着其最终运用于临床的进程。因此,阐明血管抑素抗角膜新生血管形成的机制,势必为其临床运用奠定坚实的基础,并产生巨大的社会和经济效益。 2.研究方法 众所周知,蛋白质是一类重要的生物大分子,是生命活动的主要承担者。在蛋白质组(proteome)概念提出以后,蛋白质组学的研究取得了可喜的发展。相比较传统的只针对单一蛋白质的研究,蛋白质组学更加注重的是参与特定生理或病理状态的所有蛋白质种类及其与周围环境(分子)的关系。随着相关实验技术的发展,目前已有可能对细胞在不同生理或病理条件下蛋白质表达的异同,相关蛋白质的分类和鉴定,尤其是蛋白质之间相互作用和蛋白质的功能进行探讨。因此,本课题拟以碱烧伤制备的角膜新生血管为基础,局部施加血管抑素,将角膜组织进行二维电泳,分离蛋白质并进行染色,凝胶图像分析系统对蛋白质点进行定量分析,从而明确局部施加血管抑素在抗角膜新生血管形成过程中的差异表达蛋白,进一步地,对其进行质谱分析,从而得到相关蛋白质的定性数据。 3.研究内容和过程 (1)实验动物及分组：本实验采用的是32只新西兰大白兔(购自南方医科大学动物中心),实验动物分为三组,A组为空白对照组：8只新西兰兔不做任何处理；B组为烧伤组：12只新西兰兔采用碱烧伤的方法建立角膜新生血管模型,术后第1天开始局部生理盐水点眼,每日3次,直至取角膜标本；C组为治疗组：12只新西兰兔采用同样方法建立角膜新生血管模型后,术后第1天开始局部用30μg/ml血管抑素(AS)点眼,每日3次,直至取角膜标本。 (2)角膜新生血管模型制作：碱烧伤法制备角膜新生血管模型：氯胺酮25mg/kg及氯丙嗪25mg/kg肌注新西兰兔全身麻醉,0.5%丁卡因表面麻醉,置开睑器充分暴露眼表,将浸有1.5mol/LNaOH液的10mm圆形滤纸片置于眼表中央,1.5min后取下,用50m1生理盐水冲洗眼表,滴妥布霉素眼液后涂妥布霉素眼膏。 (3)角膜新生血管的观察：术后观察角膜的新生血管,第16日使用裂隙灯(苏州66生产YZ5T型号)照相。角膜新生血管的检测：测量血管长度,以连续弯曲度小、新生血管朝向角膜混浊中心生长的最长血管为准,并计算角膜新生血管生长面积(A),按公式A=C/12×3.1416[r2-(r-1)2]。C为Nv累及角膜的圆周钟点数,1为新生血管从角膜缘深入角膜的长度,r为兔角膜半径,设为定值7mm。 (4)标本取材及全蛋白提取：术后3w取材。标本离体后迅速置入5ml冻存管,于液氮速冻后置入-80℃超低温冰箱保存备用。取-80℃保存的组织块,迅速研磨至细粉末状,加入裂解液匀浆、离心。采用Bradford定量法,计算样品蛋白质浓度。 (6)双向凝胶电泳(2-DE)：第一向(IPG-IEF)根据蛋白质等电点的不同进行等电聚焦,第二向(SDSPAGE)根据蛋白质相对分子质量大小分离样品,双向电泳后才用EMBL银染。 (7)图像分析和差异蛋白选取：将银染后的2-DE胶图扫描入电脑。应用ImageMaster2D软件分析每一张凝胶图像,计算图谱中所有蛋白质点的蛋白质相对含量,找出三块胶中均出现,并且有差异的蛋白。 (8)质谱分析：切取有差异的蛋白质点,行脱色、酶解、萃取样品,利用美国ABI4700MALDI-TOF-TOF质谱仪进行质谱分析。UV波长为355nm,重复速率为200HZ,加速电压为20000V,最优质量分辨率为1500Da。扫描质量范围为700-3200Da,收集信号。胰酶自切峰为内标校正质谱仪。所有实验样品的质谱图均以用默认模式获得。 (9)数据库检索：利用软件Mascot distiller过滤基线峰、识别信号峰。利用Matrixscience公司的Mascot软件搜索哺乳动物数据库,寻找匹配的相关蛋白质,同时查询其功能,来明确鉴定的蛋白质为何种蛋白质。查询条件：(1)肽质量控制在800-4000Da。(2)表观PI与表观Mr的误差范围：无限制。(3)肽片段分子量最大容许误差控制在±50ppm。(4)酶解片段不完全选择为1-2个。(5)物种来源选择(哺乳动物)。(6)离子选择MH和monoisotopic。(7)固定修饰为半胱氨酸碘乙酰胺化(Carbamidomethy1),可变修饰为蛋氨酸氧化(Oxsidation)。 4.主要结果 (1)成功建立兔角膜新生血管模型。 (2)兔角膜碱烧伤后第16天,血管抑素作用组(C组)的新生血管面积37.62+9.65mm2,对照组(B组)面积46.77+8.98mm2,采用两独立样本t检验,并用SPSS13.0软件进行分析,P0.05,证明血管抑素对角膜新生血管有抑制作用。 (3)各样本进行双向电泳后,将A、C两组与B组进行比较,检测到13个差异蛋白点。 (4)差异蛋白鉴定：通过质谱分析和数据库检索,成功鉴定了这13个蛋白(有两个蛋白的鉴定结果显示为同一蛋白)。其中B组较A、C组均明显降低的有6种蛋白：白蛋白前体、热休克蛋白A8、丙酮酸激酶、βB3-晶体蛋白、视黄醛蛋白1以及一个尚未命名的蛋白；B组较A、C组均明显升高的有6种蛋白：网钙结合蛋白3、角蛋白14、肌动蛋白11、免疫球蛋白λ链,免疫球蛋白K链、结合珠蛋白。 5.主要结论 在过去的几十年中,在肿瘤、角膜新生血管及其他新生血管性疾病中,抗血管治疗通过局部运用或者基因载体方式得到了巨大的发展,并且在这次实验中,我们发现经过碱烧伤的兔角膜在局部滴用血管抑素后,新生血管明显减少,这就证明了血管抑素可以有效的抑制血管内皮细胞的迁移及增殖,并且诱导这些细胞凋亡。为了了解血管抑素抗血管作用的机理,通过蛋白组学,我们发现角膜碱烧伤后局部应用血管抑素,可以抑制烧伤后升高的蛋白,如角蛋白14、网钙蛋白3、结合珠蛋白,这些蛋白都参与了新生血管的形成。血管抑素同时可以恢复烧伤后降低的蛋白,如晶体蛋白、视黄醇结合蛋白、白蛋白前体、丙酮酸激酶、HSPA8蛋白,这些蛋白与角膜上皮修复、能量代谢、蛋白合成有关。在下一步的研究中,血管抑素对各种蛋白含量的动态变化是我们研究的方向。
[Abstract]:1. purpose and significance of the study
