本文關(guān)鍵詞：重組色素上皮衍生因子對氧誘導(dǎo)大鼠視網(wǎng)膜新生血管生長的抑制作用，，由筆耕文化傳播整理發(fā)布。

        研究背景早產(chǎn)兒視網(wǎng)膜病變(retinopathy of prematurity, ROP)已經(jīng)成為全世界范圍內(nèi)致盲的重要原因,主要由于未成熟兒視網(wǎng)膜發(fā)育不完善,在多種因素影響下視網(wǎng)膜缺血,進(jìn)一步造成視網(wǎng)膜新生血管形成,然后導(dǎo)致增殖性視網(wǎng)膜病變。視網(wǎng)膜新生血管形成后可發(fā)生滲出,出血,增殖膜等一系列病變,使眼球的結(jié)構(gòu)和功能受到嚴(yán)重的損害ROP的治療主要是激光光凝術(shù)和冷凍手術(shù),晚期的患者行玻璃體切割術(shù),手術(shù)治療雖然可控制ROP的病程,防止嚴(yán)重并發(fā)癥,但治療效果仍不理想,只是延緩病變的進(jìn)展,在治療過程中破壞了視網(wǎng)膜組織,導(dǎo)致視功能的永久性損害。ROP的發(fā)生與視網(wǎng)膜新生血管有密切關(guān)系。但新生血管增殖的機(jī)制不明,新生血管的形成是一及其復(fù)雜的生物過程。研究發(fā)現(xiàn)血管內(nèi)皮生長因?yàn)榕c視網(wǎng)膜新生血管形成密切相關(guān),視網(wǎng)膜新生血管性疾病均存在這視網(wǎng)膜組織血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)生成增加。而抑制視網(wǎng)膜組織VEGF的表達(dá),能夠使視網(wǎng)膜新生血管形成減少。VEGF作為。種血管生成刺激因子被認(rèn)為在ROP血管形成中起到重要的中心調(diào)控作用,血色素上皮衍生因子(pigment epithelium-derived factor, PEDF)是VEGF的非特異性抑制劑。兩者在視網(wǎng)膜血管病變中有著密切的關(guān)系。在缺氧的視網(wǎng)膜中,PEDF(?)的含量會(huì)下降,VEGF(?)的含量增多。PEDF的產(chǎn)生和分布是很廣泛的,成人眼內(nèi)的PEDF存在于角膜,房水,玻璃體,視網(wǎng)膜等,主要由視網(wǎng)膜色素上皮分泌而來。PEDF不僅能夠影響視網(wǎng)膜分化,發(fā)育和成熟,還有神經(jīng)元保護(hù)作用,具有神經(jīng)營養(yǎng)和抗新生血管的作用。PEDF是目前最強(qiáng)大的新生血管再生抑制因子之一。另外它只針對異常新生血管而不抑制正常存在血管,而且,PEDF的神經(jīng)營養(yǎng)和保護(hù)作用,對比可能損害神經(jīng)的抑制藥物或生物因子來說,更占優(yōu)勢。因而PEDF具有廣闊的臨床應(yīng)用前景。PEDF還具有抗氧化作用,這些因素都是早產(chǎn)兒視網(wǎng)膜病變的主要因素,所以可以想象PEDF在預(yù)防和治療早產(chǎn)兒視網(wǎng)膜病變的新生血管形成及進(jìn)展方面的作用。因此,我們的實(shí)驗(yàn)通過不同的氧氣濃度建立合適的氧誘導(dǎo)視網(wǎng)膜病變(oxygen induced retinopathy, OIR)模型,模擬人類未成熟兒視網(wǎng)膜病變,觀察VEGF與視網(wǎng)膜新生血管之間的關(guān)系,采取PEDF兩種不同的注射方式(結(jié)膜下、玻璃體腔)治療,觀察PEDF對視網(wǎng)膜新生血管的抑制作用及對正常視網(wǎng)膜血管的影響,力圖尋找一種簡便、安全、有效的途徑,為未成熟兒視網(wǎng)膜病變藥物治療提供新的方法和思路。目的觀察不同氧濃度環(huán)境在新生鼠視網(wǎng)膜病的作用,建立符合早產(chǎn)兒視網(wǎng)膜病變病理特點(diǎn)的動(dòng)物模型,為研究該病的發(fā)病機(jī)制及治療提供實(shí)驗(yàn)基礎(chǔ),利用視網(wǎng)膜血管ADP酶染色及組織切片觀察大鼠視網(wǎng)膜形態(tài)及病變程度。分析新生鼠視網(wǎng)膜及氧誘導(dǎo)視網(wǎng)膜病變中VEGF蛋白質(zhì)表達(dá)的變化。方法將新生鼠40只,與母鼠共同飼養(yǎng),將其分為空氣組；波動(dòng)組1:50%-20%；波動(dòng)組2：40%-10%；波動(dòng)組3:50%-10%四組,每組各10只。正�？諝饨M正�？諝庵酗曫B(yǎng)18d,波動(dòng)組1,放入50%0224h,20%24h,循環(huán)七次,共14d,后放入空氣飼養(yǎng)4d,波動(dòng)組240%0224h,10%24h,循環(huán)七次,共14d,后放入空氣飼養(yǎng)4d,波動(dòng)組3放入50%0224h,10%24h,循環(huán)七次,共14d,后放入空氣飼養(yǎng)4d。18的時(shí),每組各取6只大鼠左眼,行視網(wǎng)膜鋪片ADP酶染色觀察視網(wǎng)膜血管形態(tài)的發(fā)育,鐘點(diǎn)法評價(jià)視網(wǎng)膜新生血管嚴(yán)重程度；每組各取6只大鼠右眼,制作眼球病理切片,常規(guī)HE染色計(jì)數(shù)突破視網(wǎng)膜內(nèi)界膜的內(nèi)皮細(xì)胞核定量分析視網(wǎng)膜新生血管增生情況,比較各組間的差異。各組剩余視網(wǎng)膜組織,利用Western-blot檢測視網(wǎng)膜血管內(nèi)皮生長因子VEGF蛋白的表達(dá)。統(tǒng)計(jì)分析：所有數(shù)據(jù)均依據(jù)SPSS13.0統(tǒng)計(jì)軟件包進(jìn)行分析,實(shí)驗(yàn)測試指標(biāo)的數(shù)據(jù)資料以x±s表示；采用One-Way ANOVA分析數(shù)據(jù),方差齊時(shí)組間采用LSD法,方差不齊時(shí)組間多重比較采用Dunnett C法,P<0.05作為差異有顯著性意義。結(jié)果視網(wǎng)膜鋪片ADP酶染色：正常空氣組新生鼠在18d時(shí)視網(wǎng)膜血管發(fā)育基本成熟,管徑較粗,全視網(wǎng)膜分深淺兩層,淺層自視盤發(fā)出,向四周呈放射狀均勻分布,深層呈多角形網(wǎng)狀形態(tài)；波動(dòng)組1初網(wǎng)膜摁管發(fā)育基本正常；波動(dòng)組2,3視網(wǎng)膜血管擴(kuò)張迂曲,中央見無灌注區(qū),血管發(fā)育未達(dá)視網(wǎng)膜周邊部,有大量新生血管形成,血管密度增高,新生血管網(wǎng)結(jié)構(gòu)紊亂,有新生血管叢和血管瘤。