【作者】 屈超义；

【导师】 唐罗生；

【作者基本信息】 中南大学 ， 眼科学， 2008， 博士

【摘要】 研究背景脉络膜新生血管(Choroidal Neovascularization,CNV)也称视网膜下新生血管,是由多种病因所致的脉络膜新生血管芽穿越bruch膜并在视网膜色素上皮下和/或上增殖形成的纤维血管组织,由于管壁的通透性高于正常血管,常伴有黄斑部视网膜下的浆液性渗出和出血,继而可形成瘢痕,造成黄斑部损伤,严重影响中心视力,甚至致盲。以CNV为主要病理特征的年龄相关性黄斑变性目前已成为国内外老年人群中首要的致盲性眼病。如何有效的抑制CNV的形成和生长是多年来一直困扰眼科学界的难题和研究热点。CNV的确切发病机制尚未阐明,缺乏理想的防治手段。目前抑制或消退CNV的途径有激光光凝、光动力疗法(PDT)、经瞳孔温热疗法(TTT)、手术剥除及放射治疗,但治疗效果不尽如人意,或复发率高,或不可避免损伤正常组织结构,造成视功能进一步恶化。基因治疗已经成为目前CNV治疗的前沿和希望。目前拮抗血管内皮生长因子(Vascular endothelial growth factor,VEGF)药物在临床实践中取得了令人鼓舞的效果。但由于VEGF及其受体具有分布广泛、生理功能复杂的特点,其一定水平的表达对眼内光感受器的生存和维持脉络膜血管对视网膜的血供是必要的,长期抑制VEGF在眼内的表达可能会有不利的后果。所以寻找更安全有效的治疗靶点势在必行。CD105作为TGF-β的受体高度表达于增生活跃的血管内皮细胞表面,而正常血管内皮细胞无或微弱表达CD105,因此CD105常被作为增生活跃的内皮细胞标记物。研究表明CD105基因在新生血管形成中是必须的。在CNV的形成中可能起重要作用,也可能成CNV治疗的靶分子之一。目的1.建立激光诱导BN(Brown Norway)大鼠脉络膜新生血管模型,研究VEGF和CD105基因在CNV形成中的表达。2.使用Pgenesil-1质粒构建CD105RNA干扰重组体,在激光诱导BN(Brown Norway)大鼠脉络膜新生血管模型中,筛选高效特异抑制CD105基因表达的短发夹样shRNA。3.研究在激光诱导BN(Brown Norway)大鼠脉络膜新生血管模型中,因CD105基因表达沉默后对CNV形成及VEGF基因表达的影响。方法1.建立BN大鼠CNV模型:采用半导体激光光凝BN大鼠单眼的视网膜作为实验组,于光凝后1h,第3,7,14,21,28d行眼底血管荧光素造影(fundus fluorescein angiography,FFA)检查,并于第14d行病理组织学检查和脉络膜铺片,观察CNV的形成情况。2.采用免疫组化和RT-PCR方法检测CD105基因在CNV模型和对照组中的表达并探讨其与CNV形成的关系。根据CD105基因序列信息设计了3条shRNA以及一条非特异阴性序列,利用含U6启动子的表达质粒成功构建其RNA干扰重组体。采用视网膜下注射的方法使shRNA表达质粒转染大鼠视网膜和脉络膜,观察质粒所携带GFP基因表达情况。根据不同的表达质粒对BN大鼠CNV模型2wk时CD105基因mRNA表达的抑制效果与空白对照及仅加转染试剂组比较分析,筛选有效的抑制序列。3.采用视网膜下注射的方法使高效CD105shRNA表达质粒转染大鼠视网膜和脉络膜,采用FFA检查和脉络膜铺片的方法观察CD105基因沉默对BN大鼠CNV形成的影响。采用RT-PCR检测实验组CD105基因沉默后VEGF基因mRNA表达情况,并与对照组进行比较。结果1.采用半导体激光光凝BN大鼠视网膜的方法可以成功诱导CNV的形成,FFA证实光凝后第7d开始形成CNV,第14d有大量CNV形成,CNV可持续至4wk。脉络膜铺片及光镜检查均证实光凝后2wkCNV形成。2.采用免疫组化和RT-PCR方法均发现CD105基因在空白对照组大鼠视网膜及脉络膜无或仅有微弱表达。光凝后1wk光凝区出现散在孤立的阳性染色,1-3wk,随着光凝后时间的延长CD 105阳性表达物逐渐增多,4wk时较3wk略有减少。CD105阳性染色IOD1wk与2wk,3wk和4wk比较差异有显著性(P＜0.01)。但2wk,3wk和4wk之间阳性染色IOD比较差异无显著性(P＞0.05)。3.基因测序证实目的序列成功插入质粒Pgenesil-1预定位置,构建了3条CD105shRNA和阳性对照Pgenesil-HK。荧光显微镜下发现质粒Pgenesil-1在治疗眼1d即可见明显的绿色荧光分布于视网膜全层包括RPE细胞层,并沿视网膜下腔成功转染了更远处的外层视网膜细胞。2～3wk荧光强度比1wk增强,荧光范围也有所扩大,可以持续表达4wk。空白对照眼无GFP表达。利用脂质体成功介导Pgenesil-1转染至视网膜全层的细胞,包括视网膜色素上皮细胞,且GFP有较高水平表达。研究发现不同的质粒转染后对CD105在CNV高峰期的表达干预效果不同。Pgenesil-eng2和Pgenesil-eng3可明显抑制CNV模型在2wk时CD105mRNA的表达,抑制效率分别为78±5%,(P＜0.01);52±3%,(P＜0.05)。其他组的CD105 RNA表达水平无明显变化。Pgenesil-eng2具有较强的基因沉默效果。4.FFA显示光凝后第14d时,对照组的荧光渗漏率为63.2%,实验组为24.6%。实验组的BN大鼠眼底渗漏点数较对照组少,渗漏强度较弱。两组间比较右显著性差异。脉络膜铺片结果显示:2wk时对照组大鼠的CNV面积为(31.22±1.46)10~3μm~2,实验组大鼠的CNV渗漏面积为(14.46±0.82)10~3μm~2,两组间比较右显著性差异。RT-PCR结果显示:光凝后7d,VEGF mRNA的表达处于高峰,2wk后表达开始下降,4wk明显减弱。实验组VEGF mRNA的表达变化规律与对照组相似,单各个时间的表达量较对照组均明显下降,其中2wk时实验组约为对照组的36.7%。对照组BN大鼠眼球在转染后第1wk CD105 mRNA表达开始升高,第2、3wk表达强度更强,到第4wk其视网膜组织中CD105mRNA表达水平开始降低。实验组CD105mRNA表达的变化趋势与对照组一致,但各个时间点上的表达水平明显降低。其中第2wk实验组约为对照组的21.68%。结论1.半导体激光光凝视网膜可以成功地建立BN大鼠CNV模型,成模时间短,成模率高,效果可靠。2.CD105基因的表达规律与CNV的形成过程一致,CD105基因是CNV的重要分子标志。3.在阳离子脂质体辅助下,Pgenesil-1质粒可以成功地将目的基因转染至大鼠视网膜各层次,且表达强度高、时间大于4wk。4.Pgenesil-eng2能明显抑制BN大鼠视网膜组织CD105 mRNA的表达。5.在CNV形成的早期沉默CD105基因的表达可以有效抑制CNV的形成。下调VEGF基因的表达可能是其作用机制之一。6.CD105基因可能在CNV形成早期发挥重要作用,它有望成为CNV治疗新的靶点。更多还原