Corneal tissue is a transparent tissue without blood vessels. One important reason for maintaining its transparency is that it has no blood vessels. In pathological conditions such as infection, trauma, immune response, rejection, wearing contact lenses, alkali burns, stromal ulcers, limbal stem cell lesions, neovascularization from the limbal vascular network gradually invades the cornea, from Certainly, CNV plays a positive role in clearance of infection and wound healing. However, CNV leads to the decrease of corneal transparency, which seriously affects visual acuity. In addition, CNV can destroy the normal corneal microenvironment and make the physiological immune immunity of the anterior segment disappear. As a high risk factor for corneal graft rejection, the treatment of corneal neovascularization has become a hot topic in ophthalmology. Recent studies have confirmed that angiostatin (AS) has a definite anti-angiogenesis effect on corneal neovascularization. Corneal epithelial cells have been shown to secrete angiostatin, but the cornea is damaged. In addition, angiostatin has been proved to inhibit corneal neovascularization by viral vectors or local application. However, the lack of research on the mechanism of action affects the clinical application of angiostatin. Therefore, clarifying the mechanism of angiostatin against corneal neovascularization is bound to lay a solid foundation for its clinical application. The foundation and generate huge social and economic benefits.
2. research methods
As we all know, proteins are a kind of important biological macromolecules and the main undertakers of life activities. Since the concept of proteome was put forward, the research of proteomics has made gratifying progress. With the development of related experimental techniques, it is possible to study the similarities and differences of protein expression in different physiological or pathological conditions, the classification and identification of related proteins, especially the interaction between proteins and the function of proteins. Based on corneal neovascularization prepared by alkali burn, local angiostatin is applied to separate the protein and dye the cornea by two-dimensional electrophoresis. The gel image analysis system is used to quantitatively analyze the protein spots, so as to identify the differential expression of local angiostatin in the process of corneal neovascularization. Further, mass spectrometry was carried out to obtain qualitative data of related proteins.