四組鐘點(diǎn)法評價(jià)新生血管鐘點(diǎn)數(shù)為：0,0.17±0.41,4.8±1.2,7.8±1.2,不同吸氧濃度導(dǎo)致的視網(wǎng)膜新生血管鐘點(diǎn)數(shù)比較,差異有顯著性意義(F=120.21,P=0.00)；空氣組波動(dòng)組2,3病變鐘點(diǎn)數(shù)明顯增多,與相應(yīng)的正�？諝饨M相比較,差異有統(tǒng)計(jì)學(xué)意義(P=0.00)；波動(dòng)組1無明顯增多,與正�？諝饨M比較差異無統(tǒng)計(jì)學(xué)意義；波動(dòng)組3比波動(dòng)組2相比較增多,相比有統(tǒng)計(jì)學(xué)意義(P=0.00)。HE染色：四組突破內(nèi)界膜內(nèi)皮細(xì)胞核數(shù)分別為0.6±0.470.87±0.6514.28±3.8322.52±4.02,四組之間比較差異有顯著性意義(F=875.366,P＝0.00);正�？諝饨M鼠幾乎沒有見到突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核,波動(dòng)組1切片中絕大多數(shù)視網(wǎng)膜內(nèi)界膜是平滑的,偶見新生血.管。波動(dòng)組2,3組所有切片均可見較多突破內(nèi)界膜的血管內(nèi)皮細(xì)胞,所有切片均可見較多突破內(nèi)界膜的血管內(nèi)皮細(xì)胞,且波動(dòng)3組突破內(nèi)界膜的血管內(nèi)皮細(xì)胞明顯比波動(dòng)2組多(P<0.01)；VEGF表達(dá)在所有波動(dòng)組中,與波動(dòng)組1,2比較,波動(dòng)組3中VEGF表達(dá)最高,空氣組表達(dá)最低。結(jié)論1.50%-10%氧氣波動(dòng)組最適合制作早產(chǎn)兒視網(wǎng)膜病變模型,具有類似ROP血管閉塞,血管無灌注區(qū),新生血管等改變,制備過程較為簡便,重復(fù)性好,是研究ROP比較適合的動(dòng)物模型；2.證實(shí)了反復(fù)血氧濃度波動(dòng)可導(dǎo)致新生鼠視網(wǎng)膜新生血管增生性病變,吸入氧分壓差(ΔFiO2)的波動(dòng)與病變程度呈正相關(guān)；3.低氧比高氧對發(fā)生視網(wǎng)膜病變可能更重要；4.VEGF在視網(wǎng)膜新生血管形成中起著重要作用,促進(jìn)視網(wǎng)膜新生血管形成。目的觀察結(jié)膜下和玻璃體注射重組人色素上皮衍生因子(pigment epithelium-derived factor, PEDF)兩種注射方式對氧誘導(dǎo)大鼠視網(wǎng)膜新生血管(Retinal neovascularization, RNV)的作用,從治療角度闡述PEDF在ROP中的作用。方法新生大鼠分別進(jìn)行氧氣或空氣暴露：新生鼠與哺乳母鼠置于密閉容器內(nèi),50%0：24h,10%/L0224h為1個(gè)循環(huán),共7個(gè)循環(huán),建立類似早產(chǎn)兒視網(wǎng)膜病變動(dòng)物模型。氧誘導(dǎo)SD新生大鼠建立類似早產(chǎn)兒視網(wǎng)膜病變動(dòng)物模型。新生鼠48只隨機(jī)分為6組(A：空氣對照組,B:高氧對照組,C:高氧+玻璃體注射PEDF2ug組,D：高氧+結(jié)膜下注射PEDF2ug組,E：高氧+結(jié)膜下注射PEDF4ug組,F：高氧+結(jié)膜下注射PEDF8ug組)。當(dāng)新生大鼠脫離氧時(shí),C,D,E和F組大鼠左眼用不同劑量和注藥次數(shù)進(jìn)行注射PEDF,共四天,18d處死取標(biāo)本,ADP酶視網(wǎng)膜血管染色觀察視網(wǎng)膜血管形態(tài),石蠟切片計(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。另新生大鼠2只進(jìn)行左眼結(jié)膜下注射PEDF, Western-blot檢測視網(wǎng)膜PEDF的表達(dá).統(tǒng)計(jì)分析：所有數(shù)據(jù)均依據(jù)SPSS13.0統(tǒng)計(jì)軟件包進(jìn)行分析,實(shí)驗(yàn)測試指標(biāo)的數(shù)據(jù)資料以x±s表示；采用One-Way ANOVA分析數(shù)據(jù),方差齊時(shí)組間比較采用Bonferroni法,方差不齊時(shí)組間多重比較采用Dunnett C法,P<0.05作為差異有顯著性意義。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果結(jié)膜下注射PEDF,可檢測到視網(wǎng)膜PEDF蛋白表達(dá)：視網(wǎng)膜鋪片結(jié)果：,A組視網(wǎng)膜血管發(fā)育正常。B組視網(wǎng)膜大量的新生血管生成。C組新生血管明顯減少。D、E、F組新生血管稍減少。六組組織病理學(xué)檢測突破視網(wǎng)膜內(nèi)界膜的新生血管內(nèi)皮細(xì)胞計(jì)數(shù)分別為：1.88±0.92,25.00±0.94,12.23±0.96,24.58±1.03,23.68±0.77,23.20±0.88,各組比較差異有統(tǒng)計(jì)意義(F=6729,P=0.00);A組視網(wǎng)膜內(nèi)界膜平滑,偶見突破的視網(wǎng)膜內(nèi)皮細(xì)胞。B組明顯高于A組(p=0.00),差異有統(tǒng)計(jì)學(xué)意義。C組明顯低于高氧塒照組B組(p=0.00),D組與B組相比無明顯差異(p=0.20),E、F組突破內(nèi)界膜的內(nèi)皮細(xì)胞核數(shù)’與C組差異有統(tǒng)計(jì)學(xué)意義(p=0.00)。結(jié)論1、結(jié)膜下注射PEDF,鞏膜和脈絡(luò)膜-色素上皮層對其是有滲透性的,可以跨越結(jié)膜下組織到達(dá)視網(wǎng)膜,但與玻璃體注射組相比,抑制新生血管作用有明顯差異；2、玻璃體注射PEDF可以有效抑制氧誘導(dǎo)大鼠視網(wǎng)膜新生血管；3、結(jié)膜下注射PEDF如何有效到達(dá)視網(wǎng)膜還需進(jìn)一步研究。

