鼻咽纤维血管瘤血管内皮细胞比正常血管内皮细胞具有更强的迁移和侵袭能力

发布时间：2018-11-14 19:57

【摘要】：鼻咽纤维血管瘤,又称青少年鼻咽纤维血管瘤(juvenile nasopharyngeal angiofibroma, JNA)是鼻咽部良性肿瘤中最常见的一种,约占总头颈部肿瘤的0.5%,主要发生于14-25岁的青少年男性。尽管JNA组织学上呈良性表型,但其没有包膜,而且具有局部侵袭和破坏性恶性表型,可以累及鼻腔、鼻咽部、鼻窦、翼腭窝、颞下窝、翼突以及前颅底组织等。 JNA没有特异性的药物治疗手段,放射疗法的使用尚存在争议,外科手术切除仍然是目前最常用的治疗方法。近年来,随着耳鼻喉科临床治疗手段的长足进步,特别是术前栓塞和鼻内镜技术的使用,使得大部分患者获得了较好的预后,但是JNA术后高复发率(可高达50%)仍是一个极大的挑战。目前关于JNA术后高复发的原因和相关的临床预测指标研究甚少,可能与JNA向周围颅底骨质侵袭,手术难以彻底切除有关。如能阐明引起JNA局部侵袭及术后复发的因素及其分子调节机制,将进一步改善JNA患者的预后。肿瘤血管生成与包括JNA在内的多种肿瘤生长和局部侵袭密切相关。但是目前基于JNA血管生成及其分子机制研究不多,特别是针对内皮细胞本身的功能研究更少,关键在于缺乏直接来源于肿瘤标本并可用于体外培养的血管内皮工具细胞。本课题首先分析血管内皮标记物CD105在JNA组织芯片(tissue microarray, TMA)微血管内皮细胞中的表达及其临床意义,进一步通过免疫磁珠分选(magnetic activated cell sorting, MACS)人JNA的血管内皮细胞进行传代培养,检测肿瘤血管内皮细胞及正常血管内皮细胞间的功能差异并探讨其差异的分子机制,以期阐明导致JNA高复发的原因及可能机制,并以此寻找新的复发干预途径,进一步提高疗效。第一部分血管内皮标记物CD105在鼻咽纤维血管瘤组织芯片微血管中的表达及其临床意义目的：通过构建组织芯片并运用免疫组织化学染色的方法明确CD105蛋白在JNA微血管中的表达,并分析其与JNA临床病理特征以及复发之间的关系。方法：通过免疫组织化学染色方法在一套独立的组织芯片(包括70例JNA患者的肿瘤组织)中检测了CD105的表达情况,进行微血管计数,并分析其与年龄、JNA手术史、手术方式、肿瘤分期、术中出血等临床病理特征的关系,同时进一步分析微血管密度(microvessel density, MVD)与JAN患者临床病理参数及至复发时间(time to recurrence, TTR)的相关性。结果：免疫组织化学检测结果显示,CD105仅表达于血管内皮细胞中,在间质中未见明显表达。卡方检验分析发现微血管数目高低与肿瘤复发显著相关(P=0.013)。Kaplan-Meier生存分析和log-rank检验显示,低微血管密度患者的TTR显著高于高微血管密度患者(P=0.009)。多因素回归模型结果也提示,微血管密度是一个决定JNA预后的独立因素(P=0.01)。结论：CD105染色的肿瘤微血管密度可以预测JNA患者术后复发,提示血管生成在JNA发生发展进程中可能起重要作用,有望成为JNA治疗靶点和预后预测指标。同时,为将CD105做为分离JNA血管内皮细胞的表面标记提供了临床依据。第二部分人鼻咽纤维血管瘤血管内皮细胞分选、鉴定与培养目的：分选、鉴定及传代培养人鼻咽纤维血管瘤血管内皮细胞。方法：采用抗CD105抗体交联免疫磁珠,通过免疫磁珠分选法,对JNA组织进行血管内皮细胞分选；采用流式细胞术检测阳性分选细胞的纯度。通过细胞免疫化学法检测von Willebrand factor (vWF,又称Ⅷ因子)的表达,并通过乙酰化低密度脂蛋白摄取实验及小管生成实验验证阳性分选细胞是否具有血管内皮细胞的功能。结果：流式检测阳性分选细胞的CD105表达高达99%以上；细胞免疫化学检测显示阳性分选细胞中Ⅷ因子表达为阳性,超过95%的阳性分选细胞乙酰化低密度脂蛋白摄取实验阳性；分选细胞在基质胶中可形成毛细管结构。同时可将阳性分选细胞进行传代培养至10代以上,其内皮细胞特性仍存在。结论：通过免疫磁珠分选(MACS)法,我们成功分选获得高纯度的CD105阳性鼻咽纤维血管瘤血管内皮细胞(CD105+ juvenile nasopharyngeal angiofibroma-derived endothelial cell, CD105+ JEC),并能够成功传代培养,为后续研究提供了细胞平台。第三部分鼻咽纤维血管瘤血管内皮细胞与正常血管内皮细胞的功能比较目的：比较JNA组织中分离的CD105阳性内皮细胞与人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)的功能差异。方法：采用CyQUANT法增殖实验、划痕实验及Transwell侵袭实验对比观察两种细胞体外的增殖、侵袭和迁移能力的异同；通过免疫荧光观察细胞骨架蛋白肌动蛋白纤维(F-actin)的表达和细胞内定位分布的差异,运用Western blot检测基质金属蛋白酶-2 (matrix metalloproteinase-2, MMP-2)及血管内皮细胞生长因子受体(Vascular endothelial growth factor receptor, VEGFR)相关信号通路蛋白表达水平异同。结果：增殖实验发现CD105+JEC的增殖能力低于HUVEC；而划痕实验、侵袭实验及F-actin荧光染色显示CD105+JEC的迁移和侵袭能力高于HUVEC; Western blot结果显示VEGFR1、p-MAPK、MMP-2在CD105+JNA血管内皮细胞中的表达高于HUVEC,而VEGFR2、p-ERK的表达则相反。结论：CD105+JEC比HUVEC具有更强的体外迁移、侵袭能力,其机制可能与VEGFR相关信号通路的差异性活化有关。
[Abstract]:Hemangioma of the nasopharynx, also known as the juvenile nasopharyngeal fibroma (JNA), is the most common one of the benign tumors of the nasopharynx, accounting for 0.5% of the total head and neck tumors, which mainly occurs in the 14-25-year-old male. Although the JNA is of a benign phenotype, it has no envelope and has a local attack and a destructive and malignant phenotype, which may involve the nasal cavity, the nasopharynx, the nose, the wing, the inferior fossa, the wing, and the anterior skull base tissue, and the like. JNA has no