3. research contents and process
(1) Experimental animals and groups: 32 New Zealand white rabbits (purchased from the Animal Center of Southern Medical University) were divided into three groups. Group A was the blank control group: 8 New Zealand rabbits did not do any treatment; Group B was the burned group: 12 New Zealand rabbits were used to establish corneal neovascularization model by alkali burns, the first one after operation. In group C, 12 New Zealand rabbits were treated with the same method to establish corneal neovascularization model, and 30 ug/ml angiostatin (AS) was used locally on the first day after operation, three times a day until corneal specimens were taken.
(2) Corneal neovascularization model: Corneal neovascularization model was prepared by alkali burn: Ketamine 25mg/kg and chlorpromazine 25mg/kg were injected intramuscularly into New Zealand rabbits for general anesthesia, 0.5% tetracaine for surface anesthesia, eyelid opener was placed to fully expose the ocular surface, 10 mm circular filter paper immersed in 1.5 mol/L NaOH solution was placed in the center of the ocular surface, and removed 1.5 minutes later, 50 M1 physiology was used. Rinse the eyedrop with saline and smear Tobramycin Eye Ointment with tobramycin eye drops.
(3) Observation of corneal neovascularization: observation of corneal neovascularization after surgery, the use of slit lamp (Suzhou 66 production YZ5T model) photography on the 16th. Corneal neovascularization detection: measurement of the length of blood vessels, small continuous curvature, neovascularization toward the center of corneal opacity growth of the longest blood vessels, and calculation of corneal neovascularization growth area (A) According to the formula A=C/12*3.1416[r2-(r-1)2], C is the number of circular clock points of Nv involved cornea, 1 is the length of neovascularization penetrating cornea from corneal limbus, and R is the radius of rabbit cornea.
(4) Sample sampling and whole protein extraction: 3 weeks after operation, the samples were quickly placed in a 5 ml cryopreservation tube, then stored in a cryopreserve refrigerator at - 80 C after liquid nitrogen freezing. The tissue blocks stored at - 80 C were quickly ground to a fine powder, then added lysate homogenate and centrifuged. The protein concentration of the samples was calculated by Bradford quantitative method.
(6) two dimensional gel electrophoresis (2-DE): first direction (IPG-IEF) isoelectric focusing was performed according to the isoelectric point of protein, and second directions (SDSPAGE) were separated according to the relative molecular weight of protein, and then stained with EMBL silver after two dimensional electrophoresis.
(7) image analysis and differential protein selection: scanning the silver stained 2-DE glue map into the computer. Each gel image was analyzed by ImageMaster2D software, and the relative protein content of all protein spots in the map was calculated to find out the protein that appeared in all three pieces of gum.
(8) Mass spectrometry analysis: the different protein spots were cut, decolorized, enzymatically hydrolyzed and extracted. The samples were analyzed by mass spectrometry using ABI 4700 MALDI-TOF-TOF mass spectrometer. The UV wavelength was 355 nm, the repetition rate was 200 HZ, the acceleration voltage was 200 000 V, the optimal mass resolution was 1500 Da. The scanning mass range was 700-3200 Da, and the signal was collected. The mass spectra of all the experimental samples were obtained by default mode.
(9) Database retrieval: Mascot distiller is used to filter baseline peaks and identify signal peaks. Matrixscience's Mascot software is used to search mammalian databases for matching proteins and query their functions to determine which proteins are identified. The error range between the apparent PI and the apparent MR is unlimited. (3) The maximum allowable error of the molecular weight of the peptide fragment is controlled at (+50 ppm.) (4) The incomplete selection of the enzymatic fragment is 1-2. (5) Species Source Selection (Mammal). (6) Ion Selection MH and monoisotopic. (7) Fixed modification to cysteine iodoacetamidation (Carbamidomethy1) can be changed to methionine. Oxidation (Oxsidation).
4. main results
(1) successful establishment of rabbit corneal neovascularization model.
(2) On the 16th day after corneal alkali burn, the area of neovascularization in angiostatin group (group C) was 37.62+9.65 mm2, and that in control group (group B) was 46.77+8.98 mm2. Two independent samples t test was used and SPSS 13.0 software was used to analyze the results. The results showed that angiostatin had inhibitory effect on corneal neovascularization.
(3) after two dimensional electrophoresis, A and C two groups were compared with B group, and 13 differential protein spots were detected.