    BackgroundROP is proliferative disease of the retinal vasculature infants that may cause severe visual loss, and it is a major cause of blindness in newborn, The disease is characterized by proliferation of abnormal fibrovascular tissue at the border of vascularised and non-vascularised retina. Retinopathy of prematurity (ROP) is a potenitially blinding disease affecting premature infants. The immature neurovascular tissues of the visual system, the retina and brain, are particularly susceptible to injury. ROP is characterized by pathological ocular angiogenesis or retinal neovasculrization (NV).This NV leads to traction retinal or further proliferation of fibrovascular tissue on the retinal surface, resulting in formation of a retrolental fibrovascular membrane, total retinal detachment, intravitreal hemorrhages and subsequent vision loss. The approved treatments consist in photocoagulation surgery, laser photocoagulation or cryotherapy. However, the visual outcomes after treatment often are not satisfactory. While guidelines for the management and treatment of ROP have recently been refined, it is still difficult to identify those infants in whom disease is more likely to progress. Preventive therapy for ROP is sorely needed.Abnormal development of the retinal vasculature is at the heart of this disease. Vascular endothelial growth factor(VEGF) has emerged as one of the most important in the development of NV. PEDF is a member of the serine protease inhibitor (serpin) family, although it does not have protease inhibitory activity. Pigment epithelium-derived factor(PEDF) is the most potent natural inhibitor of angiogenesis. PEDF has emerged as a potentially important endogenous inhibitor of ocular neovascularization. PEDF is a multifunctional serpin protein. It is PEDF also has neuroprotective activity and has been demonstrated to protect photoreceptors from degeneration. PEDF has been found in the vitreous and cornea, and it has been found to be downregulated by hypoxia in cultured cells. Its presence in the vitreous and its antiangiogenic activity suggest a possible role for PEDF in the regulation of retinal neovascularization. PEDF strongly inhibited VEGF-induced migration and proliferation of retinal endothelial cells.Therefore, our experiment used oxygen concentration variation to establish a suitable oxygen-induced retinopathy rat model for imitating human ROP. We analysed the relationship between VEGF and retinal neovascularization, We observed the effects of PEDF on OIR in rats with subconjunctival or intravitreal injection. We strived a novel, safer and more effective therapeutic approaches for the treatment of pathologic neovascular conditions.BackgroundRetinopathy of prematurity (ROP) is a causing-blindness eye disease Oxygen in premature retinal neovascularization plays an important role, but its scope of oxygen and the specific mechanism of retinopathy is not clear ObjectiveTo study the effects of different concentrations of oxygen on neovascularization in oxygen-induced retinopathy (OIR) of newborn rats and provide experimental basis for clinical oxygen therapy; To determine the roles of vascular endothelial growth factor (VEGF)in vascularization of the developing retina.Method:Forty