胶质瘤干细胞的生物学特性及其与胶质瘤血管发生的关系
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摘要
一、研究背景与目的
虽然大家都知道，血管发生（angiogenesis）的过程对于胶质瘤的生长、恶性浸润等特性来讲是一个关键性事件，但如何发生的，研究者苦苦地探索了数十年，至今仍有争论，特别是随着本世纪初才发现的肿瘤干细胞以及与之相关的肿瘤干细胞微环境(Niche)研究的深入，对肿瘤血管的生成有了新的认识，目前有如下三种观点：
A.肿瘤细胞诱导宿主血管内皮细胞增生,出芽,迁移后形成；
B.先由肿瘤细胞随机排列组成,而后刺激宿主内皮细胞加入；
C.依托肿瘤干细胞巢(Niche,微环境)转分化形成。详见英文摘要中的图1。
本研究主要在自建的胶质瘤干/祖细胞(Glioma Stem/Progenitor Cells，GSPCs)基础上探索其与肿瘤血管生成的关系，即是否存在图1中的C模式。
二、实验方法
1建立GSPCs体外生长模型：用新鲜的恶性胶质瘤临床标本消化成单细胞悬液，在无血清(含生长因子)条件下于CO2箱中培养，再从呈悬浮生长的肿瘤球中用CD133免疫磁珠分离阳性细胞，单克隆后一部分液氮冻存;另一部分在含血清(不含生长因子)条件下培养，作干祖细胞和分化细胞相关标志蛋白的流式细胞及细胞化学检测，最后用GSPCs球吹打成单个细胞进行裸小鼠脑尾状核移植，观察致瘤情况。
2建立RFP/GFP可移植性肿瘤模型与检测血管:用经病毒载体转染红色荧光蛋白(Red Fluorescent Protein，RFP)基因的GSPCs，接种于本室培育的表达绿色荧光蛋白(Green Fluorescent Protein，GFP)基因的NC裸小鼠脑内（10μl，1×106细胞），或皮下（200μl，1×106细胞），2～3周出现症状或恶病质时处死，取移植瘤组织压成簿片后在荧光显微镜下观察、或用冰冻切片在荧光激光扫描共聚焦显微镜下观察肿瘤血管.
三、结果与分析
1GSPCs的特征:克隆到的CD133~+细胞，在含生长因子条件下培养的肿瘤球中CD133~+细胞只占少数，多数细胞是Nestin~+细胞，免疫细胞化学染色后在共聚焦显微镜下还见到即使是CD133~+细胞，也同时表达Nestin，据此我们用于实验的细胞都称为胶质瘤干/祖细胞(Glioma Stem/Progenitor Cells，GSPCs)，与神经干细胞(neuralstem cell，NSCs)比，最大的不同点是在含血清条件下培养，NSCs可分化成终末细胞，GSPCs不但到不了终末分化状态，而且还可以逆向分化成前体细胞.GSPCs接种GFP裸小鼠体内，无论是脑内还是皮下，都能100％致瘤，而且呈高侵袭性生长。
2RFP/GFP模型特征:GSPCs-RFP细胞与GSPCs细胞一样，无论接种在脑内还是皮下，也都能100％致瘤，而且呈高侵袭性生长.因为接种的肿瘤细胞与宿主细胞分别表达RFP和GFP，根据显示的红色和绿色加以区别。在荧光显微镜和激光扫描共聚焦显微镜下，能清晰显示肿瘤细胞在对宿主组织重构过程中所发生的细胞和蛋白质分子事件是谁起的作用.