specific drug treatment means, the use of radiation therapy is still in dispute, and surgical resection is still the most commonly used treatment at present Methods: In recent years, with the rapid progress of the clinical treatment of otorhinolaryngology, especially the use of pre-operative embolization and nasal endoscopic technique, most patients have a good prognosis, but the high recurrence rate (up to 50%) after the operation of JNA is still a great Challenges. There are few studies on the causes of high recurrence and associated clinical predictors of postoperative high recurrence of JNA, which may be associated with the invasion of the surrounding base of the skull by the JNA, and the operation is difficult to complete with a complete resection. The factors of local invasion and post-operative recurrence of JNA and their molecular regulatory mechanisms will be further improved, which will further improve the JNA patients. Prognosis. Tumor angiogenesis and multiple tumor growth and local invasion, including JNA It is closely related to the attack, but at present, the research on the angiogenesis and molecular mechanism of the JNA is not much, especially for the function of the endothelial cell itself, the key point is the lack of a blood vessel which is directly derived from the tumor specimen and can be used for in-vitro culture. The expression of the vascular endothelial marker CD105 in the vascular endothelial cells of the tissue of the JNA tissue and its clinical significance were first analyzed. The vascular endothelial cells of the human JNA of the human JNA were further classified by the magnetic cell sorting (MACS). The functional difference between the endothelial cells of the tumor and the normal vascular endothelial cells was detected and the molecular mechanism of the difference was discussed, with a view to elucidating the causes and possible mechanisms of the high recurrence of JNA and finding a new way of recurrence intervention. step-up therapy Effect: The first part of the vascular endothelial marker CD105 is in the nasopharyngeal fibroma tissue chip. Expression of the blood vessel and its clinical significance: by constructing the tissue chip and using the immunohistochemical staining method, it is clear that the CD10 The expression of 5-protein in JNA microvessel and its interaction with JNA Methods: The expression of CD105 was detected in a set of independent tissue chips (including 70 JNA patients) by immunohistochemical staining. The microvessel counts were performed and their relationship with age was analyzed. The relationship between microvessel density (MVD) and clinical pathological parameters and time to re-recurrence in JAN patients was further analyzed. Correlation of currence, TTR). Results: The results of the immunohistochemistry showed that the CD105 only It was found that the number of microvessels was significantly correlated with the recurrence of the tumor (P = 0.013) in the vascular endothelial cells (P = 0.013). The Kaplan-Meier survival analysis and the log-rank test showed that the TTR of low microvessel density patients was significant. In patients with higher microvessel density (P = 0. 