Sprague-Dawley rats were included in the study. They were divided into four groups:Air group (10rats) was was raised in air room. Fluctation group1、2、3(10rats) was exposed to cycles of50%～20%,40%～10%,50%～10%for14days, respectively. In each case, oxygen was alternated between the two exposure concentrations every24hours. The rats were removed to room air for4days before the severity of abnormal neovascularization were measured. Retinas were dissected and stained by adenosine-diphosphatase(ADPase) histochemistry for assessmem of intraretinal vascular development and preretinal angiogenic vessel growth. The proliferative neovascularization response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. The total number of nuclei counted per retina was defined as the nucleus count. Nuclei above the internal limiting membrane were counted. Protein expressions of VEGF were studied by western-blot.Statistical analysis:The analysis was performed using SPSS13.0software package. The data was represented as the mean±tandard deviation(x±s), Comparisons of means among group were performed using one-way analysis of variance(ANOVA). If Variance were homogenous among groups, the LSD method was used for multiple comparisons, otherwise Dunnett C method was used, P<0.05were considered to be statistically significant.Result Retina flat-mounts (ADP-ase staining):The retinal vascularization had completed in normal air group, The main buanches of vessels in optic idsc are well-developed in normal air group. Retina vascular development is normal basically;Retina flat-mounts presented increasing neovascular tufts,plenty of newly born vascular plexus and disorderly arranged vessel structures in Fluctation group2and Fluctation group3.The clock number of these four groups were0,0.17±0.41,4.8±1.2,7.8±1.2,respectively. the difference was significant (F=120.21, p=0.00). The difference was statistically significant between Fluctation group2and Fluctation group3(P=0.00).HE stain results:The number of vascular nuclei extending from the internal limiting membrane into the vitreous were0.6±0.47,0.87±0.65,14.28±3.8322.52±4.02, respectively. The difference was significant (F=875.366, p=0.00). The differences in the VEGF-expression among three groups were significant (P=0.00).Conclusion1.50%～10%pattern is suitable for oxygen-induced retinopathy rat model to imitate human ROP; This animal model(50%～10%pattern) of OIR is reproducible. Because of its quantifiable characteristic, it is useful for the study of pathogenesis of retinal neovascularization and theraputic intervention for ROP.2. Changeable Oxygen concentrationa could trigger a subsequent neovascularization;△FiO2were positively correlated with the retinopathy;3. Hypoxia may be more important for retinal neovascularization on the occurrence of retinopathy than Hyperoxia;4. The over-expression of VEGF plays a important role during the formation of neovascularization. ObjectiveTo observe the inhibitive effect of intravitreal