本文關(guān)鍵詞： 早產(chǎn)兒視網(wǎng)膜病 新生血管 地塞米松 血管內(nèi)皮生長(zhǎng)因子 腫瘤壞死因子-α　出處：《廣州醫(yī)學(xué)院》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：作為世界范圍內(nèi)兒童致盲的重要原因之一，早產(chǎn)兒視網(wǎng)膜病(ROP)一直是大家關(guān)注的焦點(diǎn)，多發(fā)生于早產(chǎn)兒和低體重兒中，是一種視網(wǎng)膜血管增生性病變。ROP的發(fā)病原因與早產(chǎn)兒視網(wǎng)膜本身血管的發(fā)育不完全有關(guān)，當(dāng)視網(wǎng)膜因多種原因形成新生血管以及伴有纖維化時(shí)，即出現(xiàn)增殖性視網(wǎng)膜病變，嚴(yán)重者可發(fā)生牽引性視網(wǎng)膜脫離，所以研究新生血管的發(fā)生機(jī)制近年來(lái)成為學(xué)界重點(diǎn)關(guān)注的方向，預(yù)防及治療早產(chǎn)兒視網(wǎng)膜病的研究切入點(diǎn)是如何抑制視網(wǎng)膜新生血管的形成。 早產(chǎn)兒視網(wǎng)膜病的嚴(yán)重并發(fā)癥是因視網(wǎng)膜血管的異常增生引起，而視網(wǎng)膜血管內(nèi)皮細(xì)胞的增殖受視網(wǎng)膜血管形成刺激因子和血管形成抑制因子的共同調(diào)控，達(dá)到一種平衡狀態(tài)，該狀態(tài)一旦被打破，即可啟動(dòng)新生血管生長(zhǎng)。國(guó)外研究發(fā)現(xiàn)新生血管的生長(zhǎng)多是由于上調(diào)血管促進(jìn)因子或下調(diào)血管抑制因子所致，而這些因子主要包括有血管內(nèi)皮生長(zhǎng)因子(VEGF)、腫瘤壞死因子-α(TNF-α)、色素上皮衍生因子（PEDF）等。其中VEGF占據(jù)著至關(guān)重要的地位，無(wú)論在正常或者異常狀態(tài)下對(duì)于血管的形成均具有重要作用，在視網(wǎng)膜血管正常發(fā)育以及成熟過(guò)程中VEGF起著促進(jìn)作用，在ROP病變中視網(wǎng)膜血管質(zhì)和量的變化可以推測(cè)是由于VEGF在視網(wǎng)膜表達(dá)水平的變化所引起。除了VEGF外，TNF—α作為另外一種重要的血管誘導(dǎo)因子，在炎癥、細(xì)胞凋亡和血管形成過(guò)程中也發(fā)揮著重要作用。因此，視網(wǎng)膜新生血管抑制因子療法已成為具有頗大研究前景的ROP診治方法之一。 近年來(lái)國(guó)內(nèi)外均有報(bào)道地塞米松用于一些視網(wǎng)膜疾病如急性前葡萄膜炎、視網(wǎng)膜沖擊傷、增殖性糖尿病視網(wǎng)膜病及角膜堿燒傷等的治療。目前臨床上廣泛產(chǎn)前應(yīng)用糖皮質(zhì)激素地塞米松促進(jìn)胎肺成熟，而地塞米松對(duì)于早產(chǎn)兒視網(wǎng)膜病變的發(fā)生發(fā)展有何影響及其作用機(jī)制國(guó)內(nèi)外尚缺乏令人信服的實(shí)驗(yàn)依據(jù)。 目的 模擬早產(chǎn)兒視網(wǎng)膜病(retinopathy of prematurity，ROP)的病理特點(diǎn)，建立高氧誘導(dǎo)視網(wǎng)膜病的新生鼠模型，為研究ROP的發(fā)病機(jī)制及治療方法提供實(shí)驗(yàn)基礎(chǔ)。觀察地塞米松對(duì)高氧誘導(dǎo)小鼠視網(wǎng)膜新生血管的形成有何影響，探討地塞米松對(duì)新生血管形成是否存在抑制作用及其可能的作用機(jī)制，并研究不同劑量下地塞米松對(duì)ROP的影響，為地塞米松可能影響早產(chǎn)兒視網(wǎng)膜病的發(fā)生發(fā)展提供實(shí)驗(yàn)依據(jù)。 方法 一、高氧誘導(dǎo)新生鼠視網(wǎng)膜血管增生模型的建立：選擇7曰齡C57BL/6J新生小鼠58只，分為單純高氧誘導(dǎo)組和對(duì)照組，將29只單純高氧誘導(dǎo)組小鼠置于特制密閉容器（內(nèi)環(huán)境控制在氧濃度為75％）中飼養(yǎng)5天，隨后放回至正�？