3被荧光示踪的肿瘤血管和宿主血管:在肿瘤组织中，见到了参与组成肿瘤血管壁的细胞或血管壁，有红、绿和黄三种颜色，红、绿分别代表来自接种肿瘤细胞的子代细胞和宿主组织来源的细胞;黄色的则是红、绿两种细胞相互融合的细胞。在肿瘤组织压片，所取的组织最终压成的厚度大约在20μm，比通常5μm的病理切片要厚，因此容易看到整条的长血管，减少了病理切片取材上的局限性。在冰冻切片共聚焦显镜下见到了包括平滑肌细胞，内皮细胞等组成肿瘤血管壁的细胞共表达RFP/GFP。
四、结论与创新点
1作为以分化走向时段界定的CD133~+胶质瘤干细胞，只是指处于静止期的细胞，一旦启动了分化程序，则变为Nestin~+的祖细胞，即使是CD133~+细胞也因同时表达Nestin而被称之为胶质瘤干/祖细胞(GSPCs)。在与神经干细胞作为对照的实验中，首次证明了只有GSPCs具备逆向分化潜能和到不了终末分化阶段。
2RFP/GFP双色荧光示踪的可移植性胶质瘤模型，因其能清晰显示肿瘤组织中的肿瘤细胞与宿主细胞之间的解剖位置、组织结构关系，在研究肿瘤组织重构，特别是细胞融合方面，揭示了传统的肿瘤模型无法显示二者的新关系。在国内，这一实验平台是我们课题组首先建立。
3课题组用RFP/GFP双色荧光示踪模型，己经在国际上首先报告了GSPCs通过细胞融合和肿瘤血管拟态两方面在肿瘤组织重构中发挥了重要作用。本文则在上述基础上，在国内首先使用组织压片法展示了在传统组织切片上很少能见到的长条血管壁细胞，有单纯来源于肿瘤细胞，单纯来源于宿主细胞和既来源于肿瘤又来源于宿主的三种类型血管。第三种类型的血管不仅表现在RFP/GFP细胞水平上的镶嵌关系,而且还表现在蛋白质表达水平上的融合关系,这对进一步研究肿瘤血管的发生和转化机制有重要意义.
1, background and objective
It is well known that the angiogenesis is a key incident for the growth, infiltration ofglioma. However, the complete process of glioma angiogenesis remains to dispute, evenafter painful exploration for decades. Since the beginning of this century, with thegradually deep research on tumor stem cells and their Niche, some novel recognitionabout angiogenesis is being raised. At present, there mainly are three viewpoints:
A, Neoformed tumor vessels of tumors were thought to arise by sprouting of pre-existingbrain capillaries induce by tumor cells.
B, Tumor vessels were first arranged by tumor cells, and then host endothelial cellsjoined.
C, From transdifferentiated tumor stem cells depending on their Niche (shown asfollowing).
Figure1, Model of tumor angiogenesis: TC: tumor cells; EC: endothelial cells; GSCs: Gliomasstem cells; A：Zhang S Oncol Resp2006,15(1):15-20；B：Chang YS,Proc Natl Acad Sic USA2000,97(26):14608-14613；C：Ricc-Vitiani L,Natrue2010,133(pt4),973-982；Jun Dong, Stem CellRev and Rep2011,7(1):141-152.
This paper aims to study the GSPCs and the relationship between GSPCs and gliomaangiogenesis, i.e. to verify the existence of the Model C in Figure.
2, Methods
1) To establish the growth model of GSPCs in vitro: fresh tumor specimens weresubjected to enzymatic dissociation into cell suspension, and cultivaed in DF containingEGF and bFGF. Then CD133~+cells, after being isolated by immunomagnetic beads,were seed into caudate nucleus of nude mice. The tumorigenesis incidence was recorded.
2) To establish the RFP/GFP glioma model and detect blood vessels: GSCPs (25μl/1×106)transfected with Red Fluorescent Protein (RFP) were transplanted into NC nude miceexpressing Green Fluorescent Protein (GFP), intracranially or subcutaneously. Two to3weeks later, after tumorgenesis, tumor tissues were pressed into tabletting and tumorvessels were detected under under fluorescence microscope and confocal microscopy.
