【作者】 赵阳；

【导师】 马杰；

【作者基本信息】 上海交通大学 ， 外科学（神外）， 2020， 博士

【摘要】 背景:儿童脑肿瘤在儿童实体瘤中发病率最高,早期症状隐匿,病情发展迅速,当出现明显的临床症状时,往往病情已达中晚期。临床中的影像学检查可能会因出血、炎症等因素而影响到阅片的准确判断。此外,临床常规血清学检测缺乏儿童脑肿瘤特异性的标记物,这些限制了脑肿瘤患儿病情的诊断和疗效评估,导致无法及时调整治疗方案,进而延误诊疗。因此,在临床中寻找更合适的检测手段来判断和评估脑肿瘤患儿的病情,已逐渐成为提高儿童脑肿瘤诊疗水平的重要方向。目的:本研究是基于表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)和胶质原纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP)抗体免疫脂质磁球技术与免疫荧光鉴定技术,构建免疫磁球/免疫荧光为核心的儿童脑肿瘤循环肿瘤细胞(Circulating Tumor Cell,CTC)分离鉴定系统,用于对儿童脑肿瘤患者外周血和脑脊液中的CTC进行高效的捕获与鉴定。通过细胞实验、动物实验、临床验证和基因检测等方法,评估该系统的稳定性、安全性和有效性,并验证通过该分离鉴定系统获得的细胞为循环肿瘤细胞。方法:通过反向蒸发法制备EGFR免疫脂质磁球(EGFR Magnetic liposome,EG-ML)和GFAP免疫脂质磁球(GFAP Magnetic liposome,G-ML),然后用粒径电位分析仪、蛋白电泳仪、原子力显微镜、紫外分光光度仪、磁性能分析仪、X射线衍射仪等对其分子结构和结晶性能进行表征和研究,建立一套儿童脑肿瘤CTC分离鉴定系统,对此进行细胞实验、动物实验、临床验证和基因检测,并做出相应的评估。通过EG-ML、G-ML磁分选获得的细胞分别提取DNA和RNA,然后进行荧光定量PCR、RNA-seq、Sanger测序和全基因外显子测序(Whole Exome Sequencing,WES),并结合实体肿瘤标本中明确的突变基因,分析CTC中的突变基因表达和RNA表达差异情况,进而验证循环肿瘤细胞。对患儿进行随访,探究CTC捕获量与生存时间的关系。结果:(1)免疫脂质磁球的制备和表征。成功制备了EG-ML和G-ML两种免疫脂质磁球。制备的EG-ML和G-ML的粒径约为122nm,电位为30m V,其在水溶液中稳定,分散均匀,呈单分散分布,不易团聚。免疫脂质磁球表面抗体纯度较高,磁滞回曲线闭合,具有超顺磁性,饱和磁化强度为32.0 Am~2/Kg,具有磁性颗粒的结晶性能。细胞毒性实验表明,EG-ML和G-ML的细胞毒性较低,生物相容性较好,能够很好的达到临床使用的要求。(2)儿童脑肿瘤CTC新型分离鉴定系统的构建及研究。通过对构建分离鉴定过程的试剂用量和反应条件等各项参数进行逐步优化,初步构建出一套CTC检测系统。然后用免疫荧光法来鉴定细胞,即用DAPI确定是完整细胞,CD45-PE排除白细胞,EGFR-FITC和GFAP-FITC初步拟定是循环肿瘤细胞。此外,进行的细胞实验和动物实验表明,两种磁球对儿童脑肿瘤细胞均有较高的捕获能力,两者依次捕获的回收率可达到95%。(3)临床验证表明,EG-ML和G-ML可有效地捕获脑肿瘤患儿外周血和脑脊液中的CTC,染色效果清晰可辨。临床数据分析可得:无论是在脑肿瘤患儿的脑脊液中还是在其血液中,CTC数量与肿瘤的WHO分级、患儿年龄均无显著相关性。(4)对患儿进行随访,死亡的患儿有15例,1年生存率为82%。结果显示,年龄、性别和WHO分级与生存时间没有显著性差异。而脑脊液中CTC数量与生存时间有显著相关性,CTC数量越多,生存时间越短,预后越差。(5)本研究共筛选出CTC中的RNA表达有显著性差异的有2551个,其中上调表达的1238个,下调表达的有1313个。(6)部分髓母细胞瘤患儿(Group 3)的血液和脑脊液CTC中的NPR3基因表达水平高表达,其对应的肿瘤标本组织肿瘤细胞的NPR3免疫组化呈阳性,两者结果一致。(7)部分弥漫性内源性桥脑胶质瘤患儿进行Sanger测序,在脑脊液和组织中均检测到H3K27M突变,提示基因检测在脑脊液标本中可能更有效,有特异性遗传改变的患者需要进行CTC单细胞测序。(8)本研究对一例脑肿瘤患儿血液中分选出的CTC和组织进行全外显子组测序(WES),结果显示均发生了KMT2A和TMPRSS2突变,这又一次说明了自制的免疫脂质磁球分选的细胞是脑肿瘤细胞。肿瘤突变负荷(Tumor Mutation Burden,TMB)低于平均TMB,对免疫治疗响应不大。结论:本研究选用细胞毒性低、生物相容性高的脂质材料DOPC作为磁性微球的基质,利用GHDC表面的氨基与GFAP、EGFR抗体结合,制备出G-ML和EG-ML,其性质稳定,表面抗体含量可控,具有磁性颗粒的结晶性能,可与儿童脑肿瘤细胞反应形成抗原抗体特异性结合,捕获效率较高。由此形成了一套高效精准的儿童脑肿瘤CTC检测体系。该体系具有无创、便捷、动态监测的优点。与基因检测相结合,对儿童脑肿瘤的早期筛查与诊断、疗效检测和预后评估都具有重要的临床参考价值。更多还原