諝猸h(huán)境下飼養(yǎng)，17日齡時(shí)處死；另29只對(duì)照組小鼠持續(xù)在正�？諝猸h(huán)境中生長(zhǎng)到17日齡。兩組新生鼠在鼠齡分別為P12(生后第12天)、P14時(shí)各處死8只，其余的在P17時(shí)處死，處死前稱(chēng)重。兩組視網(wǎng)膜鋪片ADP酶染色后，比較視網(wǎng)膜血管形態(tài)、走向及分布上的差異；兩組視網(wǎng)膜切片HE染色后，觀察并計(jì)數(shù)突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù)目，從而定量反映視網(wǎng)膜血管的增生情況。 二、地塞米松影響鼠視網(wǎng)膜新生血管生成和VEGF、TNF—α表達(dá)的研究：建立高氧大劑量地塞米松組、高氧小劑量地塞米松組、高氧對(duì)照組及評(píng)價(jià)組，每組小鼠各15只。在P12、P14和P17時(shí)稱(chēng)重，并于17日齡時(shí)處死。各組視網(wǎng)膜鋪片經(jīng)ADP酶染色后，觀察視網(wǎng)膜血管的改變；視網(wǎng)膜病理切片HE染色后，計(jì)數(shù)突破視網(wǎng)膜內(nèi)界膜的新生血管內(nèi)皮細(xì)胞核數(shù)目；VEGF蛋白及TNF—α蛋白在各組小鼠視網(wǎng)膜中表達(dá)的強(qiáng)度及分布。 結(jié)果 一、高氧誘導(dǎo)新生鼠視網(wǎng)膜血管增生模型的建立： 1.對(duì)照組在P17時(shí)視網(wǎng)膜血管的發(fā)育大致趨向成熟，血管網(wǎng)結(jié)構(gòu)清晰，由視盤(pán)發(fā)出的大血管呈放射狀，向四周均勻分布散開(kāi)，管徑逐漸由粗變細(xì)直至視網(wǎng)膜周邊部，未見(jiàn)血管閉塞。單純高氧誘導(dǎo)組在P17時(shí)由視盤(pán)發(fā)出的視網(wǎng)膜血管出現(xiàn)明顯擴(kuò)張、迂曲，在中周部可見(jiàn)較多新生血管，這些血管的密度增高，血管網(wǎng)的結(jié)構(gòu)和分布均比較紊亂，大片無(wú)灌注區(qū)存在于后極部視網(wǎng)膜。 2.對(duì)照組未發(fā)現(xiàn)或僅在非常少數(shù)的切片中發(fā)現(xiàn)有突破視網(wǎng)膜內(nèi)界膜血管內(nèi)皮細(xì)胞核，而單純高氧誘導(dǎo)組可見(jiàn)較多突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核，有的單獨(dú)出現(xiàn)，有的成簇狀，有的形成新生血管管腔；高氧誘導(dǎo)組內(nèi)突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù)P14、P17分別與P12比較均有統(tǒng)計(jì)學(xué)意義（P0.01），而兩組比較在P12時(shí)無(wú)差異，在P14、P17時(shí)差異有統(tǒng)計(jì)學(xué)意義（P0.01）。 3.單純高氧誘導(dǎo)組在P17時(shí)新生鼠視網(wǎng)膜切片中新生血管內(nèi)皮細(xì)胞核數(shù)左右眼相比較無(wú)統(tǒng)計(jì)學(xué)意義。 二、地塞米松影響鼠視網(wǎng)膜新生血管生成和VEGF、TNF—α表達(dá)的研究： 1. ADP酶染色：大、小劑量地塞米松組視網(wǎng)膜血管形態(tài)與高氧對(duì)照組相比明顯正常，更接近正常對(duì)照組的血管形態(tài)，無(wú)灌注區(qū)明顯縮小，新生血管不明顯，說(shuō)明兩組不同劑量的地塞米松對(duì)新生血管的生長(zhǎng)均有明顯抑制作用。評(píng)價(jià)組的視網(wǎng)膜血管與對(duì)照組相比，差異無(wú)統(tǒng)計(jì)學(xué)意義，說(shuō)明地塞米松對(duì)正常血管的生長(zhǎng)抑制作用不明顯。 2.HE染色：兩組不同劑量地塞米松組眼組織切片中僅見(jiàn)少量突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核，高氧對(duì)照組所有切片均可見(jiàn)突破內(nèi)界膜的內(nèi)皮細(xì)胞核，與高氧誘導(dǎo)組相類(lèi)似。