【摘要】：1.研究目的及意義 角膜組織沒有血管的透明組織。維持其透明性的一個很重要的原因是它沒有血管。在感染、外傷、免疫反應、排斥反應、佩戴角膜接觸鏡、堿燒傷、基質潰瘍、角膜緣干細胞病變等病理情況下,從角膜緣血管網(wǎng)形成的新生血管逐漸侵入角膜,從而形成角膜新生血管(corneal neovascularization, CNV)。誠然,CNV在感染清除、傷口愈合等方面具有一定的積極作用。但是,CNV導致角膜的透明性降低,從而嚴重的影響視力,另外,CNV可破壞角膜正常微環(huán)境,使眼前節(jié)生理性的免疫豁免消失,因而成為角膜移植排斥反應的高危因素。因此,針對角膜新生血管的治療業(yè)已成為眼科的研究熱點。近年來的研究證實,血管抑素(angiostatin, AS)存在著明確的抗角膜新生血管形成的作用,角膜上皮細胞被證明可以分泌血管抑素,但在角膜受到損傷后分泌減少。另外,血管抑素被證實通過病毒載體或者局部運用可以抑制角膜新生血管形成。但相關作用機制研究的不足,影響著其最終運用于臨床的進程。因此,闡明血管抑素抗角膜新生血管形成的機制,勢必為其臨床運用奠定堅實的基礎,并產(chǎn)生巨大的社會和經(jīng)濟效益。 2.研究方法 眾所周知,蛋白質是一類重要的生物大分子,是生命活動的主要承擔者。在蛋白質組(proteome)概念提出以后,蛋白質組學的研究取得了可喜的發(fā)展。相比較傳統(tǒng)的只針對單一蛋白質的研究,蛋白質組學更加注重的是參與特定生理或病理狀態(tài)的所有蛋白質種類及其與周圍環(huán)境(分子)的關系。隨著相關實驗技術的發(fā)展,目前已有可能對細胞在不同生理或病理條件下蛋白質表達的異同,相關蛋白質的分類和鑒定,尤其是蛋白質之間相互作用和蛋白質的功能進行探討。因此,本課題擬以堿燒傷制備的角膜新生血管為基礎,局部施加血管抑素,將角膜組織進行二維電泳,分離蛋白質并進行染色,凝膠圖像分析系統(tǒng)對蛋白質點進行定量分析,從而明確局部施加血管抑素在抗角膜新生血管形成過程中的差異表達蛋白,進一步地,對其進行質譜分析,從而得到相關蛋白質的定性數(shù)據(jù)。 3.研究內容和過程 (1)實驗動物及分組：本實驗采用的是32只新西蘭大白兔(購自南方醫(yī)科大學動物中心),實驗動物分為三組,A組為空白對照組：8只新西蘭兔不做任何處理；B組為燒傷組：12只新西蘭兔采用堿燒傷的方法建立角膜新生血管模型,術后第1天開始局部生理鹽水點眼,每日3次,直至取角膜標本；C組為治療組：12只新西蘭兔采用同樣方法建立角膜新生血管模型后,術后第1天開始局部用30μg/ml血管抑素(AS)點眼,每日3次,直至取角膜標本。 (2)角膜新生血管模型制作：堿燒傷法制備角膜新生血管模型：氯胺酮25mg/kg及氯丙嗪25mg/kg肌注新西蘭兔全身麻醉,0.5%丁卡因表面麻醉,置開瞼器充分暴露眼表,將浸有1.5mol/LNaOH液的10mm圓形濾紙片置于眼表中央,1.5min后取下,用50m1生理鹽水沖洗眼表,滴妥布霉素眼液后涂妥布霉素眼膏。 (3)角膜新生血管的觀察：術后觀察角膜的新生血管,第16日使用裂隙燈(蘇州66生產(chǎn)YZ5T型號)照相。角膜新生血管的檢測：測量血管長度,以連續(xù)彎曲度小、新生血管朝向角膜混濁中心生長的最長血管為準,并計算角膜新生血管生長面積(A),按公式A=C/12×3.1416[r2-(r-1)2]。C為Nv累及角膜的圓周鐘點數(shù),1為新生血管從角膜緣深入角膜的長度,r為兔角膜半徑,設為定值7mm。 (4)標本取材及全蛋白提�。盒g后3w取材。標本離體后迅速置入5ml凍存管,于液氮速凍后置入-80℃超低溫冰箱保存?zhèn)溆谩Ｈ?80℃保存的組織塊,迅速研磨至細粉末狀,加入裂解液勻漿、離心。