【Abstract】 BackgroundsChoroidal neovascularization(CNV) is the main cause of severe vision loss associated age-related macular degeneration(AMD) and some other fundus diseases.Choroidal neovascularization(CNV) also named subretinal neovascularization.It derived from choroidal vessels and breaks through Bruch’s membrane then grows into the subretinal pigmental epithelial space.Because of higher permeability of the new vessels wall,it cause exudation,hemorrhage,cicatrix forming and impair central vision at last. CNV is the leading cause of blindness in individuals aged 55 years and older in developed countries.How to inhibit CNV is a problem to ophthalmologists and researchers for a long time.The exact mechanism of CNV is not clear,there is no ideal therapy to it.The outcomes of current treatments such as Laser photocoagulation, PDT(photodynamic therapy),TTT(through pupil thermotherapy), surgery excision,radiotherapy are not satisfactory for recurrence or additional damage to ocular tissues.Gene therapy provides prospect to treatment of CNV.Anti-VEGF (vascular endothelial growth factor) agents have dramatically changed the treatment and prognosis of patients with CNV.VEGF and it’s receptor expressed widely at ocular tissues and it is proved that normal level of VEGF expression is vital for photoreceptor survive and maintaining chorodial vasculature.Long term use of anti-VEGF agents may has potential risk.Seek new target and method is necessary.CD105 is a TGF-βsuperfamily receptor and expressed predominantly on vascular endothelial cells.CD 105 was regard as a specific marker of proliferating endothelium.Researches showed CD105 is essential for endothelium angiogenic activity and it may plays a critical role in CNV.CD105 can be a target for anti- angiogenesis therapy.Objective1.Estabish the CNV model in BN(brown norway) rats,then investigate the expression of CD105 and VEGF(vascular endothelial growth factor) in it.2.Construct the recombinant plasmids expressing CD105 short hairpin RNA(shRNA) by Pgenesil-1 plasmid,and to screen the highly efficient shRNA in BN rat CNV model.3.Research the effect of CD 105 gene silencing to CNV on BN rat CNV model.Methods1.Choroidal neovascularization was induced in Brown Norway rats by diode-laser photocoagulation.The formation and nature process of CNV was evaluated by fundus fluorescein angiography on 0,3,7,14, 21and 28 days after photocoagulation.Light microscopy examination and quantitive analysis of CNV was performed on 14 days.2.The expression of CD105 was evaluated by immunohistochemistry and semi-quantitive reverse transcription polymerise chain reaction in CNV model,and the relationship between CD105 and CNV was studied. Four recombinant Pgenesil-1 with U6 promoter was constructed based on the segnence information of CD105,three targeting rat CD105 and one unspecific.Metafectene containing Pgenesil-1 was injected into subretinal space in the eyes of BN rat.Localization of GFP was oberserved by fluorescence microscopy.Three recombinant plasmids being transfected into BN rat CNV model,the expression of CD105 mRNA level was determined by reverse transcription polymerise chain reaction 2 weeks later.3.The effective CD105 shRNA express plasmid was transinfectd in BN rat CNV model by subretina injection.The result of CD105 gene silencing to CNV model was evaluated dy FFA,quantitive analysis of CNV and reverse transcription-polymerise chain reaction.The expression of VEGF at mRNA level was evaluated by reverse