各組鏡下觀察突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù)，大、小劑量地塞米松組分別和高氧對(duì)照組比較，差異均有統(tǒng)計(jì)學(xué)意義。但地塞米松不同劑量組之間比較，P0.05，兩組差異無(wú)統(tǒng)計(jì)學(xué)意義。 3.免疫組化分析：在P17時(shí)，對(duì)照組VEGF蛋白主要在視網(wǎng)膜神經(jīng)纖維層、神經(jīng)節(jié)細(xì)胞層、色素上皮層（RPE層）呈弱陽(yáng)性表達(dá)；TNF—α蛋白也呈弱陽(yáng)性，，陽(yáng)性信號(hào)主要分布于內(nèi)核層、視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層。單純高氧誘導(dǎo)組和高氧對(duì)照組VEGF蛋白及TNF—α蛋白均呈強(qiáng)陽(yáng)性表達(dá)，其中VEGF蛋白在視網(wǎng)膜各層均有表達(dá)，而TNF—α蛋白則主要分布于新生血管內(nèi)皮細(xì)胞、內(nèi)界膜層、神經(jīng)節(jié)細(xì)胞層和內(nèi)核層。大、小劑量地塞米松組中VEGF及TNF—α的表達(dá)較單純高氧誘導(dǎo)組明顯減弱。 4.大、小劑量地塞米松組與對(duì)照組相比較，在P7時(shí)體重?zé)o明顯差異，而在P12、P17時(shí)體重差異均有統(tǒng)計(jì)學(xué)意義。 結(jié)論 1.成功建立了ROP的小鼠模型，為進(jìn)一步研究藥物治療早產(chǎn)兒視網(wǎng)膜病提供了比較合適的動(dòng)物模型。 2.在高氧誘導(dǎo)視網(wǎng)膜病變的小鼠模型中，VEGF蛋白、TNF—α蛋白在視網(wǎng)膜上的表達(dá)增強(qiáng)，作為氧誘導(dǎo)視網(wǎng)膜病變的重要血管影響因子，它們的表達(dá)與視網(wǎng)膜血管新生有著密切的關(guān)系。 3.地塞米松對(duì)視網(wǎng)膜新生血管的形成可能有抑制作用，影響高氧誘導(dǎo)新生鼠視網(wǎng)膜新生血管生成過(guò)程，對(duì)視網(wǎng)膜起著一定的保護(hù)作用。 4.地塞米松影響視網(wǎng)膜新生血管形成的作用機(jī)制可能與其抑制了視網(wǎng)膜VEGF蛋白及TNF—α蛋白表達(dá)有關(guān)，這為進(jìn)一步研究ROP的預(yù)防和治療開(kāi)辟了新方法。
[Abstract]:As one of the important causes of blindness in children all over the world, retinopathy of prematurity (ROP) has been the focus of attention, mainly occurs in premature and low birth weight infants, is a cause of disease and retinopathy of prematurity of vascular proliferative retinopathy.ROP itself vascular development related to incomplete, when retinal neovascularization and fibrosis form for various reasons, namely proliferative retinopathy, serious person can produce traction retinal detachment, so the study of mechanism of angiogenesis in recent years has become the academic focus on the direction of research, prevention and treatment of retinopathy of prematurity is the cut in point form how to inhibit retinal neovascularization.
