【作者】 赵耀东；

【导师】 黄强；

【作者基本信息】 苏州大学 ， 神经外科， 2009， 博士

【摘要】 第一部分胶质瘤干祖细胞分化抑制的超微结构机制【目的】在先前的研究中已经见到,体外培养的胶质瘤干祖细胞(glioma stem progenitor cells, GSPCs)与神经干祖细胞(neural stem/progenitor cells, NSPCs)相比的最大不同点是GSPCs始终处于分化抑制状态,还时有逆向分化发生,为了寻找产生这种区别的原因,本实验比较两者超微结构的异同,旨在亚细胞水平阐明GSPCs分化抑制的机制。【方法】GSPCs由我们实验室保存。NSPCs来源于流产的人胚脑,经分离、培养、扩增后,予以鉴定。分别收集GSPCs和NSPCs,培养于经多聚赖氨酸预处理的无菌玻片上,2小时后结束培养,取贴敷有细胞的玻片行扫描电镜检查。同时,收集GSPCs和NSPCs行透射电镜观察,定性或定量比较二者单细胞的细胞膜、细胞器、细胞核以及核内容物的异同,分析细胞球间结构的异同。【结果】GSPCs和NSPCs共同点如下:细胞器不发达,少数细胞中可见神经微丝、微管,核质比高,核内常染色质多,异染色质少。二者形成的细胞球内的细胞间均存在特定的细胞连接。不同点有:GSPCs的细胞体积比NSPCs大;GSPCs的微绒毛丰富;GSPCs中核糖体、内质网、高尔基体等与蛋白质合成分泌相关的细胞器较NSPCs发达,GSPCs中线粒体数量较NSPCs多;部分NSPCs中自噬小体多见,而GSPCs罕见自噬小体;GSPCs胞核体积较NSPCs大,但核质比NSPCs小,GSPCs核内异染色质的相对含量比NSPCs多,GSPCs多有两个或以上核仁。NSPCs球的相邻细胞间可见突触样连接、胞膜融合等结构,GSPCs球内相邻细胞间仅可见桥粒样结构。需要特别指出的是GSPCs比NSPCs的自噬小体数量明显少。【结论】GSPC和NSPC在超微结构上的异同点,是我们先前见到的GSPCs始终处于分化抑制状态的亚细胞形态结构基础,也有可能是GSPCs产生逆向分化的原因,对进一步研究GSPCs与NSPCs相比之所以具有独特的的肿瘤生物学特征有重要意义,特别是自噬小体数量的变化值得进一步研究。第二部分胶质瘤干祖细胞分化抑制与其自噬活性低下相关【目的】在第一部分研究中观察到了GSPCs中自噬小体的数量明显少于NSPCs,本研究观察其是否存在自噬活性的变化,以及这种变化与GSPCs分化抑制的关系。【方法】利用第一部分中培养所得的GSPCs和NSPCs,分别用单丹(磺)酰戊二胺MDC染色、微管相关蛋白轻链3(microtubule-associated protein light chain-3,LC3, LC3)免疫荧光染色、western-blot法检测LC3-Ⅱ/LC3-Ⅰ等多种方法检测GSPCs与NSPCs中自噬活性的差异。检测GSPCs分化后的自噬活性,在诱导GSPCs分化同时加入自噬抑制剂3-甲基腺嘌呤(methyladenine,3-MA)或Bafilomycin A1(BFA),观察抑制自噬是否可以抑制GSPCs的分化。自噬激活剂雷帕霉素(rapamycin,RPM)被用于检测是否能促进GSPCs的分化。【结果】自噬在GSPCs中的活性明显低于其在NSPCs中的活性。当诱导GSPCs分化后,其自噬活性增高;自噬的抑制剂3-MA或BFA作用于GSPCs可以抑制后者贴壁分化,western blot分析提示经上述自噬抑制剂作用后的GSPCs的GFAP以及MAP-2的表达量较未加抑制剂的对照组降低。自噬的活化剂RPM在适当的条件下可以促进GSPCs的贴壁分化。【结论】通过自噬抑制剂或活化剂从正反两方面证实GSPCs的自噬活性与其分化抑制相关,为进一步探索发生这种变化的分子机制打下了基础。第三部分胶质瘤干祖细胞自噬活性低下与PTEN基因失活相关【目的】PTEN是一种与自噬密切相关又常常在神经系统肿瘤中失活的抑癌基因,通过第二部分研究明确了GSPCs分化抑制与其自噬活性低下有关。本部分的研究欲探讨是否GSPCs的自噬活性低下与PTEN基因失活相关。【方法】对比GSPCs和NSPCs中PTEN基因序列与genbank中报道的序列的异同,以及翻译后蛋白质多肽链中氨基酸残基序列的差异。通过腺病毒介导将外源的野生型PTEN转导入GSPCs,检测自噬活性的变化。【结果】与genbank报道的序列相比较,GSPCs中PTEN存在多数碱基的点突变,而NSPCs中PTEN基因则完全与genbank中报道的序列相吻合;翻译成为多肽链后,可见GSPCs的PTEN蛋白质在N端存在连续多个氨基酸残基(第8-14个氨基酸)变异,同时,第238和398两个位点上的氨基酸也发生突变。转导野生型的PTEN后,GSPCs的自噬活性显著增强。【结论】GSPCs内PTEN基因失活是其自噬活性低下的原因之一,对进一步研究与自噬性相关的靶分子具有重要价值。第四部分胶质瘤干祖细胞分化抑制后向血管内皮细胞转分化研究【目的】GSPCs分化抑制是胶质瘤的一种恶性表现。在第一至第三部分研究中,已从细胞、亚细胞和分子水平上证明了GSPCs的分化抑制与其自噬活性低下、PTEN基因失活相关;同时,胶质瘤内丰富的血供、活跃的血管形成也同样是胶质瘤恶性表现,但是否与GSPCs相关尚不清楚。所以,在第四部分我们旨在研究GSPCs是否可以通过向血管内皮细胞转分化进而参与胶质瘤的血管形成。【方法】将GSPCs培养于转分化培养基内(含多种生长因子),并以培养在普通条件下(仅含10%小牛血清)的GSPCs作为对照,10天后观察细胞的形态学变化。将GSPCs培养于Matrigel上,动态观察GSPCs的形态、结构的变化,10天后终止培养,免疫组织化学法检测CD31的表达;并取少许含有细胞的Matrigel行透射电镜检测,观察胶中细胞的超微形态学特征。同时,将GSPCs培养于缺氧或缺氧合并缺糖的条件下4小时,然后通过RT-PCR法、实时定量PCR以及免疫细胞化学荧光染色法检测CD31,CD34,KDR和vWF等血管内皮细胞标志物在转录以及翻译水平的变化。【结果】培养于分化培养基内的GSPCs在10天后呈典型的“铺路石”样的表型,而对照组则仅贴壁分化呈星形或梭形细胞;培养在Matrigel上的GSPCs随着时间的延长呈现一系列的变化,从单细胞逐渐变成管状结构;后者为CD31阳性,在透射电镜下呈中空的管腔,管腔周围的细胞具有血管内皮细胞样的超微结构特点。当GSPCs培养于缺氧或缺氧合并缺糖的条件下时,其多种血管内皮细胞的标志分子在转录和翻译水平显著上调。【结论】GSPCs在摸拟体内常见的缺血后引起缺氧条件下培养,可以向血管内皮细胞方向发生转分化。为进一步在动物体内原位接种GSPCs研究胶质瘤细胞自主产生新生血管找到了依据,对阐明肿瘤血管发生机理研究具有重要意义。更多还原

【Abstract】 PartⅠUltrastructural Mechanism of Differential Inhibition in Glioma Stem/Progenitor CellsObjective: In previous study, it has been demonstrated that the most significant difference of glioma stem/ progenitor cells (GSPCs) cultivated in vitro is consecutive differential inhibition comparing with neural stem/ progenitor cells (NSPCs), even occasional retrodifferentiation. In order to explore the underlying mechanism at subcellsular levels, we study the similarity and difference of ultrastructures between GSPCs and NSPCs.Methods: GSPCs were kept by our laboratory. NSPCs