【Abstract】 Background: Brain tumors are of the highest morbidity among solid tumors in children.The early symptoms of the patients are concealed.The disease has become severe when obvious clinical symptoms appear.At present,clinical imaging examinations will affect the accurate judgment of reading due to bleeding,inflammation and other factors.In addition,the lack of specific serum markers in pediatric brain tumors has limited the diagnosis and evaluation of the efficacy of children with brain tumors,resulting in the inability to adjust the treatment plan in time and delay in treatment.Therefore,finding more suitable detection methods in clinic to judge and evaluate the conditions of children with brain tumors has gradually become an important topic to improve the level of diagnosis and treatment of children with brain tumors.Objective: This study was based on Epidermal Growth Factor Receptor(EGFR)and Glial Fibrillary Acidic Protein(GFAP)antibody immunolipid magnetic sphere technology and immunofluorescence identification technology to construct an immunomagnetic spherical / immunofluorescent children’s brain tumor circulating tumor cell(CTC)separation and identification system,used to efficiently capture and identify CTC in peripheral blood and cerebrospinal fluid of children with brain tumors.The stability,security and effectiveness of the CTC isolation and identification system for children’s brain tumors are evaluated through cell experiments,animal experiments,clinical validation and genetic testing.It was verified that the cells obtained by this system were circulating tumor cells.Methods: In this paper,EGFR magnetic liposome(EG-ML)and GFAP magnetic liposome(G-ML)were prepared by reverse evaporation.We used particle size potential analyzer,protein electrophoresis instrument,atomic force microscope,ultraviolet spectrophotometer,magnetic analyzer,and X-ray diffraction to characterize and study its molecular structure and crystalline properties,and to establish a brain tumor CTC isolation and identification system.We make evaluations through cell experiments,animal experiments,and clinical validations.Cells obtained by EG-ML and G-ML magnetic sorting were used to extract DNA and RNA respectively,and then performed quantitative PCR 、RNA-seq 、Sanger sequencing and WES.Combined with the mutant genes identified in solid tumor samples,the differences between the mutant gene and RNA expressions in CTC were analyzed.We also followed up the children to analyze the factors affecting survival time.Results:(1)Preparation and characterization of immunolipid magnetic spheres.EG-ML and G-ML immunolipid magnetic spheres were successfully prepared.The prepared EG-ML and G-ML have a particle diameter of about 122 nm and a potential of about 30 m V.They are relatively stable in aqueous solution,uniformly dispersed,basically monodisperse and difficult to agglomerate.The antibody on the surface of the immunolipid magnetic sphere has high purity,closed hysteresis curve,superparamagnetism,saturation magnetization of 32.0 Am2/Kg,and crystalline properties of magnetic particles.Cytotoxicity experiments show that EG-ML and GML have low cytotoxicity and good biocompatibility,and can meet the requirements of clinical use.(2)Construction and research of a new CTC isolation and identification system for children’s brain tumors.By gradually optimizing the parameters such as the amount of reagents and reaction conditions during the construction of the separation and identification process,a set of CTC detection systems was initially constructed.Then immunofluorescence was used to identify the cells,DAPI for intact cells,CD45-PE for leukocytes,and EGFR-FITC/GFAP-FITC for brain tumor cells.In addition,cell experiments and animal experiments have shown that the two magnetic spheres have a high ability to capture children’s brain tumor cells,and their recovery rate of capture can reach 95%.(3)Clinical verification shows that EG-ML and G-ML can effectively capture CTC in peripheral blood and cerebrospinal fluid of children with brain tumors,and the staining effect is clear and discernible.According to the analysis of clinical data,in cerebrospinal fluid or blood of children with brain tumors,there was no significant correlation between CTC quantity and WHO grade of tumors and age of children.(4)Follow-up of the children,15 children died,and the 1-year survival rate was 82%.The results showed no significant differences in age,gender,and WHO classification with survival time.However,the amount of CTC in CSF was significantly correlated with survival time.With the increase of CTC,the survival time is shorter and the prognosis is worse.(5)We screened 2,551 RNAs with significant differences in CTC,of which1,238 were up-regulated and 1,313 were down-regulated.(6)The expression of NPR3 gene in the blood and cerebrospinal fluid CTC of some children with medullary tumor(group 3)is highly expressed,and the NPR3 cytoplasmic nucleus of tumor cells in the corresponding tumor specimen tissue is positive.(7)We performed Sanger sequencing on some children with diffuse endogenous pontine glioma.Mutations in H3K27 M were detected in cerebrospinal fluid and tissue.The results showed that genetic testing may be more effective in cerebrospinal fluid samples.Patients with specific genetic changes need to undergo CTC single-cell sequencing.(8)We also performed WES sequencing on glioma patients.The results showed that KMT2 A and TMPRSS2 mutations in blood and tissue.This indicated that the cells selected by the self-made immunolipid magnetic spheres are brain tumor cells.TMB was lower than the average TMB,and the response to immunotherapy was not obvious.Conclusion: In this study,the lipid material DOPC with low cytotoxicity and high biocompatibility was used as the matrix of magnetic microspheres.The amino group on the surface of GHDC was combined with GFAP and EGFR antibodies to prepare EG-ML and G-ML.It is stable,the surface antibody content is controllable,and it has the crystallization properties of magnetic particles.It can react with children’s brain tumor cells to form antigen-antibody specific binding,and has high capture efficiency.A high-efficiency and accurate CTC detection system for children’s brain tumors was formed.This system has the advantages of non-invasiveness,convenience and dynamic monitoring.Combined with genetic testing,it has important clinical reference value for early screening and diagnosis,curative effect detection and prognostic evaluation of brain tumors in children.更多还原