3, results and discussion
1) Characteristics of GSPCs: minority of cells in tumor spheres were CD133~+, but mostwere Nestin~+; in addition, some cells co-expressed both CD133and Nestin, accordingly,cells used in our research were called Glioma Stem/Progenitor Cells (GSPCs). The mostdifference between GSPCs and neural stem cells (NSCs) was that NSCs coulddifferentiate into mature end cells, but GSPCs could not, for the differentiated GSPCscould even differentiate backwards into progenitor cells. The tumorigenesis ratio were100%no matter GSPCs were transplanted intracranially or subcutaneously, and thetransplanted tumor grows at a highly-invasive pattern.
2) Characteristics of RFP/GFP glioma models: the tumorigenesis ratio of GSPCs-RFP were100%, like GSPCs, no matter being transplanted intracranially or subcutaneously,with a highly-invasive growth pattern. The different fluorescence expressed by tumorcells (RFP) and host cells (GFP) make it very convenient to show their different rolesduring the tissue remodel process when observed under a fluorescence microscope orconfocal microscopy.
3) Tumor vessels and host vessels: tumor vessels were made up of cells with red, greenor yellow fluorescence under a confocal microscopy, indicating the cellular originrespectively being tumor cells, host cells and their fusional cells. The thickness of tumortissue tabletting was about20μl, which was much thicker than common pathologicalsection, and it made the strip vessels much more visible.
4, conclusion and Innovation
1) CD133+glioma stem cells are only cells at quiescent periods, and once thedifferentiation program started, CD133+glioma stem cells will become Nestin+evenco-expressing CD133. We first demonstrated that GSPCs could not differentiate intomature end cells but have a potential to retro-differentiate.
2) RFP/GFP bio-fluorescence labelled glioma models could clearly show the anatomyand histological relationships between tumor cells and host cells, which has an advantageat the research field of tumor tissue remodel and micro-environments. And theexperimental platform was reported first by us at home.
3) With the RFP/GFP bio-fluorescence labelled glioma models, we first took advantageof tissue squash technique at home to show strip vessels, which could seldom be viewedby traditionally histological section. Our results showed that there were three types ofvessels cellular origins in tumor tissues: tumor cells, host cells and their fusional cells.
These results had an important significance to the further study on tumor angiogenesisand transformation.
虽然大家都知道，血管发生（angiogenesis）的过程对于胶质瘤的生长、恶性浸润等特性来讲是一个关键性事件，但如何发生的，研究者苦苦地探索了数十年，至今仍有争论，特别是随着本世纪初才发现的肿瘤干细胞以及与之相关的肿瘤干细胞微环境(Niche)研究的深入，对肿瘤血管的生成有了新的认识，目前有如下三种观点：
A.肿瘤细胞诱导宿主血管内皮细胞增生,出芽,迁移后形成；
B.先由肿瘤细胞随机排列组成,而后刺激宿主内皮细胞加入；
C.依托肿瘤干细胞巢(Niche,微环境)转分化形成。详见英文摘要中的图1。
本研究主要在自建的胶质瘤干/祖细胞(Glioma Stem/Progenitor Cells，GSPCs)基础上探索其与肿瘤血管生成的关系，即是否存在图1中的C模式。
二、实验方法
1建立GSPCs体外生长模型：用新鲜的恶性胶质瘤临床标本消化成单细胞悬液，在无血清(含生长因子)条件下于CO2箱中培养，再从呈悬浮生长的肿瘤球中用CD133免疫磁珠分离阳性细胞，单克隆后一部分液氮冻存;另一部分在含血清(不含生长因子)条件下培养，作干祖细胞和分化细胞相关标志蛋白的流式细胞及细胞化学检测，最后用GSPCs球吹打成单个细胞进行裸小鼠脑尾状核移植，观察致瘤情况。
2建立RFP/GFP可移植性肿瘤模型与检测血管:用经病毒载体转染红色荧光蛋白(Red Fluorescent Protein，RFP)基因的GSPCs，接种于本室培育的表达绿色荧光蛋白(Green Fluorescent Protein，GFP)基因的NC裸小鼠脑内（10μl，1×106细胞），或皮下（200μl，1×106细胞），2～3周出现症状或恶病质时处死，取移植瘤组织压成簿片后在荧光显微镜下观察、或用冰冻切片在荧光激光扫描共聚焦显微镜下观察肿瘤血管.
