视网膜母细胞瘤中HIF-1α的区域性表达及其与增殖、凋亡和新生血管生成的关系

发布时间：2018-05-29 06:32

本文选题：视网膜母细胞瘤 + 免疫组织化学　；参考：《复旦大学》2011年硕士论文

【摘要】：背景：视网膜母细胞瘤(retinoblastoma, Rb)是婴幼儿中最常见的眼内恶性肿瘤,虽然目前化学缩瘤辅助局部治疗(经瞳孔温热疗法TTT、冷冻治疗,结膜下注射卡铂SCC,表层巩膜敷贴放疗等)的综合治疗方法不仅可以挽救患儿生命,甚至能保留一部分有用视力。但是,仍然存在部分患者接受治疗后出现复发,甚至转移的现象。一方面,化疗耐药现象仍是困扰眼科医生的棘手课题：研究提示Rb内部的缺氧现象可能是Rb化疗耐药的原因之一,这些缺氧区中的肿瘤细胞由于主要通过糖酵解途径获取能量,使它们对普通的化疗药物不敏感,成为化疗耐药和复发的潜在原因。另一方面,由于Rb各种治疗方法均具有自身的特性,使瘤体不同部位在接受治疗的方式和程度上有所不同,故可能出现容易被忽略的治疗盲点,最终导致复发和耐药现象。因此,通过对肿瘤内不同区域缺氧现象研究,不仅有利于研究Rb耐药的可能机制,也有助于揭示综合治疗后局部复发的原因,进而探索一种综合治疗的最佳模式,达到优化互补、取长补短的目的,并最终提高疗效,改善患儿预后。第一部分HIF-1α在视网膜母细胞瘤中的区域性表达及其与VEGF、Bax、Ki-67的关系目的通过研究缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)在Rb的表达及分布,以及其与VEGF、Ki-67、Bax的关系,从而探讨Rb中的缺氧现象及特点,并尝试寻找局部治疗后仍导致复发的潜在原因,为最终指导临床治疗提供理论依据。方法采用免疫组织化学EnVision法检测HIF-1α、VEGF、Bax、Ki-67在39例(39只眼)Rb石蜡标本中的表达。按照肿瘤区域分为表面区、中央区、基地部、脉络膜区及子瘤五个区域,分析以上指标的表达及分布差异。结果39例Rb标本中,HIF-1α阳性者29例,占74.4%,其中,按整体表达强度计算,表达(-)有10例(25.6%),(+)有17例(43.6%),(++)有12例(30.8%)；按区域计算,表面区、中央区、基地部、脉络膜区及子瘤区染色阳性者分别占71.0%,36.8%,84.2%,45.4%,82.1%,其分布具有明显差异(P0.001),在表面区、基底部及子瘤区HIF-1α表达阳性率较高。VEGF、Bax和Ki-67表达阳性者分别占53.8%,66.7%,59.0%,其中,VEGF和Bax在不同区域内表达有差异(均为P0.001),Ki-67的阳性率无明显分布差异(P=0.976)。整体上,HTF-1α的表达与VEGF和Bax的表达呈正相关(rs=0.51,P=0.001和rs=0.32,P=0.046),而与Ki-67的表达无明显相关(P=0.441)。在不同区域,VEGF和Bax的表达均与HIF-1α有较好的一致性,而Ki-67与HIF-1α的表达在不同区域分布无明显一致性。结论缺氧现象多分布在视网膜母细胞瘤瘤体的边缘地带,这可能与肿瘤形成过程中边缘相对缺乏血管分布有关,这提示化疗药物应当有足够的穿透力或者更具靶向性才能彻底杀灭这些细胞：另一方面,缺氧可能通过诱导凋亡增加,新生血管生成增加,进一步耐受缺氧环境,达到逃脱化疗药物杀伤的目的,这将对理解并解决视网膜母细胞瘤的耐药现象有重要的指导意义。第二部分刀切实验排除边缘效应目的通过刀切实验,排除边缘效应及其他人为因素造成的结果偏差。方法根据第一部分结果,选取HIF-1α染色阳性的标本5例(表面区染色较中央区染色深)各三张,其中一张用尖手术刀片刮除肿瘤游离缘约0.5mm区域,称作刀切组；一张不做刀切处理,称未刀切实验组；余下一张作空白对照。在相同条件下重复HIF-1α染色,观察结果结果通过刀切实验,刀切组新的表面区与未处理组的肿瘤表面区相比,HIF-1α的染色在后者明显比前者更深,且刀切组新的表面区与二者肿瘤中央区的HIF-1α染色结果基本一致。结论通过刀切实验、操作中铺平抗体液、防止干片、随机阅片等操作,避免了实验过程中边缘效应及其他人为因素造成的结果偏差,因此该课题结果的可靠性得到验证：即在Rb中缺氧现象的分布特征不是实验和人为因素造成的,而是由瘤体自身特点决定的。
[Abstract]:Background: retinoblastoma (Rb) is the most common intraocular malignant tumor in infants and children. Although the combined therapy of chemical contraction assisted local treatment (TTT, cryotherapy, subconjunctival injection of carboplatin SCC, surface scleral compress and radiotherapy) can not only save the life of the children, but can even preserve it. There are some useful eyesight. However, there is still a recurrence and even metastasis of some patients after treatment. On the one hand, the phenomenon of chemotherapeutic resistance is still a difficult problem for ophthalmologists: the study suggests that the hypoxia in Rb may be one of the reasons for the resistance of Rb to chemotherapy, and the tumor cells in these anoxic areas are mainly passed through Glycolysis, which makes them insensitive to common chemotherapeutic drugs, is a potential cause of chemotherapeutic resistance and relapse. On the other hand, because Rb has its own characteristics, different parts of the tumor are different in the manner and degree of treatment, so it may be easily ignored in the treatment of blind spots. Therefore, the study of hypoxia in different regions of the tumor is not only conducive to the study of the possible mechanism of Rb resistance, but also helps to reveal the causes of local recurrence after comprehensive treatment, and then explore the best mode of comprehensive treatment to optimize the complementarity, make up for short, and ultimately improve the curative effect. The prognosis of good children.
The first part is the regional expression of HIF-1 alpha in retinoblastoma and its relationship with VEGF, Bax and Ki-67.
Objective to study the expression and distribution of hypoxia inducible factor -1 alpha (hypoxia inducible factor-1 a (HIF-1 a), HIF-1 alpha) in Rb, as well as its relationship with VEGF, Ki-67, Bax, and to explore the phenomenon and characteristics of hypoxia in Rb, and try to find the potential cause of recurrence after local treatment, and provide a theoretical basis for guiding clinical treatment.