Serious complications of retinopathy of prematurity is caused by abnormal proliferation of retinal vessels and retinal vascular endothelial cell proliferation by retinal angiogenesis stimulating factor and angiogenesis inhibiting factor co regulation, to reach a balance state, the state once it is broken, you can start the growth of new blood vessels. The foreign study found that the growth of new blood vessels is due to the upregulation of vascular promoting factor or downregulation of angiogenesis inhibitory factor caused by these factors including vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF- alpha), pigment epithelium derived factor (PEDF). The VEGF plays a crucial role, either in normal or abnormal condition for the formation of blood vessels have an important role, VEGF plays a role in promoting the normal development of the retinal vessels and the maturation of the lesions in ROP in vascular net film quality and quantity The change may be speculated to be due to changes in the expression level of VEGF in the retina caused. In addition to VEGF, TNF - alpha as another important factor in angiogenesis, inflammation, apoptosis and angiogenesis also plays an important role. Therefore, retinal angiogenesis inhibitors therapy has become one of the methods of diagnosis and treatment of ROP with considerable research prospect.
In recent years at home and abroad have reported dexamethasone for some retinal diseases such as acute anterior uveitis, retinal blast injury, the treatment of proliferative diabetic retinopathy and corneal alkali burn. The widely clinical application of prenatal dexamethasone promoting fetal lung maturation, and dexamethasone for the occurrence and development of retinopathy of prematurity and the impact of domestic role the mechanism is still a lack of convincingevidence.
objective
Simulation of retinopathy of prematurity (retinopathy of, prematurity, ROP) of the pathological characteristics, establishment of neonatal rat model of oxygen induced retinopathy, provide the experimental basis for studying the pathogenesis and treatment of ROP. What is the effect of dexamethasone on the formation of high oxygen induced retinal neovascularization in mice, to investigate whether dexamethasone inhibitory effect and mechanism of existence the possible formation of new blood vessels, and to study the effects of different doses of dexamethasone on ROP, provide experimental basis for the effect of the occurrence and development of retinopathy of prematurity for dexamethasone.
Method
First, establish a neonatal rat model of oxygen induced retinal neovascularization: 7 day old neonatal C57BL/6J 58 mice were divided into pure oxygen induced group and control group, 29 pure oxygen induced mice were placed in a special airtight container (environmental control in 75% oxygen concentration) in the feeding for 5 days. Then back to normal air feeding, were sacrificed at the age of 17 days; the other 29 mice in control group continued growth to 17 days of age in the normal environment. The two groups of neonatal rats in rat age were P12 (postnatal day twelfth), P14 8 rats of each group were sacrificed, the rest were killed at P17 before death, weighing two group. Retinal flatmount ADP staining after comparing the morphology of retinal vessels, and to the difference of distribution of the two groups; retinal sections after HE staining to observe and count the number of vascular endothelial cells in retinal, and quantitatively reflect the proliferation of retinal vessels.
Two, the effect of dexamethasone in retinal angiogenesis and VEGF expression of TNF alpha: a high oxygen high dose dexamethasone group, high oxygen and low dose dexamethasone group, hyperoxia group and evaluation group, each group of 15 mice each. Only in P12, weighing P14 and P17, and were sacrificed at 17 days of age each. The retinas by ADP staining, observe the changes of retinal vessels; retinal pathological HE staining, neovascularization of endothelial cell count the number of retinal VEGF protein; and the expression of TNF protein in mice retina in intensity and distribution.
Result
1. The establishment of hyperoxia induced retinal vascular proliferation model in neonatal rats.
1. P17 in the control group when the development of retinal vessels generally mature vascular network, clear structure, large vessels from the optic disc from the radial, around to evenly spread, the diameter gradually tapering to peripheral retinal vascular occlusion, no. Pure oxygen induced retinal blood vessels in P17 group by a disc significant expansion in the periphery, tortuous, more visible neovascularization, the vascular density, structure and distribution of vascular network was disorder, avascular area in the posterior retina.