三、结果与分析
1GSPCs的特征:克隆到的CD133~+细胞，在含生长因子条件下培养的肿瘤球中CD133~+细胞只占少数，多数细胞是Nestin~+细胞，免疫细胞化学染色后在共聚焦显微镜下还见到即使是CD133~+细胞，也同时表达Nestin，据此我们用于实验的细胞都称为胶质瘤干/祖细胞(Glioma Stem/Progenitor Cells，GSPCs)，与神经干细胞(neuralstem cell，NSCs)比，最大的不同点是在含血清条件下培养，NSCs可分化成终末细胞，GSPCs不但到不了终末分化状态，而且还可以逆向分化成前体细胞.GSPCs接种GFP裸小鼠体内，无论是脑内还是皮下，都能100％致瘤，而且呈高侵袭性生长。
2RFP/GFP模型特征:GSPCs-RFP细胞与GSPCs细胞一样，无论接种在脑内还是皮下，也都能100％致瘤，而且呈高侵袭性生长.因为接种的肿瘤细胞与宿主细胞分别表达RFP和GFP，根据显示的红色和绿色加以区别。在荧光显微镜和激光扫描共聚焦显微镜下，能清晰显示肿瘤细胞在对宿主组织重构过程中所发生的细胞和蛋白质分子事件是谁起的作用.
3被荧光示踪的肿瘤血管和宿主血管:在肿瘤组织中，见到了参与组成肿瘤血管壁的细胞或血管壁，有红、绿和黄三种颜色，红、绿分别代表来自接种肿瘤细胞的子代细胞和宿主组织来源的细胞;黄色的则是红、绿两种细胞相互融合的细胞。在肿瘤组织压片，所取的组织最终压成的厚度大约在20μm，比通常5μm的病理切片要厚，因此容易看到整条的长血管，减少了病理切片取材上的局限性。在冰冻切片共聚焦显镜下见到了包括平滑肌细胞，内皮细胞等组成肿瘤血管壁的细胞共表达RFP/GFP。
四、结论与创新点
1作为以分化走向时段界定的CD133~+胶质瘤干细胞，只是指处于静止期的细胞，一旦启动了分化程序，则变为Nestin~+的祖细胞，即使是CD133~+细胞也因同时表达Nestin而被称之为胶质瘤干/祖细胞(GSPCs)。在与神经干细胞作为对照的实验中，首次证明了只有GSPCs具备逆向分化潜能和到不了终末分化阶段。
2RFP/GFP双色荧光示踪的可移植性胶质瘤模型，因其能清晰显示肿瘤组织中的肿瘤细胞与宿主细胞之间的解剖位置、组织结构关系，在研究肿瘤组织重构，特别是细胞融合方面，揭示了传统的肿瘤模型无法显示二者的新关系。在国内，这一实验平台是我们课题组首先建立。
3课题组用RFP/GFP双色荧光示踪模型，己经在国际上首先报告了GSPCs通过细胞融合和肿瘤血管拟态两方面在肿瘤组织重构中发挥了重要作用。本文则在上述基础上，在国内首先使用组织压片法展示了在传统组织切片上很少能见到的长条血管壁细胞，有单纯来源于肿瘤细胞，单纯来源于宿主细胞和既来源于肿瘤又来源于宿主的三种类型血管。第三种类型的血管不仅表现在RFP/GFP细胞水平上的镶嵌关系,而且还表现在蛋白质表达水平上的融合关系,这对进一步研究肿瘤血管的发生和转化机制有重要意义.
1, background and objective
It is well known that the angiogenesis is a key incident for the growth, infiltration ofglioma. However, the complete process of glioma angiogenesis remains to dispute, evenafter painful exploration for decades. Since the beginning of this century, with thegradually deep research on tumor stem cells and their Niche, some novel recognitionabout angiogenesis is being raised. At present, there mainly are three viewpoints:
A, Neoformed tumor vessels of tumors were thought to arise by sprouting of pre-existingbrain capillaries induce by tumor cells.