were isolated from human fetal brain tissue, and were cultivated, amplificated in vitro under the same conditions as that of GSPCs. After being identified, NSPCs, as well as GSPCs, were collected and prepared for the ultrastructural detections under scanning electron microscope (SEM) and transmission electron microscope (TEM), and their ultrastructures were compared qualitatively or quantitively.Results: Their common characteristics were undeveloped organelles, neurofilaments and microtubules in minority of cells, high nuclear-cytoplasmic ratio, and more euchromatin and less heterochromatin; some certain kinds of cell junctions could be seen between adjacent cells in both kinds of cellular spheres with lucent and dark cells according to electron densities. Differentia: a GSPC, which was bigger than a NSPC, had more microvilli, more developed ribosomes, endoplasmic reticulums, Golgi apparatus and mitochondrias, all of which were closely related to protein synthesis and secretion. Autophagosomes could be seen in some NSPCs but hardly in GSPCs. A GSPC frequently had two or more chromatospherites in a bigger nucleus than that of a NSPC. And the relative quantity of heterochromatin in a GSPC was higher than that in a NSPC. Synapsis-like structures and cellular membrane fusion could be found in neural spheres, but not in the spheres of GSPCs, where existed desmosome-like structures.Conclusions: Between GSPCs and NSPCs there were extensive similarities, as well as a lot of difference, which were the structural bases for the differential inhibition of GSPCs, and were also probably the mechanisms of retrodifferentiation of GSPCs, especially the different quantities of autophagosomes was worth of further study.PartⅡDifferential Inhibition of Glioma Stem/Progenitor Cells Were Related to Autophagy DepletionObjective: In PartⅠ, we found that there were much less autophagosomes in GSPCs than in NSPCs. In this part, we studied whether the autophagic activities between GSPCs and NSPCs were also different, as well, whether the difference was related to differential inhibition of GSPCs.Methods: MDC staining, immunofluorescence staining against microtubule-associated protein light chain-3 (LC3) and the expression of LC3 measured by western-blot were used to detect the autophagic activities of both GSPCs and NSPCs. The autophagic activities of GSPCs induced to differentiate were also detected. As well, autophagy inhibitors including 3- methyladenine (3-MA) and Bafilomycin A1 (BFA) were exerted to inhibit the autophagy of GSPCs when being induced to differentiate to study the effect of inhibition of autophagy on GSPCs’differentiation. Rapamycin (RPM), an autophagy promoter, was used to study its effects on the differentiation of GSPCs.Results: By comparative studies, we found that the autophagic activities of GSPCs were significantly lower than that of NSPCs. However, the autophagic activities increased markedly after GSPCs were induced to differentiate, and autophagy inhibitors, 3-MA or BFA, could inhibit the adherence and differentiation of GSPCs, and western-blot showed that the expressions of GFAP and MAP-2 were much lower than that of control group without autophagy inhibitors. What’s more, under special condition, RPM could promote the differentiation of GSPCs.Conclusion: The autophagic activity in NSPCs was much higher than that in GSPCs, which probably leads to the differential inhibition of GSPCs.PartⅢDeactivation