(4) Differential protein identification: 13 proteins were identified by mass spectrometry and database searching. Among them, 6 proteins were significantly lower in group B than in group A and C: albumin precursor, heat shock protein A8, pyruvate kinase, beta B3-crystallin, retinal protein 1 and one not yet identified. Named proteins; group B was significantly higher than group A and C in six proteins: reticulum calcium binding protein 3, keratin 14, actin 11, immunoglobulin lambda chain, immunoglobulin K chain, binding globin.
5. main conclusions
In the past few decades, anti-angiogenic therapy has developed dramatically in tumor, corneal neovascularization and other neovascular diseases by local use or gene delivery. In this experiment, we found that the corneal neovascularization in alkali-burned rabbits decreased significantly after topical drip of angiostatin. In order to understand the mechanism of angiostatin's anti-angiogenic effect, we found that angiostatin could inhibit the elevated proteins such as keratin 14, reticulin 3 and nodule after alkali burn in cornea. Angiostatin also restores reduced proteins such as crystallin, retinol binding protein, albumin precursor, pyruvate kinase, and HSPA8, which are involved in corneal epithelial repair, energy metabolism, and protein synthesis. The dynamic change of protein content is the direction of our research.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R772.2
本文编号：2245466
[Abstract]:1. purpose and significance of the study
Corneal tissue is a transparent tissue without blood vessels. One important reason for maintaining its transparency is that it has no blood vessels. In pathological conditions such as infection, trauma, immune response, rejection, wearing contact lenses, alkali burns, stromal ulcers, limbal stem cell lesions, neovascularization from the limbal vascular network gradually invades the cornea, from Certainly, CNV plays a positive role in clearance of infection and wound healing. However, CNV leads to the decrease of corneal transparency, which seriously affects visual acuity. In addition, CNV can destroy the normal corneal microenvironment and make the physiological immune immunity of the anterior segment disappear. As a high risk factor for corneal graft rejection, the treatment of corneal neovascularization has become a hot topic in ophthalmology. Recent studies have confirmed that angiostatin (AS) has a definite anti-angiogenesis effect on corneal neovascularization. Corneal epithelial cells have been shown to secrete angiostatin, but the cornea is damaged. In addition, angiostatin has been proved to inhibit corneal neovascularization by viral vectors or local application. However, the lack of research on the mechanism of action affects the clinical application of angiostatin. Therefore, clarifying the mechanism of angiostatin against corneal neovascularization is bound to lay a solid foundation for its clinical application. The foundation and generate huge social and economic benefits.
2. research methods
As we all know, proteins are a kind of important biological macromolecules and the main undertakers of life activities. Since the concept of proteome was put forward, the research of proteomics has made gratifying progress. With the development of related experimental techniques, it is possible to study the similarities and differences of protein expression in different physiological or pathological conditions, the classification and identification of related proteins, especially the interaction between proteins and the function of proteins. Based on corneal neovascularization prepared by alkali burn, local angiostatin is applied to separate the protein and dye the cornea by two-dimensional electrophoresis. The gel image analysis system is used to quantitatively analyze the protein spots, so as to identify the differential expression of local angiostatin in the process of corneal neovascularization. Further, mass spectrometry was carried out to obtain qualitative data of related proteins.
3. research contents and process
(1) Experimental animals and groups: 32 New Zealand white rabbits (purchased from the Animal Center of Southern Medical University) were divided into three groups. Group A was the blank control group: 8 New Zealand rabbits did not do any treatment; Group B was the burned group: 12 New Zealand rabbits were used to establish corneal neovascularization model by alkali burns, the first one after operation. In group C, 12 New Zealand rabbits were treated with the same method to establish corneal neovascularization model, and 30 ug/ml angiostatin (AS) was used locally on the first day after operation, three times a day until corneal specimens were taken.
(2) Corneal neovascularization model: Corneal neovascularization model was prepared by alkali burn: Ketamine 25mg/kg and chlorpromazine 25mg/kg were injected intramuscularly into New Zealand rabbits for general anesthesia, 0.5% tetracaine for surface anesthesia, eyelid opener was placed to fully expose the ocular surface, 10 mm circular filter paper immersed in 1.5 mol/L NaOH solution was placed in the center of the ocular surface, and removed 1.5 minutes later, 50 M1 physiology was used. Rinse the eyedrop with saline and smear Tobramycin Eye Ointment with tobramycin eye drops.
