诱导性一氧化氮合成酶iNOS在大鼠穿透性角膜移植中的表达及意义
发布时间：2018-08-03 20:27
【摘要】： 目的 同种异体角膜移植是治疗角膜盲最为有效的方法,而角膜移植排斥反应仍然是移植失败的主要原因；角膜新生血管是角膜盲的主要原因,亦是角膜移植排斥反应的高危因素。因此,如何防治角膜移植术后排斥反应及新生血管一直是角膜病研究领域的重点和难点。 目前的研究表明,角膜的炎症、感染、变性、外伤都会使血管生成因子和抗血管发生因子之间的平衡倾斜,刺激原有角膜缘血管生成CNV。一氧化氮不仅是一种内源性血管扩张剂还是一种炎症介质。iNOS是一氧化氮生成的合成酶,催化机体产生一氧化氮参与血管生成、肿瘤发展和转移等过程。有关iNOS在CNV发生过程中的作用报道很少,且有完全相反结论的报道。我们可以认为iNOS与CNV的发生发展有密切关系。此外,一系列体内和体外试验提示NO具有免疫作用。因此,iNOS有可能参与了角膜移植排斥反应及新生血管形成的过程。 方法 随机选取健康成年雌性Wistar大鼠15只为供体,雄性SD大鼠30只为受体,做异体角膜移植为实验组,另取18只做自体角膜移植为移植对照组。余3只SD大鼠(6眼)作为正常对照组。术后,每日于裂隙灯显微镜下观察角膜以及眼前段组织的变化。于术后第1d,3d,7d,14d,21d,28d各随机处死5只异体角膜移植实验组大鼠,2只自体角膜移植对照组大鼠正常组3只大鼠于实验结束前处死,取角膜标本行石蜡切片、苏木素-伊红(hemotoxylin and eosin, HE)染色观察新生血管生长和炎性浸润情况；以免疫组织化学方法检测角膜移植片iNOS的表达水平。每张切片角膜中央区随机取5个400倍视野,使用计算机图像分析软件计算平均灰密度值,取其平均值作为该标本各检测指标的表达率。 结果 (一)角膜新生血管及植片存活情况 异体角膜移植术后1d可见明显水肿,术后7d可见角膜新生血管长入植片,14d为高峰,21d以后逐渐消退。自体对照组新生血管及排斥反应均未及异体移植组明显。 (二)组织病理学检查 术后1天时异体角膜移植实验组切片可见轻度水肿,少量炎性细胞浸润,7d时可见大量炎性细胞及新生血管,14d时达到高峰,21d以后炎性细胞减少新生血管萎缩。自体移植对照组无论是炎性细胞数还是新生血管数量均未及异体移植实验组。 (三)免疫组织化学 iNOS在正常对照组大鼠角膜上皮层及基质层仅有少量表达,术后各时间点自体、异体角膜移植实验组均较正常对照组表达明显增强(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),表达范围主要在基质层炎性细胞、角膜上皮细胞及新生血管内皮细胞胞浆内。异体移植实验组及自体移植对照组均在术后第14d表达最强烈。3d后异体移植实验组iNOS的表达较同期自体移植对照组明显增强(P=0.002、0.025、0.042、0.027,0.034,F=19.912、7.804、6.043、8.645、6.527)。 结论 iNOS在大鼠异体角膜移植模型中的表达增加,并参与了大鼠异体角膜移植排斥反应及新生血管形成的过程。
[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.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R779.65
本文编号：2162911
[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.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R779.65
【参考文献】
相关期刊论文 前2条
1 司良毅,陈运贞;NO、NOS与缺血再灌注损伤的研究进展[J];国外医学(内科学分册);1999年05期
2 谢立信;我国角膜手术的现状和发展策略[J];中华眼科杂志;2005年08期
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