早产儿视网膜病图文诊断及与血管新生细胞因子关系研究

发布时间：2018-06-30 20:53

本文选题：早产儿视网膜病变 + 图文诊断系统　；参考：《南方医科大学》2011年博士论文

【摘要】：研究背景： ROP是目前国内外儿科和眼科研究的热点之一。1984年国际ROP会议制定了ROP的分期标准,2003年早产儿视网膜病早期协作治疗组进一步完善ROP的分期标准。近几年来,国外研究学者已就ROP的高危因素、病因与治疗等方面开展了相关的基础试验与临床实践的研究,并取得可喜的研究成果。然而,ROP检查手段的研究仍然不十分成熟。目前不仅缺乏统一、高效、安全、可普及的检查工具,从临床角度,对ROP的发病规律、眼底视网膜的形态学变化等研究也无法深入开展。临床研究发现,大约85%未经治疗的ROP自行消退并发育成完全血管化的视网膜,标准的治疗可以预防剩下5%的儿童视力丧失,大约6%到10%的ROP患者最终出现严重的视力丧失。到底在什么样情况下ROP会自行消退,什么样的情况它会发展乃至出现视网膜脱离最终导致失明?对这些ROP发病规律的研究,将使我们对ROP的发展趋势进一步了解,从而准确地对ROP病人选择性进行早期干预性的治疗,更加有效地把握治疗的最佳时机,既可以防治过度治疗,又可以避免因把握不当而导致病程恶化乃至无法控制的局面出现。因此,如何确定早期ROP的病变特点,从而客观、准确地对早期ROP做出诊断,并进行早期追踪和有效的干预性治疗已成为了国内外学者研究的方向。目前,国内外眼科医生主要使用双目间接检眼镜进行ROP的筛查,它具有立体感、照明强度强、视野宽、成像清晰等特点,被公认为是ROP筛查的“金标准”和首选检查工具。但是,由于双目间接检眼镜下眼底图像的采集与输出困难,眼底检查结果无法记录,一直以来采用画图描述的方法保存资料,缺乏客观性结果。为了解决这个问题,虽然国外学者进行了多方面的尝试,但是始终没有获得最佳办法。在广州市、广东省科研机构的支持下,我们采用双目间接检眼镜成像与眼底图像输出技术为检查手段,建立了计算机辅助的双目间接检验镜ROP检查体系。成熟的视网膜出现新生血管常导致视力下降,甚至视力丧失。视网膜新生血管(retinal neovascularization, RNV)多见于糖尿病性视网膜病变、早产儿视网膜病变、缺血性视网膜静脉栓塞,病因复杂,发病机制尚未明确。随着新生儿重症监护的建立及医疗护理技术的进步,早产低出生体重儿的成活率大大提高,早产儿视网膜病(ROP)的发生率也呈上升趋势,在出生体重小于1000g的超低出生体重儿中发病率高达80%以上,占儿童视力损害的比例逐年增加,已引起人们的重视。但是,该病的发病机制仍未明确,目前研究表明新生血管的形成可能起主导作用。抑制新生血管形成,可达到防治ROP的目的。因而越来越多的学者从该病的发病机制着手,希望通过抑制新生血管的生长阻断该病的进展,这在动物实验中被证实是可行的。但是,新生血管抑制剂同时也可抑制正常的血管发育,阻碍眼球正常的解剖和功能发育,这也是尚未解决的问题。目前大多数对ROP的基础研究多停留在单基因以及基因水平。但是,机体生理机能的真正执行者是蛋白质,有研究表明细胞内mRNA水平和蛋白质的表达峰度并不完全一致,因此从蛋白质水平对疾病进行研究显得特别重要。然而,传统的对单个蛋白质或某几种蛋白质进行研究的方式难以系统透彻地整体阐释ROP形成及病情发展的基本机制。要对其复杂活动有全面深入的认识,必然要在整体、动态、网络的水平上对蛋白质进行研究。蛋白质组是一个基因组所能编码的所有蛋白的组合。蛋白质组学是对一个基因组、一种生物、一种细胞、组织所表达的全部蛋白质及其相互作用的研究,提供的资料更真实地反映基因、组织、器官的实际功能状态。细胞因子抗体芯片技术是在蛋白质组概念上发展起来的高通量实验检测方法。在疾病的诊断、病程观察、疗效判断及细胞因子治疗监测方面有重要意义。研究细胞因子有助于阐明分子水平的功能调节机制,特别是利用细胞因子治疗肿瘤、感染、造血功能障碍以及自身免疫病等已收到初步疗效,具有非常广阔的应用前景。研究目的 1.本研究拟在前期工作基础上,建立ROP图文报告系统,并通过视网膜图像的分析,了解早产儿视网膜解剖结构的形态学特点,探索早产儿视网膜发育的特点,掌握ROP视网膜的病变特点,进一步阐明ROP的早期发病特点及规律,寻求ROP的临床治疗干预时机。同时通过对该图文报告系统的统计与研究分析,可以了解广州地区早产儿视网膜病变的发生情况,对发现的早产儿视网膜病变进行早期治疗,摸索出对该病预防、临床治疗的经验。 2.本研究拟将利用新生血管相关细胞因子抗体芯片技术检测正常早产儿、ROP患儿术前及术后血清中55种相关细胞因子的表达,有助于从全局及整体角度考虑新生血管相关细胞因子对ROP形成及发展过程的影响。研究方法 1.利用计算机辅助双目间接检眼镜录制早产儿视网膜病变筛查视频； 2.构建图文诊断系统,在录制视频后能及时截图,打印报告 3.从获得的大量图文报告中分析早产儿视网膜解剖学及形态学特点； 4.随机抽取正常早产儿、ROP术前及术后一周患儿血清,取血后血液在冰上静置1h,0℃离心5000r/min 10 min。分离血清,并冻存于-80℃以备后用。 