采用Bradford定量法,計算樣品蛋白質濃度。 (6)雙向凝膠電泳(2-DE)：第一向(IPG-IEF)根據(jù)蛋白質等電點的不同進行等電聚焦,第二向(SDSPAGE)根據(jù)蛋白質相對分子質量大小分離樣品,雙向電泳后才用EMBL銀染。 (7)圖像分析和差異蛋白選�。簩€y染后的2-DE膠圖掃描入電腦。應用ImageMaster2D軟件分析每一張凝膠圖像,計算圖譜中所有蛋白質點的蛋白質相對含量,找出三塊膠中均出現(xiàn),并且有差異的蛋白。 (8)質譜分析：切取有差異的蛋白質點,行脫色、酶解、萃取樣品,利用美國ABI4700MALDI-TOF-TOF質譜儀進行質譜分析。UV波長為355nm,重復速率為200HZ,加速電壓為20000V,最優(yōu)質量分辨率為1500Da。掃描質量范圍為700-3200Da,收集信號。胰酶自切峰為內標校正質譜儀。所有實驗樣品的質譜圖均以用默認模式獲得。 (9)數(shù)據(jù)庫檢索：利用軟件Mascot distiller過濾基線峰、識別信號峰。利用Matrixscience公司的Mascot軟件搜索哺乳動物數(shù)據(jù)庫,尋找匹配的相關蛋白質,同時查詢其功能,來明確鑒定的蛋白質為何種蛋白質。查詢條件：(1)肽質量控制在800-4000Da。(2)表觀PI與表觀Mr的誤差范圍：無限制。(3)肽片段分子量最大容許誤差控制在±50ppm。(4)酶解片段不完全選擇為1-2個。(5)物種來源選擇(哺乳動物)。(6)離子選擇MH和monoisotopic。(7)固定修飾為半胱氨酸碘乙酰胺化(Carbamidomethy1),可變修飾為蛋氨酸氧化(Oxsidation)。 4.主要結果 (1)成功建立兔角膜新生血管模型。 (2)兔角膜堿燒傷后第16天,血管抑素作用組(C組)的新生血管面積37.62+9.65mm2,對照組(B組)面積46.77+8.98mm2,采用兩獨立樣本t檢驗,并用SPSS13.0軟件進行分析,P0.05,證明血管抑素對角膜新生血管有抑制作用。 (3)各樣本進行雙向電泳后,將A、C兩組與B組進行比較,檢測到13個差異蛋白點。 (4)差異蛋白鑒定：通過質譜分析和數(shù)據(jù)庫檢索,成功鑒定了這13個蛋白(有兩個蛋白的鑒定結果顯示為同一蛋白)。其中B組較A、C組均明顯降低的有6種蛋白：白蛋白前體、熱休克蛋白A8、丙酮酸激酶、βB3-晶體蛋白、視黃醛蛋白1以及一個尚未命名的蛋白；B組較A、C組均明顯升高的有6種蛋白：網(wǎng)鈣結合蛋白3、角蛋白14、肌動蛋白11、免疫球蛋白λ鏈,免疫球蛋白K鏈、結合珠蛋白。 5.主要結論 在過去的幾十年中,在腫瘤、角膜新生血管及其他新生血管性疾病中,抗血管治療通過局部運用或者基因載體方式得到了巨大的發(fā)展,并且在這次實驗中,我們發(fā)現(xiàn)經(jīng)過堿燒傷的兔角膜在局部滴用血管抑素后,新生血管明顯減少,這就證明了血管抑素可以有效的抑制血管內皮細胞的遷移及增殖,并且誘導這些細胞凋亡。為了了解血管抑素抗血管作用的機理,通過蛋白組學,我們發(fā)現(xiàn)角膜堿燒傷后局部應用血管抑素,可以抑制燒傷后升高的蛋白,如角蛋白14、網(wǎng)鈣蛋白3、結合珠蛋白,這些蛋白都參與了新生血管的形成。血管抑素同時可以恢復燒傷后降低的蛋白,如晶體蛋白、視黃醇結合蛋白、白蛋白前體、丙酮酸激酶、HSPA8蛋白,這些蛋白與角膜上皮修復、能量代謝、蛋白合成有關。在下一步的研究中,血管抑素對各種蛋白含量的動態(tài)變化是我們研究的方向。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R772.2

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