B, Tumor vessels were first arranged by tumor cells, and then host endothelial cellsjoined.
C, From transdifferentiated tumor stem cells depending on their Niche (shown asfollowing).
Figure1, Model of tumor angiogenesis: TC: tumor cells; EC: endothelial cells; GSCs: Gliomasstem cells; A：Zhang S Oncol Resp2006,15(1):15-20；B：Chang YS,Proc Natl Acad Sic USA2000,97(26):14608-14613；C：Ricc-Vitiani L,Natrue2010,133(pt4),973-982；Jun Dong, Stem CellRev and Rep2011,7(1):141-152.
This paper aims to study the GSPCs and the relationship between GSPCs and gliomaangiogenesis, i.e. to verify the existence of the Model C in Figure.
2, Methods
1) To establish the growth model of GSPCs in vitro: fresh tumor specimens weresubjected to enzymatic dissociation into cell suspension, and cultivaed in DF containingEGF and bFGF. Then CD133~+cells, after being isolated by immunomagnetic beads,were seed into caudate nucleus of nude mice. The tumorigenesis incidence was recorded.
2) To establish the RFP/GFP glioma model and detect blood vessels: GSCPs (25μl/1×106)transfected with Red Fluorescent Protein (RFP) were transplanted into NC nude miceexpressing Green Fluorescent Protein (GFP), intracranially or subcutaneously. Two to3weeks later, after tumorgenesis, tumor tissues were pressed into tabletting and tumorvessels were detected under under fluorescence microscope and confocal microscopy.
3, results and discussion
1) Characteristics of GSPCs: minority of cells in tumor spheres were CD133~+, but mostwere Nestin~+; in addition, some cells co-expressed both CD133and Nestin, accordingly,cells used in our research were called Glioma Stem/Progenitor Cells (GSPCs). The mostdifference between GSPCs and neural stem cells (NSCs) was that NSCs coulddifferentiate into mature end cells, but GSPCs could not, for the differentiated GSPCscould even differentiate backwards into progenitor cells. The tumorigenesis ratio were100%no matter GSPCs were transplanted intracranially or subcutaneously, and thetransplanted tumor grows at a highly-invasive pattern.
2) Characteristics of RFP/GFP glioma models: the tumorigenesis ratio of GSPCs-RFP were100%, like GSPCs, no matter being transplanted intracranially or subcutaneously,with a highly-invasive growth pattern. The different fluorescence expressed by tumorcells (RFP) and host cells (GFP) make it very convenient to show their different rolesduring the tissue remodel process when observed under a fluorescence microscope orconfocal microscopy.
3) Tumor vessels and host vessels: tumor vessels were made up of cells with red, greenor yellow fluorescence under a confocal microscopy, indicating the cellular originrespectively being tumor cells, host cells and their fusional cells. The thickness of tumortissue tabletting was about20μl, which was much thicker than common pathologicalsection, and it made the strip vessels much more visible.
4, conclusion and Innovation
1) CD133+glioma stem cells are only cells at quiescent periods, and once thedifferentiation program started, CD133+glioma stem cells will become Nestin+evenco-expressing CD133. We first demonstrated that GSPCs could not differentiate intomature end cells but have a potential to retro-differentiate.
2) RFP/GFP bio-fluorescence labelled glioma models could clearly show the anatomyand histological relationships between tumor cells and host cells, which has an advantageat the research field of tumor tissue remodel and micro-environments. And theexperimental platform was reported first by us at home.
3) With the RFP/GFP bio-fluorescence labelled glioma models, we first took advantageof tissue squash technique at home to show strip vessels, which could seldom be viewedby traditionally histological section. Our results showed that there were three types ofvessels cellular origins in tumor tissues: tumor cells, host cells and their fusional cells.
These results had an important significance to the further study on tumor angiogenesisand transformation.
引文
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