5. RayBioTM人血管新生细胞因子抗体芯片检测血清中55种可能相关的细胞因子表达；结果： 1、计算机辅助ROP图文报告系统的临床应用 1)、检查前的准备工作：早产儿需要使用五次复方托吡卡胺滴眼液,才可以达到理想状态。进行ROP筛查工作需要医院眼科、新生儿科及妇幼保健新生儿科合作进行。 2)、计算机辅助ROP图文报告系统结果分析我们的研究结果表明：“计算机辅助双目间接检眼镜成像技术”能够有把握地让我们回答早产儿视网膜病变的重要指标存在与否：周边部视网膜是否存在无血管区；血管区与无血管区之间是否存在明显的分界线；分界线是否有隆起；隆起处血管是否有增生和血管纤维增殖。因此,我们能够有把握地诊断该早产儿是否ROP患者。 3)、同时,与其他疾病鉴别(牵牛花综合征、视网膜母细胞瘤、早产儿眼底出血、正常周边灰白色视网膜)中,该系统可以提供永久的确凿的证据支持诊断。 4)、通过研究双目间接检眼镜成像技术下获得的眼底检查录像、典型眼底病变图像回顾,可以在ROP工作开展中对医务人员进行教学培训。 2、正常早产儿与ROP患儿血清中血管相关细胞因子检测发现有14组蛋白表达差异显著,而正常早产儿与ROP术后一周时间的血清学检查发现了14种蛋白表达差异。其中： 1)：相对于正常早产儿及术前,激光光凝术后1周ROP患儿FGF basic、IGFBP-3表达水平从异常增高到正常表达,证明了这两个细胞因子在ROP病理生理过程中具有重要作用：视网膜细胞的凋亡与血清内大量表达的IGFBP-3可能相关,bFGF的存在可能对于视网膜神经节细胞进一步损伤有预防作用?这提示我们在ROP中要考虑到神经发育与血管新生的相互影响,考虑神经系统与ROP形成的相关性。 2)：一部分炎性反应相关因子Amphiregulin、GMCSF, MMP-8表达水平由异常降低到正常,及IL-1β由正常表达到轻度升高,说明ROP患儿术后产生了炎性反应,但是严重程度不足以对患儿产生不利影响。 3):PIGF可以减轻病理性血管新生而并不影响正常生理性血管新生、Vaschibin唯一的以负反馈为作用机制调节血管新生的蛋白。PIGF及Vaschibin由术前的低表达变为术后正常表达。提示PIGF、Vasohibin有可能成为治疗ROP的有效手段。结论： 1.整理出一套行之有效的ROP的图文诊断体系； 2.睫状后长神经是ROP筛查中要观察的重要解剖部位； 3.应用新生血管形成相关细胞因子芯片技术获得正常早产儿、ROP患儿术前及术后一周血清标志蛋白表达的实验方法稳定可行； 4.提出在ROP中要考虑到神经发育与血管新生的相互影响,考虑神经系统与ROP形成的相关性； 5.从炎性细胞因子表达角度证明视网膜激光光凝术的有效性及安全性； 6. PIGF、Vasohibin有可能成为治疗ROP的有效手段。 7.ROP的病理生理过程是一个复杂的血管新生细胞因子间相互调节过程。创新 1.构建了行之有效的ROP图文诊断报告系统； 2.首次提出神经系统可能通过分泌神经肽刺激血管新生细胞因子的激活参与机体的新生血管形成,影响ROP发生发展。提出神经系统参与了ROP生理病理过程的观点。 3.提出PIGF、Vasohibin有可能成为治疗ROP的新靶标。
[Abstract]:Research background:
ROP is one of the hotspots in the research of Pediatrics and ophthalmology at home and abroad at present.1984 international ROP meeting set up the staging standard of ROP. In 2003, the early cooperative treatment group of retinopathy of preterm infants further perfected the staging standard of ROP. In recent years, foreign researchers have carried out the basic test of the high risk factors, etiology and treatment of ROP. The research of clinical practice and clinical practice has been studied. However, the research of ROP examination is still not very mature. At present, there is not only a lack of unified, efficient, safe and universal inspection tools. From the clinical point of view, the research on the regularity of ROP and the morphological changes of the retina of the fundus can not be carried out in depth.