(3) Observation of corneal neovascularization: observation of corneal neovascularization after surgery, the use of slit lamp (Suzhou 66 production YZ5T model) photography on the 16th. Corneal neovascularization detection: measurement of the length of blood vessels, small continuous curvature, neovascularization toward the center of corneal opacity growth of the longest blood vessels, and calculation of corneal neovascularization growth area (A) According to the formula A=C/12*3.1416[r2-(r-1)2], C is the number of circular clock points of Nv involved cornea, 1 is the length of neovascularization penetrating cornea from corneal limbus, and R is the radius of rabbit cornea.
(4) Sample sampling and whole protein extraction: 3 weeks after operation, the samples were quickly placed in a 5 ml cryopreservation tube, then stored in a cryopreserve refrigerator at - 80 C after liquid nitrogen freezing. The tissue blocks stored at - 80 C were quickly ground to a fine powder, then added lysate homogenate and centrifuged. The protein concentration of the samples was calculated by Bradford quantitative method.
(6) two dimensional gel electrophoresis (2-DE): first direction (IPG-IEF) isoelectric focusing was performed according to the isoelectric point of protein, and second directions (SDSPAGE) were separated according to the relative molecular weight of protein, and then stained with EMBL silver after two dimensional electrophoresis.
(7) image analysis and differential protein selection: scanning the silver stained 2-DE glue map into the computer. Each gel image was analyzed by ImageMaster2D software, and the relative protein content of all protein spots in the map was calculated to find out the protein that appeared in all three pieces of gum.
(8) Mass spectrometry analysis: the different protein spots were cut, decolorized, enzymatically hydrolyzed and extracted. The samples were analyzed by mass spectrometry using ABI 4700 MALDI-TOF-TOF mass spectrometer. The UV wavelength was 355 nm, the repetition rate was 200 HZ, the acceleration voltage was 200 000 V, the optimal mass resolution was 1500 Da. The scanning mass range was 700-3200 Da, and the signal was collected. The mass spectra of all the experimental samples were obtained by default mode.
(9) Database retrieval: Mascot distiller is used to filter baseline peaks and identify signal peaks. Matrixscience's Mascot software is used to search mammalian databases for matching proteins and query their functions to determine which proteins are identified. The error range between the apparent PI and the apparent MR is unlimited. (3) The maximum allowable error of the molecular weight of the peptide fragment is controlled at (+50 ppm.) (4) The incomplete selection of the enzymatic fragment is 1-2. (5) Species Source Selection (Mammal). (6) Ion Selection MH and monoisotopic. (7) Fixed modification to cysteine iodoacetamidation (Carbamidomethy1) can be changed to methionine. Oxidation (Oxsidation).
4. main results
(1) successful establishment of rabbit corneal neovascularization model.
(2) On the 16th day after corneal alkali burn, the area of neovascularization in angiostatin group (group C) was 37.62+9.65 mm2, and that in control group (group B) was 46.77+8.98 mm2. Two independent samples t test was used and SPSS 13.0 software was used to analyze the results. The results showed that angiostatin had inhibitory effect on corneal neovascularization.
(3) after two dimensional electrophoresis, A and C two groups were compared with B group, and 13 differential protein spots were detected.
(4) Differential protein identification: 13 proteins were identified by mass spectrometry and database searching. Among them, 6 proteins were significantly lower in group B than in group A and C: albumin precursor, heat shock protein A8, pyruvate kinase, beta B3-crystallin, retinal protein 1 and one not yet identified. Named proteins; group B was significantly higher than group A and C in six proteins: reticulum calcium binding protein 3, keratin 14, actin 11, immunoglobulin lambda chain, immunoglobulin K chain, binding globin.
5. main conclusions
In the past few decades, anti-angiogenic therapy has developed dramatically in tumor, corneal neovascularization and other neovascular diseases by local use or gene delivery. In this experiment, we found that the corneal neovascularization in alkali-burned rabbits decreased significantly after topical drip of angiostatin. In order to understand the mechanism of angiostatin's anti-angiogenic effect, we found that angiostatin could inhibit the elevated proteins such as keratin 14, reticulin 3 and nodule after alkali burn in cornea. Angiostatin also restores reduced proteins such as crystallin, retinol binding protein, albumin precursor, pyruvate kinase, and HSPA8, which are involved in corneal epithelial repair, energy metabolism, and protein synthesis. The dynamic change of protein content is the direction of our research.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R772.2
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