【摘要】： 目的 同種異體角膜移植是治療角膜盲最為有效的方法,而角膜移植排斥反應仍然是移植失敗的主要原因；角膜新生血管是角膜盲的主要原因,亦是角膜移植排斥反應的高危因素。因此,如何防治角膜移植術后排斥反應及新生血管一直是角膜病研究領域的重點和難點。 目前的研究表明,角膜的炎癥、感染、變性、外傷都會使血管生成因子和抗血管發(fā)生因子之間的平衡傾斜,刺激原有角膜緣血管生成CNV。一氧化氮不僅是一種內(nèi)源性血管擴張劑還是一種炎癥介質(zhì)。iNOS是一氧化氮生成的合成酶,催化機體產(chǎn)生一氧化氮參與血管生成、腫瘤發(fā)展和轉(zhuǎn)移等過程。有關iNOS在CNV發(fā)生過程中的作用報道很少,且有完全相反結論的報道。我們可以認為iNOS與CNV的發(fā)生發(fā)展有密切關系。此外,一系列體內(nèi)和體外試驗提示NO具有免疫作用。因此,iNOS有可能參與了角膜移植排斥反應及新生血管形成的過程。 方法 隨機選取健康成年雌性Wistar大鼠15只為供體,雄性SD大鼠30只為受體,做異體角膜移植為實驗組,另取18只做自體角膜移植為移植對照組。余3只SD大鼠(6眼)作為正常對照組。術后,每日于裂隙燈顯微鏡下觀察角膜以及眼前段組織的變化。于術后第1d,3d,7d,14d,21d,28d各隨機處死5只異體角膜移植實驗組大鼠,2只自體角膜移植對照組大鼠正常組3只大鼠于實驗結束前處死,取角膜標本行石蠟切片、蘇木素-伊紅(hemotoxylin and eosin, HE)染色觀察新生血管生長和炎性浸潤情況；以免疫組織化學方法檢測角膜移植片iNOS的表達水平。每張切片角膜中央?yún)^(qū)隨機取5個400倍視野,使用計算機圖像分析軟件計算平均灰密度值,取其平均值作為該標本各檢測指標的表達率。 結果 (一)角膜新生血管及植片存活情況 異體角膜移植術后1d可見明顯水腫,術后7d可見角膜新生血管長入植片,14d為高峰,21d以后逐漸消退。自體對照組新生血管及排斥反應均未及異體移植組明顯。 (二)組織病理學檢查 術后1天時異體角膜移植實驗組切片可見輕度水腫,少量炎性細胞浸潤,7d時可見大量炎性細胞及新生血管,14d時達到高峰,21d以后炎性細胞減少新生血管萎縮。自體移植對照組無論是炎性細胞數(shù)還是新生血管數(shù)量均未及異體移植實驗組。 (三)免疫組織化學 iNOS在正常對照組大鼠角膜上皮層及基質(zhì)層僅有少量表達,術后各時間點自體、異體角膜移植實驗組均較正常對照組表達明顯增強(P=0.000、0.000、0.000、0.000、0.000、0.000、0.001、0.000、0.000、0.000、0.000、0.000、0.000,F=236.453、132.785、144.727、326.412、163.883、53.116、25.868、140.831、123.902、64.913、264.474、378.131),表達范圍主要在基質(zhì)層炎性細胞、角膜上皮細胞及新生血管內(nèi)皮細胞胞漿內(nèi)。異體移植實驗組及自體移植對照組均在術后第14d表達最強烈。3d后異體移植實驗組iNOS的表達較同期自體移植對照組明顯增強(P=0.002、0.025、0.042、0.027,0.034,F=19.912、7.804、6.043、8.645、6.527)。 結論 iNOS在大鼠異體角膜移植模型中的表達增加,并參與了大鼠異體角膜移植排斥反應及新生血管形成的過程。
[Abstract]:objective
Corneal allograft is the most effective method for the treatment of corneal blindness, and corneal graft rejection is still the main cause of graft failure. Corneal neovascularization is the main cause of corneal blindness and a high risk factor for corneal graft rejection. Therefore, how to prevent corneal graft rejection and neovascularization has always been a corner. The key and difficult points in the field of membrane disease.
The current research shows that the inflammation, infection, degeneration and injury of the cornea will make the balance between the angiogenic factor and the antiangiogenic factor, and stimulate the original corneal limbus to produce CNV. nitric oxide not only an endogenous vasodilator or an inflammatory mediator,.INOS, a synthetase produced by nitric oxide, which catalyzes the production of the body. There are few reports on the role of nitric oxide in angiogenesis, tumor development and metastasis. There are few reports about the role of iNOS in the occurrence of CNV, and there is a complete contrary conclusion. We can consider that iNOS is closely related to the development of CNV. In addition, a series of in vivo and in vitro experiments suggest that NO has immune function. Therefore, iNOS may be involved in the development of the immune system. With corneal transplant rejection and neovascularization.
Method
15 healthy adult female Wistar rats were randomly selected as donor, 30 of male SD rats were used as receptors, corneal allograft was used as experimental group, and 18 rats were transplanted into control group with autologous corneal transplantation. The remaining 3 SD rats (6 eyes) were used as normal control group. The changes of cornea and anterior segment tissue were observed daily by slit lamp microscopes. After the operation, 1D, 3D, 7d, 14d, 21d, 28d were randomly killed in 5 corneal allograft rats, and 2 rats in the control group of autologous corneal transplantation were killed before the end of the experiment, and the paraffin section was taken. The growth of neovascularization and inflammatory infiltration were observed by hematoxylin eosin (hemotoxylin and eosin, HE). The expression level of iNOS in corneal graft was detected by histochemical method. 5 400 times of visual field were taken at the central region of each slice of cornea. The average grey density was calculated by computer image analysis software, and the average value was taken as the expression rate of each test index of the specimen.
Result
(1) survival of corneal neovascularization and graft
After corneal allograft, obvious edema was seen in 1D, and corneal neovascularization was seen in 7d after operation. 14d was the peak and 21d gradually subsided after 21d. The neovascularization and rejection in the control group were not obvious in the allograft group.
(two) histopathological examination
At 1 days after the operation, there were slight edema and small amount of inflammatory cell infiltration in the experimental group of corneal allograft, a large number of inflammatory cells and neovascularization were found at 7d, when 14d reached the peak, after 21d, inflammatory cells reduced neovascularization, and the number of inflammatory cells and the number of new blood vessels in the autotransplantation control group were not in the experimental group.
(three) immuno histochemistry
INOS was only a small amount of expression in the corneal epithelium and stroma layer of normal control rats, and the expression of corneal allograft in the experimental group was significantly enhanced (P=0.000,0.000,0.000,0.000,0.000,0.000,0.001,0.000,0.000,0.000,0.000,0.000,0.000, F =236.453132.785144.727326.412163.883,53.116,25.8) at all time points after the operation. 68140.831123.902,64.913264.474378.131), the expression range was mainly in the matrix layer inflammatory cells, corneal epithelial cells and neovascular endothelial cells. Both the allotransplantation experimental group and the autotransplantation control group had the most intense expression of 14d after the operation, and the expression of iNOS in the allograft group was significantly higher than that of the autotransplantation control group. Strong (P=0.002,0.025,0.042,0.027,0.034, F=19.912,7.804,6.043,8.645,6.527).
conclusion
The expression of iNOS was increased in rat corneal allograft model, and it was involved in the process of corneal allograft rejection and neovascularization.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R779.65

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