Methods immunohistochemical EnVision method was used to detect the expression of HIF-1 alpha, VEGF, Bax, Ki-67 in 39 cases (39 eyes) of Rb paraffin specimens. According to the tumor area, the expression and distribution of the above indexes were analyzed in five regions, central region, base, choroidal area and subtumor.
Results of the 39 Rb specimens, 29 cases were positive for HIF-1 alpha, accounting for 74.4%. Among them, 10 cases (25.6%) were expressed (25.6%), 17 (43.6%) and 12 (30.8%) were expressed (+) in the total expression intensity (+ +). According to the region, the positive staining in the surface area, central area, base, choroidal area and subtumor area accounted for 71%, 36.8%, 84.2%, 45.4%, 82.1%, respectively. The positive rate of HIF-1 alpha expression in the surface area, the basal part and the subtumor region was higher than that of.VEGF, and the positive rates of Bax and Ki-67 were 53.8%, 66.7%, 59% respectively in the surface area, and the positive rates of VEGF and Bax were different in different regions (P0.001), and the positive rate of Ki-67 was not significantly different (P=0.976).
On the whole, the expression of HTF-1 alpha was positively correlated with the expression of VEGF and Bax (rs=0.51, P=0.001 and rs=0.32, P=0.046), but there was no significant correlation with Ki-67 expression (P=0.441). In different regions, the expression of VEGF and Bax was in good agreement with HIF-1 alpha, but there was no obvious consistency in the distribution of the tables in different regions.
Conclusions hypoxia is mostly distributed in the marginal zone of the retinoblastoma, which may be related to the relative lack of blood vessel distribution during the formation of the tumor. This suggests that the chemotherapeutic drugs should have sufficient penetration or more targeted to kill these cells. On the other hand, hypoxia may increase apoptosis by inducing apoptosis. It is important to understand and solve the drug resistance of retinoblastoma by increasing the angiogenesis and further tolerance to the anoxic environment.
The second part cutting experiment excludes the edge effect
Objective to eliminate the margin of effect and other factors caused by other factors through knife cut experiments.
Methods according to the results of the first part, 5 specimens of positive HIF-1 alpha staining were selected (the surface area staining was compared with the deep staining of the central region) in three pieces, one of which was used to scrape the tumor free margin about 0.5mm area, called the knife cutting group, and the other was called the non knife test group, and the remaining one was a blank control. Under the same condition, the rest was weighed under the same condition. Complex HIF-1 alpha staining, observation results
Results compared with the tumor surface area in the untreated group, the new surface area of the knife cut group was much deeper than the former in the tumor surface area of the untreated group. The new surface area of the knife cut group was basically the same as the results of the HIF-1 alpha staining in the two central region of the tumor.
Conclusion through the knife cutting experiment, the antibody liquid was paved in the operation to prevent the dry slice and the random reading of the film. The results were proved to be reliable, that is, the distribution characteristics of the anoxia phenomenon in Rb were not caused by the experiment and human factors, but by the tumor. The character of the body is determined by its own characteristics.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.7

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