2. the control group was not found or only in very few sections were found in retinal vascular endothelial cells, while the group showed more retinal oxygen induced simple vascular endothelial cells, some alone, some tufted, some form of blood vessels; retinal vascular endothelial the number of nuclei of P14 induced by hyperoxia group, P17 and P12 were statistically significant (P0.01), and the comparison between the two groups in P12, no differences in P14, P17 had significant difference (P0.01).
3. there was no significant difference in the number of the nuclei of the neovascular endothelial cells in the retinal slices of the newborn rats at P17.
Two, dexamethasone affects the formation of retinal neovascularization and the expression of VEGF, TNF - alpha in the retina of rats.
1. ADP staining: large, small doses of dexamethasone group, retinal vascular morphology and high oxygen control group significantly than normal vascular morphology, close to the normal control group, no significantly reduced perfusion and neovascularization is not obvious, that the growth of both the two groups of different doses of dexamethasone on angiogenesis is inhibited. Compared to the evaluation group retinal blood vessels and the control group, the difference was not statistically significant, indicating the growth inhibitory effect of dexamethasone on normal blood vessels is not obvious.
2.HE staining: only two groups of different doses of dexamethasone eye tissue sections in small retinal vascular endothelial cells in hyperoxia group, all sections were visible through the internal limiting membrane of endothelial cells, and high oxygen induced group of similar groups. Under microscope through endothelial cell nuclei, retinal inner limiting membrane the large and small dose dexamethasone group and hyperoxia compared to the control group, there were statistically significant differences between the different doses of dexamethasone. But compared to P0.05, there was no significant difference between the two groups.
3. immunohistochemical analysis: in P17, the control group of VEGF protein mainly in the retinal nerve fiber layer, ganglion cell layer and pigment epithelium layer (RPE layer) showed weak positive expression; TNF protein was weakly positive, the positive signals were mainly distributed in the inner nuclear layer, retinal ganglion cell layer with high oxygen induced group. And the hyperoxia group VEGF protein and TNF protein showed strong positive expression, the expression of VEGF protein in retinal layers were TNF protein mainly distributed in vascular endothelial cells, inner layer, ganglion cell layer and kernel layer. Large group was significantly decreased induced by low-dose dexamethasone group VEGF and TNF expression compared with hyperoxia.
The 4. major, small dose of dexamethasone group compared with the control group, there was no significant difference in weight at P7, but the weight difference was statistically significant at P12 and P17.
conclusion
1. the mouse model of ROP was successfully established, which provides a more suitable animal model for the further study of the treatment of retinopathy in preterm infants.
2., in the hyperoxia induced retinopathy model, the expression of VEGF protein and TNF - alpha protein on the retina is enhanced. As an important vascular factor of oxygen induced retinopathy, their expression is closely related to retinal angiogenesis.
3., dexamethasone may inhibit the formation of retinal neovascularization, influence the process of retinal angiogenesis induced by hyperoxia, and play a protective role in retina.
4., the mechanism of the effect of dexamethasone on retinal neovascularization may be related to the inhibition of the expression of retinal VEGF protein and TNF - alpha protein, which opens a new way for further research on the prevention and treatment of ROP.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R774.1

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相關(guān)期刊論文 前3條

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2 石文靜;陳超;周?chē)?guó)民;胡寶洋;肖虹蕾;;血管內(nèi)皮生長(zhǎng)因子、堿性成纖維細(xì)胞生長(zhǎng)因子-2及雌激素受體在早產(chǎn)兒視網(wǎng)膜病發(fā)病機(jī)制中的作用[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2007年02期

3 侯瑋瑋;蔣犁;喬立興;;血管內(nèi)皮生長(zhǎng)因子、色素上皮衍生因子在酸中毒誘導(dǎo)的早產(chǎn)兒視網(wǎng)膜病大鼠動(dòng)物模型中的表達(dá)[J];現(xiàn)代醫(yī)學(xué);2009年01期

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