or Subconjunctival injecting PEDF for retinal neovascularization in Oxygen-induced animal models.MethodMouse models of oxygen-induced retinopathy of prematurity were established.48newborn rats were randomly diveded into6groups (A: Air control group; B:Hyperoxygen control guoup; C:Hyperoxygen+intravitreal injection of2ugPEDFgroup; D:Hyperoxygen+injection of2ugPEDFgroup; E:Hyperoxygen+injection of4ugPEDFgroup; F:Hyperoxygen+injection of8ugPEDFgroup). When the rats were taken off the high oxygen, the PBS and corresponding dosage of PEDF were intravitreal or Subconjunctival injected in group C, D, E and F, respectively. ADP-ase stained retina flat-mounts was used to evaluate the retinal vascular modality and development, Eyeball pathological section was used to count the pre-retinal endothelial nucleus in retinal new vessel. Retinal PEDF expression were detected by western-blot.Statistical analysis:The analysis was performed using SPSS13.0software package. The data was represented as the mean±standard deviation(x±s), Comparisons of means among group were performed using one-way analysis of variance(ANOVA). If Variance were homogenous among groups, the Bonferroni method was used for multiple comparisons, otherwise Dunnett C method was used, P<0.05were considered to be statistically significant.ResultsPEDF were achieved in retinal after a periocular injection. Retinal flat-mounts: A group:The retina vessels of the normal had a fine radial branching pattern that extend from the optic nerve to the periphery; B group:There were more retinal neovascularization compared with A group.The density and shape of retinal neovascularization were disordered; Comparing with B group, retinal neovascularization decreased obviously in C group while D, E, F group decreased mildly. HE staining:A group:Retinal internal limiting membrane was smooth, and occasionally the endothelial cells break through the inner retinal. B group was significantly more than group A(p<0.05). C group was significantly less than the B group (p<0.05), D, E, F group had significant difference between group C (all p <0.05).Conclusion1. PEDF are able to penetrate the sclera into retinal after a periocular injection, however, compared with intravitreal injection, the effect reduced significantly.2. Intravitreal injection of PEDF can arrest retinal neovascularization in SD rat models;3. How to reach the retina effectively requires further study by subconjunctival injection.

        重組色素上皮衍生因子對氧誘導(dǎo)大鼠視網(wǎng)膜新生血管生長的抑制作用

摘要3-8ABSTRACT8-13前言15-21    參考文獻(xiàn)17-21第一部分 不同吸氧濃度誘導(dǎo)視網(wǎng)膜病變大鼠模型21-45    背景內(nèi)容21    1 材料21-24    2 實(shí)驗(yàn)方法24-28    3 結(jié)果28-35    4 討論35-41    5 參考文獻(xiàn)41-45第二部分 PEDF抑制ROP新生血管形成的實(shí)驗(yàn)研究45-61    1 材料45    2 實(shí)驗(yàn)方法45-48    3 結(jié)果48-54    4 討論54-58    5 參考文獻(xiàn)58-61全文總結(jié)與展望61-62附錄一 縮略詞表62-63攻讀學(xué)位期間的研究成果63-64致謝64-66統(tǒng)計(jì)學(xué)審稿證明66

  本文關(guān)鍵詞：重組色素上皮衍生因子對氧誘導(dǎo)大鼠視網(wǎng)膜新生血管生長的抑制作用，由筆耕文化傳播整理發(fā)布。