009), the multi-factor regression model results also suggest that the microvessel density is one The independent factor of the prognosis of JNA was determined (P = 0.01). Conclusion: The tumor microvessel density of CD105 can predict the postoperative recurrence of JNA, and it is suggested that angiogenesis can play a role in the development of JNA. It is expected to be a target for the treatment of JNA and to predict the prognosis of the prognosis. Separation of JN A clinical basis is provided for surface labeling of vascular endothelial cells. The classification, identification and identification of vascular endothelial cells in the second part of human nasopharyngeal fibroma in that method, the anti-CD105 antibody cross-linked immunomagnetic bead is adopted, The expression of von Willebrand factor (vWF, also called factor VIII) was detected by flow cytometry, and the low density of von Willebrand factor (vWF, also called factor VIII) was detected by cell immunochemical method. The results showed that the expression of CD105 in the flow-type test-positive sorted cells was higher than 99%, and the immunochemical test of the cells showed that the positive separation was fine. The expression of the factor VIII in the cell is positive, and more than 95% of the male is positive. Positive for low-density lipoprotein uptake in sex-sorted cells, and the ability of sorting cells to form a hair in a matrix gel It is concluded that by the method of immunomagnetic bead sorting (MACS), we have successfully sorted the vascular endothelial cells (CD105 + juvenile nasopharyngeal angio-derivedenthel) with high-purity CD105-positive nasopharyngeal fibroma. (c) ell, CD105 + JEC) and can be successfully subcultured, after The functional comparison of the vascular endothelial cells of the third part of the nasopharyngeal fibroma and the normal vascular endothelial cells is to compare the CD105-positive endothelial cells isolated from the JNA tissues with the human umbilical vein. Methods: The proliferation and invasion of two cells in vitro were observed by Cyquant method proliferation experiment, scratch test and Transwell invasion experiment. Western blot was used to detect the expression of the matrix metalloproteinases-2 (MMP-2) and the vascular endothelial growth factor receptor (Vasca). The results showed that the proliferation ability of CD105 + JEC was lower than that of HUVEC. The results showed that the proliferation ability of CD105 + JEC was lower than that of HUVEC. The results showed that the migration and invasion ability of CD105 + JEC was higher than that of HUVEC. Western blot showed that VEGFR1, p-M The expression of APK and MMP-2 in CD105 + JNA vascular endothelial cells was higher than that of HUVEC, while the expression of VEGFR2, p-ERK was opposite.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.63

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