胶质瘤干细胞微环境间质细胞恶性转化及相关分子特征分析

Relevant molecular characteristics analysis on malignant transformation of interstitial cells induced by tumor stem cells in glioma microenvironment

目的探讨胶质瘤干细胞（GSC）诱导TME中多种间质细胞的恶性转化及其相关分子特征。方法将红色荧光蛋白（RFP）基因稳定转染人SU3一RFP的GSC与增强型绿色荧光蛋白（EGFP）转基因裸小鼠间质细胞体外共培养，将SU3．RFP细胞接种于EFGP-Balb／c裸小鼠不同部位，从体外培养基与体内肿瘤组织中分别单克隆具永生化特征的EGFP’细胞，经染色体分析和致瘤试验证实其具有肿瘤细胞特征，再以RT-PCR、流式细胞术和细胞免疫化学染色等分子手段检测相关分子表型。结果（1）体外培养共得到2株EGFP‘细胞系，体内致瘤实验共得到5株EGFP^＋细胞系，所克隆的7株EGFP^＋细胞具自我更新、呈异倍体染色体核型和100％致瘤率；（2）根据细胞标志物表达状况鉴定其分别起源于巨噬细胞（tMφl和tMcP2）、树突状细胞（tDCl和tDC2）、成纤维细胞（tFB）、少突胶质细胞（tOG）和骨髓间充质干细胞（tBMSC）；（3）根据七株细胞共同表达Sca-1（髓源性干细胞标志物）和c-myc，分别表达Soχ2、Nanog等诱导性多能干细胞（iPS）标志物，确定这些恶性转化细胞不同程度上兼具髓源干细胞和iPS细胞的分子特征。结论（1）TME中肿瘤间质细胞自身恶变与GSC对TME组织重构相关，与其寄生地组织类型关系不大；（2）源于GSC的肿瘤细胞和GSCTME中恶变的间质细胞，是GSC重构的肿瘤组织内两大类不同起源的肿瘤细胞，由此构成广义肿瘤异质性概念的细胞学基础，后者有潜力作为靶向诊疗的新靶标。

Objective A variety of interstitial cells in tumor microenvironment （TME） based on glioma stem cells （ GSC ） have the function to promote malignant progression of tumors, but whether these interstitial cells have already undergone malignant transformation and their related molecular characteristics are still poorly understood. Methods Human SU3-RFP glioma stem cells （GSC） stably transfected with red fluorescent protein （RFP） and interstitial cells from enhanced green fluorescent protein （EGFP） transgenic nude mice were co-cultured in vitro. SU3-RFP cells were also inoculated in different tissues of EGFP-Balb/c nude mice. Immortal EGFP ~ cells were monocloned either from co-culture cells in vitro, or from their xenografts in vivo. These immortal EGFP^＋~ cells were confirmed to bear characteristics of tumor cell via chromosomal analysis and tumorigenicity assay. Related molecular phenotypes of these cells were further detected through RT-PCR, flow cytometry and immunochemistry （ IHC ） techniques. Results （ 1 ） Two EGFP^＋ cell lines were obtained in vitro, and 5 EGFP^＋ cell lines were obtained in vivo tumorigenic experiments. Seven EGFP^ ＋ cell lines all have characteristics of self-renewal, heteroploid of chromosomes and 100% tumorigenicity. （2） Cell surface marker analysis showed cell origin of these cell lines were macrophages （tM^l and tM^2 ）, dendritic cells （ tDC1 and tDC2）, fibroblasts （ tFB）, oligodendrocytes （tOG） and BMSC cells （tBMSC）, respectively. （3）All of these seven cell lines co-expressed Sca-1 and c- myc, and have Sox-2 or Nanog expression also, which suggest that they may bear molecular characteristics of mesenchymal stem cells or pluripotent stem ceils. Conclusions （ 1 ） Tumor stromal ceils in TME have undergone malignant transformation, which is related to the tissue remodeling of TME by GSCs, and not limit to the specific type of their parasitic tissues. （2） Tumor ceils originated from GSC and tumor interstitial cells, respectively, are two major types of tumor cells with different origins in glioma parenchyma, can not be simply regarded as tumor heterogeneity, transformed interstitial cells of TME may have the potential to serve as new targets for target diagnosis and therapy.