Clinical studies have found that about 85% of the untreated ROP subsided and developed into a fully vascularized retina. Standard treatment can prevent the loss of vision in 5% of the children, and about 6% to 10% of ROP patients eventually have severe loss of vision. In what case, ROP will fade away, and what kind of situation it will develop or even be. The occurrence of retinal detachment eventually leads to blindness? The study of these ROP pathogenesis will enable us to further understand the trend of the development of ROP, and thus accurately treat ROP patients with early intervention, and more effectively grasp the best time for treatment, not only to prevent over treatment, but also to avoid the improper control of the treatment. Therefore, how to determine the characteristics of early ROP disease, so as to make an objective and accurate diagnosis of early ROP, and to carry out early tracking and effective intervention therapy have become the research direction of domestic and foreign scholars. At present, ophthalmologists at home and abroad mainly use binocular indirect ophthalmoscope for R OP screening, which has the characteristics of stereoscopic sense, strong lighting intensity, wide field of vision, clear imaging and so on, is recognized as the "gold standard" and the preferred inspection tool for ROP screening. However, because of the difficulty in collecting and exporting of the ocular fundus image under binocular indirect ophthalmoscope, the results of fundus inspection cannot be recorded, and the method of drawing description has been used to save the data. In order to solve this problem, in order to solve this problem, although foreign scholars have tried many aspects, the best method has never been obtained. Under the support of scientific research institutions in Guangzhou, Guangdong Province, we used binocular indirect ophthalmoscope imaging and fundus image output technology as inspection means to establish a computer aided binocular indirect method. The inspection mirror ROP inspection system.
The emergence of neovascularization in mature retina often leads to visual loss and even loss of vision. Retinal neovascularization (retinal neovascularization, RNV) is often seen in diabetic retinopathy, retinopathy of prematurity, ischemic retinal vein embolism, complicated pathogeny, and the pathogenesis is not clear. With the establishment of neonatal intensive care system As well as the progress of medical and nursing technology, the survival rate of premature birth weight infants is greatly increased. The incidence of retinopathy of preterm infants (ROP) is also rising. The incidence of the infant birth weight is up to 80% in the ultra low birth weight infants less than 1000g, which is increasing year by year in children's visual impairment. However, the disease has been paid more attention. The pathogenesis is still not clear. The current research shows that the formation of new blood vessels may play a leading role. Inhibition of the formation of new blood vessels can be used to prevent and control ROP. Therefore, more and more scholars start from the pathogenesis of the disease and hope to block the progress of the disease by inhibiting the growth of the neovascularization, which is proved to be feasible in animal experiments. However, neovascularization inhibitors can also inhibit normal vascular development and prevent normal anatomical and functional development of the eyeball, which is also an unsolved problem.
Most of the basic studies on ROP remain at the single gene and gene level. However, the real executors of the body's physiological functions are proteins. Studies have shown that the intracellular mRNA level and protein expression kurtosis are not completely consistent. Therefore, it is particularly important to study the disease from protein level. It is difficult to systematically and thoroughly interpret the basic mechanism of ROP formation and disease development in a systematic and comprehensive way. It is necessary to understand the complex activities of a protein in an overall, dynamic, and network level. The protein group is all the proteins that a genome can encode. Proteomics is a study of all proteins and their interactions expressed in a genome, a species, a cell, a tissue, and the information provided more truly to reflect the actual functional state of genes, tissues and organs. Cytokine antibody chip technology is a high throughput test developed in the concept of proteome. It is of great significance in the diagnosis of disease, the observation of the course of the disease, the evaluation of the curative effect and the monitoring of the cytokine treatment. It is helpful to elucidate the functional regulation mechanism of the molecular level, especially the treatment of tumor, infection, hematopoiesis and self - free disease by the use of cytokine. The prospect of application.
research objective
1. on the basis of the earlier work, this study is to establish the ROP graphic and text reporting system, and through the analysis of retinal images to understand the morphological features of the retina anatomy of preterm infants, explore the characteristics of retina development in preterm infants, grasp the characteristics of ROP retinopathy, further elucidate the characteristics and laws of the early onset of ROP, and seek the clinical practice of ROP. At the same time, we can understand the occurrence of retinopathy in preterm infants in Guangzhou, and make early treatment for the retinopathy of preterm infants, and find out the experience of prevention and treatment of the disease.