transcription-olymerise chain reaction.Result1.Diode laser photocoagulation on BN rat’s retina can induce the CNV successfully.The CNV occurred on day 7,reached the peak on day 14 and sustained more than 28 day after photocoagulation.CNV was confirmed by light microscopy examination and quantitive analysis.2.CD105 expresses weakly at mRNA and protein level in normal BN rats retina.In CNV model,CD105 expression increased at 7day, gradually increased to 21 day,and decreased at 28 day after photocoagulation.3.One day after transfection,green fluorescence was observed in the rat’s retina including RPE cells under fluorescent microscope.The intensity became stronger on the second and third week than the first week,and sustained four weeks.The CD105 shRNAs were successfully inserted into plasmid Pgenesil-1.The recombinants were identified by sequencing.Compared with the controls,the expression of CD105 mRNA level in BN rat CNV model transfected with CD105 shRNA recombinants was markedly down-regulated 78±5%(P＜0.01) and 52±3%(P＜0.01) respectively in two groups of recombinant plasmids, whereas it had not any significant changes in another shRNA and unspecific shRNA-transfected and only with transfection reagent to BN rat CNV model.4.In CD105shRNA-treated group and CNV model control group, the incidence of fluorescein leakage was 24.6%and 63.2%at 14 day after photocoagulation.Significant difference was found between the two groups.The result of quantitive analysis of CNV showed the area of CNV leakage is(14.46±0.82)×10~3μm~2and(31.22±1.46)×10~3μm~2 in CD105 shRNA-treated group and CNV model control group,significant difference was found between the two groups.The expression of VEGF at mRNA level in CNV model control group reached the peak at 7 day after photocoagulation,then decreased gradually,at 28 day it decreased significantly.It is similar in CD105shRNA-treated group,but the expression decreased significantly at each time point.At 14 day,the expression of VEGF at mRNA level in CD105shRNA-treated group is 36.7%of it in CNV model control group.The expression of CD105 at mRNA level in CNV model control group was found at 7 day after photocoagulation,reached the peak at 14 day,and then decreased gradually,at 28 day it decreased significantly.It is similar in CD105 shRNA-treated group,but the expression decreased significantly at each time point.At 14 day,the expression of VEGF at mRNA level in CD105shRNA-treated group is 21.68%of it in CNV model control group.Conclusion1.To estabish the CNV animal model by diode laser photocoagulation on BN rat’s retina is feasible.2.The expression pattern of CD105 in CNV is consistent to the formation of CNV.CD105 is an important marker of CNV.3.Metafectene can transfect Pgenesil-1 into the retina of the BN rat effectively,and their expression can maintain 4 weeks after transfection.4.Three recombinants expressing CD105 shRNA are successfully constructed.Pgenesil-ENG2 could silence CD105 expression in vivo potently.5.CD105 gene silencing at early stage could inhibit the formation of CNV in vivo.Down-regulation of VEGF expression may be one of it’s mechanisms.6.CD105 plays an important role in early stage of CNV,it is hopeful to be a target of CNV therapy.更多还原