原代食管癌干细胞的分离及初步鉴定

发布时间：2018-08-31 15:54

【摘要】：背景和目的:食管癌是最常见的消化道恶性肿瘤之一,全球每一年大约有30万人死于食管癌。我国作为食管癌的高发区之一,食管鳞状细胞癌占所有病理类型的的百分之九十。随着对肿瘤干细胞的研究不断深入,通过有效简便的方法筛选鉴定肿瘤干细胞成为目前肿瘤干细胞研究的前提。近期有相关报道已经鉴定了不同种类的实体瘤的肿瘤干细胞中的一种固有的自发荧光表型,运用这种标记物作为一种新颖和功能性的相关工具去分离和鉴定肿瘤干细胞,特别的是在胰腺导管腺癌、结肠癌、肝细胞癌、非小细胞癌中,可以运用流式细胞仪技术对自发荧光细胞进行跟踪和分离。本研究根据这项新的方法将食管癌原代细胞进行分离,并通过流式细胞仪检测自发荧光细胞亚群在食管癌原代细胞中的表达,并对其进行筛选及初步鉴定,以丰富食管癌实体瘤干细胞筛选及鉴定的相关研究。材料和方法:收集2015年3月1日至2016年3月1日,于郑州大学第一附属医院胸外科行食管癌根治术的新鲜肿瘤组织标本48例,组织病理类型均经过三位病理专家鉴定为食管鳞状细胞癌。患者病史完整且术前均未经放化疗等治疗。首先运用酶消化法进行食管癌原代细胞的分离,然后通过流式细胞仪检测自发荧光细胞亚群在食管癌原代细胞中的表达并对其进行筛选。最后通过平板克隆形成实验、Transwell侵袭迁移实验、MTT法测增值能力试验和成球试验来初步鉴定自发荧光细胞亚群具有肿瘤干细胞特性。结果:1.运用酶消化法对食管癌原代细胞进行分离,成功分离了能够满足后期实验的10例标本,成功率达到20.8%(10/48)。2.运用流式仪进行分选,细胞自发荧光在488nm蓝色激光激发,通过530/40和580/30间交叉的滤光片接收荧光信号并进行检测分选。未添加核黄素组自发荧光细胞百分比约为0.325±0.077,添加30μM核黄素组自发荧光细胞百分比约为4.875±0.498,两组之间t=9.013,P0.001,差异具有统计学意义。3.通过添加不同浓度的核黄素观察食管癌原代细胞中自发荧光细胞的百分比,发现自发荧光百分比和培养基核黄素浓度之间有很强的联系,并证明了在核黄素浓度在30μM的自发荧光细胞亚群比例达到平衡。4.通过平板克隆形成实验其结果显示:自发荧光细胞组克隆形成率是0.325±0.031,非自发荧光细胞组克隆形成率是0.083±0.015,两组间克隆率有显著性差异,具有统计学意义(t=7.106,P0.001)。5.通过Transwell迁移实验其结果显示:自发荧光细胞的迁移细胞数为136±5.51,非自发荧光细胞的迁移细胞数为66±2.08,有显著性差异,有统计学意义(t=11.89,P0.01)。在检测两组细胞侵袭能力的结果显示:自发荧光细胞的侵袭细胞数为45.33±1.45,非自发荧光细胞的迁移细胞数为20±2.52,有显著性差异,有统计学意义(t=8.718,P0.01)。6.通过MTT法测增值实验其结果显示:自发荧光细胞组的增值能力显著高于非自发荧光细胞组。在第一天两组之间的增值能力没有明显差异,无统计学意义(P0.05)。在第二天到第七天两组之间的增值能力差异显著,均具有统计学意义(P0.05)。7.通过细胞成球实验其结果显示:观察第1,3,7天的细胞变化情况,自发荧光细胞的成球能力明显高于非自发荧光细胞。两组细胞成球数差异有显著性,有统计学意义(P0.05)。结论:1.采用酶消化法可以成功对食管癌原代细胞进行有效分离培养。2.运用食管癌原代细胞中表达的自发荧光细胞表型,可以对原代食管癌细胞进行干细胞的筛选。3.食管癌自发荧光细胞亚群具有自我更新、高增值能力、高侵袭迁移能力的肿瘤干细胞生物特性。
[Abstract]:BACKGROUND AND OBJECTIVE: Esophageal cancer is one of the most common malignant tumors of the digestive tract, and about 300,000 people die of esophageal cancer every year. As one of the high incidence areas of esophageal cancer, esophageal squamous cell carcinoma (ESCC) accounts for 90% of all pathological types in China. Identification of cancer stem cells is a prerequisite for cancer stem cell research. Recent reports have identified an intrinsic autofluorescent phenotype in cancer stem cells from different types of solid tumors. The use of this marker as a novel and functional tool for isolating and identifying cancer stem cells, particularly in the context of cancer stem cell research. In pancreatic ductal adenocarcinoma, colon cancer, hepatocellular carcinoma and non-small cell carcinoma, autofluorescent cells can be tracked and isolated by flow cytometry. Materials and Methods: From March 1, 2015 to March 1, 2016, 48 fresh specimens of esophageal carcinoma were collected from thoracic surgery of the First Affiliated Hospital of Zhengzhou University. The histopathological types of the specimens were all specialized in three pathological types. Esophageal squamous cell carcinoma (ESCC) was identified by our laboratory. The patient had a complete history and had not been treated by radiotherapy or chemotherapy before operation. First, the primary cells of ESCC were isolated by enzymatic digestion, then the expression of autofluorescent cell subsets in ESCC was detected by flow cytometry and screened by plate cloning. Results: 1. The primary cells of esophageal cancer were separated by enzymatic digestion, and 10 specimens were successfully separated. The success rate was 20.8% (10/48). 2. The flow rate was used to isolate the primary cells of esophageal cancer. The percentage of spontaneous fluorescent cells in the non-riboflavin group was about 0.325 [0.077] and the percentage of spontaneous fluorescent cells in the riboflavin group was about 4.875 [0.498] and T = 9.0 between the two groups. 13, P 0.001, the difference was statistically significant. 4. The results of plate cloning experiment showed that the cloning rate of spontaneous fluorescent cells was 0.325 (+ 0.031) and that of non-spontaneous fluorescent cells was 0.083 (+ 0.015). There was a significant difference in the cloning rate between the two groups (t = 7.106, P 0.001). 5. The results of Transwell migration experiment showed that spontaneous fluorescent cells could form spontaneous fluorescent cells. The number of migrating cells was 136 [5.51] and that of non-spontaneous fluorescent cells was 66 [2.08]. There was significant difference between the two groups (t = 11.89, P 0.01). The results showed that the number of invasive cells of spontaneous fluorescent cells was 45.33 [1.45] and that of non-spontaneous fluorescent cells was 20 [2.52], which was significantly worse. The results of MTT assay showed that the proliferation ability of spontaneous fluorescent cells was significantly higher than that of non-spontaneous fluorescent cells. Spontaneous fluorescent cells were significantly higher than non-spontaneous fluorescent cells on the 1st, 3rd and 7th day of observation. There was a significant difference in the number of globules between the two groups, with statistical significance (P 0.05). Conclusion: 1. Enzymatic digestion can successfully eat food. Esophageal cancer primary cells can be effectively isolated and cultured. 2. Esophageal cancer primary cells can be screened by autofluorescent cell phenotype. 3. Esophageal cancer autofluorescent cell subsets have the biological characteristics of self-renewal, high proliferation, high invasion and migration.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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