2. the purpose of this study is to detect normal preterm infants by using new blood vessel related cytokine antibody chip technology. The expression of 55 related cytokines in serum of ROP children before and after operation is helpful to consider the influence of neovascular related cytokines on the formation and development of ROP from the global and overall perspective.
research method
1. using computer-assisted binocular indirect ophthalmoscope to record video screening for retinopathy of prematurity.
2. build a graphic diagnostic system. After recording the video, it can capture and print the report in time.
3. to analyze the anatomical and morphological characteristics of retina in premature infants from a large number of graphic reports.
4. the normal preterm infants were randomly selected. Before ROP and one week after the operation, the blood serum was collected on the ice and 1H was placed on the ice. The serum was centrifuged and 5000r/min 10 min. was centrifuged at 0 centigrade. The serum was frozen at -80.
5. RayBioTM human angiogenesis factor antibody chip was used to detect 55 possible cytokines expression in serum.
Result:
1, the clinical application of computer-aided ROP graphic reporting system.
1) pre examination: preterm infants need to use five Compound Tropicamide Eye Drops to achieve their ideal status. ROP screening needs to be conducted in the Department of Ophthalmology, newborn pediatrics and maternity and child care.
2) computer aided ROP graphic report system results analysis
Our results show that "computer assisted binocular ophthalmoscope" can give us a good grasp of the existence of an important indicator of retinopathy in preterm infants: whether there is a vascular area in the peripheral retina; whether there is a clear dividing line between the vascular area and the vascular area; whether there is a boundary line or not. There is a proliferation of blood vessels and proliferation of blood vessels in the uplift. Therefore, we can be able to diagnose whether the premature infant is ROP.
3), at the same time, the system can provide permanent and conclusive evidence to support the diagnosis of other diseases (morning glory syndrome, retinoblastoma, preterm fundus hemorrhage, and normal peripheral white retina).
4), through the study of the fundus examination video obtained under the binocular indirect ophthalmoscope imaging technique, the typical fundus lesion image review can be used in the training of medical staff in the work of ROP.
2, the serum levels of vascular related cytokines in normal preterm infants and ROP children showed that there were significant differences in the expression of 14 groups of proteins, and 14 kinds of protein expression differences were found in normal preterm infants and one week after ROP.
1): compared with normal preterm infants and preoperative, FGF basic in children with ROP, 1 weeks after laser photocoagulation, the expression level of IGFBP-3 from abnormal to normal expression, proves that these two cytokines play an important role in the pathophysiological process of ROP: the apoptosis of retina cells may be related to the large amount of IGFBP-3 in the serum, and the existence of bFGF may be possible. There is a preventive effect on further damage to retinal ganglion cells, which suggests that we should consider the interaction between neurodevelopment and angiogenesis in ROP, and consider the correlation between the nervous system and the formation of ROP.
2): a part of the inflammatory response related factors Amphiregulin, GMCSF, MMP-8 expression level from abnormal to normal, and IL-1 beta from normal to mild elevation, indicating that ROP children have an inflammatory response after operation, but the severity is not enough to have adverse effects on the children.
3): PIGF can reduce the pathological angiogenesis without affecting the normal physiological angiogenesis. The only negative feedback as the mechanism to regulate the angiogenesis of the protein.PIGF and Vaschibin from the preoperative low expression to normal expression after the operation, suggesting that PIGF, Vasohibin may be an effective means for the treatment of ROP.
Conclusion:
1. sort out a set of effective ROP diagnostic system.
2. the long posterior ciliary nerve is an important anatomical site to be observed in ROP screening.
3. the normal preterm infants were obtained by the technique of neovascularization related cytokine microarray. The experimental methods for the expression of serum markers before and after the operation of ROP were stable and feasible.
4. it is proposed that the interaction between neural development and angiogenesis should be considered in ROP, and the correlation between nervous system and ROP formation should be considered.
5. to demonstrate the effectiveness and safety of retinal laser photocoagulation from the perspective of inflammatory cytokines expression.
6. PIGF, Vasohibin may become an effective way to treat ROP.
The pathophysiology of 7.ROP is a complex process of regulation of angiogenesis factors.
innovate
1. an effective ROP graphic diagnostic report system has been constructed.
2. it is the first time that the nervous system may be stimulated by the activation of neuropeptides to stimulate neovascularization of the body and affect the development of ROP. It is suggested that the nervous system is involved in the physiological and pathological process of ROP.
3. it is suggested that PIGF and Vasohibin may become new targets for the treatment of ROP.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1;R722.6

【参考文献】