【作者】 王林；

【导师】 董军；

【作者基本信息】 苏州大学 ， 神经外科学（专业学位）， 2015， 硕士

【摘要】 第一部分:胶质瘤干祖细胞与小鼠巨噬细胞共培养产生的融合细胞可转化成恶性巨噬细胞目的我们曾推测胶质瘤间质中的巨噬细胞(macrophage,MP)恶性转化是通过胶质瘤干祖细胞(Glioma Stem Progenitor cells,GSPCs)与MP融合导致的。本文旨在通过GSPCs与MP共培养来验证这种推测是否正确。方法将转有红色荧光蛋白基因(RFP)的GSPCs(SU3)与表达增强型绿色荧光蛋白(EGFP)的小鼠腹腔冲洗出的MP进行体外共培养;用单克隆方法建立表达RFP+/GFP+双色荧光(黄色)的细胞系,然后进行癌细胞相关表型分析、致瘤性试验、鼠巨噬细胞标记物检测。结果(1)这两种细胞共培养后,细胞群中出现为数不多的黄色细胞,并成功单克隆到C3,C4和C12三株细胞。(2)C12继续传代培养后分化出RFP+、EGFP+和RFP+/EGFP+三种类型细胞,但随着传代次数增多,EGFP+(绿色)细胞比例逐渐升高而成为主体,RFP+/EGFP+(黄色)细胞比例逐渐下降并维持在较低水平,而RFP+(红色)细胞基本消失。(3)C12细胞株中的EGFP+细胞具有:①生长接触抑制消失,增殖速度快,染色体异倍体等癌细胞特征;②在裸小鼠体内具有高致瘤率(5/5);③表达巨噬细胞特异性标记CD68,大部分染色体为鼠端着丝粒。结论体外模型证明我们之前在实体瘤中观察到的人脑胶质瘤干祖细胞诱导宿主巨噬细胞恶性转化可通过细胞融合途径实现。与我们先前从荷瘤鼠实体瘤组织中克隆到恶性转化的巨噬细胞株(ih CTC)的体内实验一起,相互印证了肿瘤微环境中宿主巨噬细胞恶性转化是客观存在的。宿主细胞与肿瘤干祖细胞融合后发生的恶变既为肿瘤恶性进展和肿瘤异质性增添了新的内涵;又为肿瘤靶向治疗增加了新的靶标。第二部分:胶质瘤干祖细胞与巨噬细胞共培养时的社会活动:细胞连接、融合及胞释事件的缩时摄影观察目的已有用细胞社会学去理解实时监测到的单个免疫细胞分泌的因子在免疫疾病中所起作用的报告,通过分析免疫细胞社会中的肿瘤细胞与宿主巨噬细胞之间的关系,旨在探寻肿瘤细胞社会中打破免疫平衡的大事件。方法釆用红绿双色荧光示踪胶质瘤干/祖细胞与宿主巨噬细胞共培养,单克隆其中的黄色细胞,体外扩増培养后于活细胞工作站实时摄影缩时成像并视频分析目标细胞的社会行为。结果相关细胞实时动态连续观察发现:①细胞间传递信息的连接管道的6种类型。②两个活跃细胞相互作用后变成一个细胞,即细胞融合;已融合的细胞与另一个细胞再融合。③融合细胞的命运有对称分裂产生子细胞和瞬间凋亡二种。④存在一个细胞进入另一细胞内(cell in cell)后胞释(entosis)现象。结论胶质瘤干/祖细胞与宿主巨噬细胞间连接方式,融合、分裂及胞释过程,可作为胶质瘤干/祖细胞诱导宿主巨噬细胞恶变的细胞行为学依据,对进一步理解肿瘤细胞社会成员间的复杂关系有重要意义。更多还原

【Abstract】 Part I: Malignant transformation of fusion cells derived from theco-cultured glioma stem progenitor cells and macrophagesObjective We have hypothesized that the malignant transformation of macrophages(MPs) in glioma was induced by fusion of glioma stem progenitor cells(GSPCs) with MPs.The purpose of this study was to verify this hypothesis by co-culturing GSPCs and MPs.Methods Human GSPCs cell line SU3 transfected with red fluorescent protein(RFP)gene was co-cultured with peritoneal lavage MPs from EGFP mice(whole body expression of enhanced green fluorescent protein) in vitro.RFP+/GFP+dual-color fluorescent cell lineages were established with monoclonal pipetting techniques, then were analyzed on their biological phenotypes, including tumorigenic assay, surface marker expression.Results(1)A few dual-color fluorescent cells were observed several days later in the co-culture model later. Three GFP and RFP double positive monoclonal cell lineages were cloned and named after C3,C4 and C12.(2)Three types of cell subpopulation arose during continuous passage of C12 cells namely,RFP+cells, EGFP+cells and RFP+/EGFP+cells.But the proportion of EGFP+ cells increased gradually and they became the main population of C12 cells,meanwhile the proportion of RFP+/EGFP+cells decreased and maintained at a low level,the RFP+ cells almost disappeared.(3)EGFP+ cells in C12 had the following characteristics: ① malignant biological characteristics,such as lost of contact inhibition,rapid proliferation and chromosome aneuploidy;②high tumorigenic rate in nude mice(5/5);③expression of macrophage specific marker CD68,and most of chromosomes were telocentric chromosomes.Conclusion In vitro studies support our previous finding that human glioma stem progenitor cells could induce malignant transformation of host macrophages in xenografttumors via cell fusion.Along with the malignant transformed macrophages(ih CTC) in vivo obtained from solid tumor tissue of the tumor-bearing mice,our results further confirmed the possibility of malignant transformation of host macrophages in vitro in tumor microenvironment.The malignant transformation of host cells induced by fusion with tumor stem progenitor cells not only provided new finding to understand the progression of tumor and cancer heterogeneity,but also offered new targets for therapy against glioma.Part II: Social activities of co-cultured glioma stem progenitorcells and macrophages under live cell imagingsystem:cell conjunction,cell fusion,and entosisObjective :The roles of real-time monitoring of cytokine secretion at single immunocyte level,following the concept of immune cells sociology has been reported recently.However, the mutual interactions between glioma stem/progenitors cells and host macrophages in the immune cells society has never been reported previously, which deserve further investigations discover the important events which broke the immune balance of the cancer cells society.Methods: The dual fluorescence tracing method was applied to observe the co-cultured glioma stem/progenitor cells and host macrophages,interactions between the two kinds of cells were studied under live cell imaging system,fusion cells(yellow) in the co-culture system were mono-cloned,and cultured in vitro.Then their social activities were observed and recorded under the live cell imaging system,too.Results: Via real-time dynamic observation of the target cells,we noticed that①there were 6 types of conjunction pipes can be found, which functioned as transfering intercellular information; ② two activating cells formed hybrid cell after mutual interactions,and the hybrid cell can fuse with another cell again;③the fates of hybrid cell were to generate offspring cells by symmetrical division, or to undergo apoptosis;④the phenomenon of cell in cell,which can release out another cell, namely entosis,can be found,too.Conclusion:The research revealed the patterns of cell conjunction between glioma stem/progenitor cells and host macrophages,the processes of cell fusion,division and entosis.These were the cell sociology evidences from direct visualization of mutual interaction between glioma stem/progenitor cells and host macrophages,which offered basic knowledges for exploring the complex relationships among the residents in the tumor cell society.更多还原