of PTEN in GSPCs is related to Autophagy DepletionObjective: PTEN, an anti-oncogene, has a close relationship with autophagy, and often deactivated in tumors of nervous system. In partⅡ, we demonstrated the correlativity of differential inhibition of GSPCs and autophagy depletion. In this part, we want to explore the autophagy depletion in GSPCs was related to the deactivation of PTEN.Methods: The genetic sequences of PTEN, as well as the sequence of PTEN protein, in both GSPCs and NSPCs were compared with that reported by genbank. The wild-type PTEN was introduced into GSPCs by adenovirus, and then the autophagic activities of GSPCs were detected.Results: There were a lot of base mutations of PTEN in GSPCs comparing with the sequence of PTEN reported by genbank, but no base mutations in NSPCss. As well, for the pten protein sequences in GSPC, there were several mutations of amino-acid residues (8th-14th) locating at the N-end, the 238th and the 398th amino-acid residue. After the wild-type PTEN were introduced into GSPCs, the autophagic activities increased significantly.Conclusion: The mutation of PTEN in GSPCs is one of the mechanisms of low atuophagic activities in GSPCs, which is valuable for the further studies on target molecules related to autophagy.PartⅣEndothelial Cell Transdifferentiation of Glioma Stem/ Progenitor Cells In vitroObjective: Differential inhibition of GSPCs is a malignant characteristic of glioma. And in PartⅠtoⅢ, we have demonstrated, from cellular, subcellular, and molecular levels, that cellular autophagy depletion of GSPCs was involved in differential inhibition. Meanwhile, affluent blood supply and active angiogenesis are also malignant characteristics of glioma. In this part, we aimed to investigate whether glioma stem/ progenitor cells (GSPCs) have the capabilities to participate in angiogenesis by trans-differentiating into vascular endothelial cell-like cells.Methods: we cultivated GSPCs in endothelial differentiation medium for 10 days and to observe the changes of appearance. We also cultivated GSPCs on Matrigel for 10 days, and the process of morphological changes was observed. Ten days later, part of the Matrigel containing cells was detected for the expression of CD31 by immunohistochemistry, and part of them were used for the ultrastructural study. GSPCs were also cultivated under hypoxia or oxygen-glucose deprivation (OGD) for 4 hours, and the transcriptions and expressions of numerous types of molecular markers of vascular endothelial cell (VEC), including CD31, CD34, KDR and vWF were detected respectively with RT-PCR and immuocytochemistry.Results: Ten days after GSPCs were cultivated in endothelial differenetiation medium they present to be the typical "flagstone" appearance of VEC; when cultured on Matrigel, GSPCs gradually formed tubular-like structures in vitro, and cells, which formed the tubular-like structures, were positive to CD31 (a marker of VEC), and had similar ultrastructural characteristics of VEC under a transmission electron microscope. What’s more, when cultured in hypoxia or OGD for 4hours, the transcriptions and expressions of these VEC markers increased a lot.Conclusion: GSPC could transdifferentiate into VEC-like cells in vitro, which enlightened the further studies on the independent formation of new vessels by GSPCs in orthotopic implantation tumors, and was significant for the illumination of angiogenesis mechanism in tumors.更多还原