利用人脑类器官构建胶质瘤侵袭模型

目的利用人脑类器官建立胶质瘤侵袭模型并用于评价胶质瘤细胞侵袭能力。方法通过神经定向诱导分化形成人胚胎干细胞（embryonicstemcells,ESCs）来源的脑类器官;将人脑类器官与人胶质瘤干细胞（gliomastemcells,GSCs）共培养,构建胶质瘤侵袭模型;检测不同类型GSCs在脑类器官中的侵袭深度,评价其侵袭能力。结果（1）人ESCs经诱导分化,于第31天形成大量神经上皮芽结构,免疫荧光染色显示神经干细胞特异性蛋白Sox2、Pax6和nestin表达,提示脑类器官形成;（2）与脑类器官共培养后,胶质瘤细胞侵入脑类器官中生长,成功建立胶质瘤侵袭模型;（3）不同类型GSCs在脑类器官中的侵袭深度存在差别。结论本研究建立了胶质瘤侵袭类器官模型,该模型可用于评价GSCs的侵袭能力。     

实时荧光定量PCR方法检测人正常组织及癌组织中CD109蛋白的表达

目的CD109是最近克隆到的一种细胞表面抗原,为糖基化磷脂酰肌醇联结的糖蛋白,属于含硫酯的啦巨球蛋白家族成员,本研究的目的是探讨CD109基因在人正常组织及癌组织中的分布。方法使用实时荧光定量PER方法分析了CD109在人正常组织及癌组织中的表达。结果研究结果表明,该蛋白的mRNA丰度在睾丸组织中最高,而在其他组织中较低,在癌组织标本中检测到CD109表达水平上调。结论以上结果提示表达在细胞表面的CD109蛋白可能是治疗恶性肿瘤的一个潜在的分子靶点。     

Feeding cancer's sweet tooth: specialized tumour vasculature shuttles glucose in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal neoplasm characterized by a ‘fortress’ of thick collagen fibres, abundant myofibroblasts, and paradoxically reduced vascularization compared to normal pancreas. Despite these features, PDAC shows no reduction in the uptake of glucose that fuels tumour cell survival. In new work published in The Journal of Pathology, Saiyin and colleagues have identified a novel adaptation of PDAC tumour endothelium; namely, ‘hairy‐like’ basal microvilli that increase the total vascular surface area and correlate with regions of highest glucose uptake. Since basal microvilli are not present on normal pancreatic blood vessels, their presence may add diagnostic value and blocking their function is a potential new treatment strategy for PDAC. This novel finding of basal microvilli on PDAC endothelium is a striking example of how phenotypic plasticity in tumour blood vessels contributes to tumour growth and progression, independent of conventional modes of angiogenesis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

From expert opinion to evidence-based: changes in the gold standard of primary brain tumour diagnosis

Since 1997, significant progress has been made in the WHO guidelines for the diagnosis of primary tumours of the central nervous system. A large group of international experts was involved in editing the content; consensus on definitions and classifications was sought; and updated findings of genomic investigations were included. Nevertheless, significant inter-observer variability still exists in the histopathological diagnosis of several tumour types. The challenge for the near future is to identify histological and genotypic characteristics with prognostic or predictive value. With that aim, testing histological or molecular parameters in prospective clinical studies is indicated.     

Glioblastoma, cancer stem cells and hypoxia

Glioblastoma (GBM) prognosis remains dismal, with most patients succumbing to disease within 1 or 2 years of diagnosis. Recent studies have suggested that many solid tumors, including GBM, are maintained by a subset of cells termed cancer stem cells (CSCs). It has been shown that these cells are inherently radio- and chemotherapy resistant, and may be maintained in vivo in a niche characterized by reduced oxygen tension (hypoxia). This review examines the recently described effects of hypoxia on CSC in GBM, and the potential promise in targeting the hypoxic pathway therapeutically.     

Stromal regulation of hemopoietic stem cells in long-term human bone marrow tissue cultures under the effect of parathyroid hormone

We studied the interaction between different categories of hemopoietic precursors with parathyroid hormone-activated stromal microenvironment. Improved survival of early precursors capable long-term hemopoiesis maintenance and increased number of later short-term repopulating precursors was demonstrated on the model of co-culturing of human bone marrow cells on a layer of adherent cells of long-term bone marrow cultures treated with parathyroid hormone. These changes correlate with increased expression of genes involved in the maintenance of the hemopoietic stem cells in the sublayer activated by parathyroid hormone. Simultaneously, the expression of some stromal differentiation genes, adhesion molecules for hemopoietic stem cells, and growth factors increased in adherent cell layers treated with parathyroid hormone. These findings attest to activating effect of parathyroid hormone on cells forming the niches for both early and later hemopoietic precursors, and hence parathyroid hormone can be used as a potential agent promoting expansion of early hemopoietic stem cells ex vivo. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 4, pp. 218–222, December, 2006     

C6胶质瘤细胞诱导神经干细胞分化的初步研究

为了探讨C6胶质瘤细胞分泌的因子对神经干细胞分化的作用及机制。本研究从Wistar孕鼠(E17～E18)纹状体组织中分离、培养神经干细胞,将其分离纯化后进行nestin免疫荧光染色鉴定后分为两组进行诱导分化实验。对照组:RPMI1640培养液+基础培养基(1∶1配制);处理组:C6胶质瘤细胞培养上清+基础培养基(1∶1配制)。采用免疫印迹法分析胶质瘤细胞条件培养液作用于神经干细胞后的不同时相(1、3、5d)MAP-2蛋白的表达情况;通过RT-PCR检测不同时相(1、3、5d)MAP-2的表达及调控神经干细胞向神经元分化的多种转录因子如:neurogenin3(Ngn3)、neuroD和neuroD2的mRNA的表达情况。结果显示:处理组MAP-2在蛋白和mRNA水平的表达量均明显高于对照组(P<0.01),并且处理组的Ngn3、neuroD和neuroD2的mRNA水平比对照组也有明显升高。上述结果提示,C6胶质瘤细胞条件培养液能够促进神经干细胞向神经元分化,此作用可能与Ngn3、neuroD和neuroD2等转录调控因子表达水平的提高有关。     

体外模拟肿瘤微环境中致大鼠MSCs瘤化因子分析

 目的 探讨模拟肿瘤微环境中骨髓间充质干细胞（MSCs）肿瘤转化的发生与HGF、IL-6、BFGF关系。方法 实验组为C6胶质瘤与MSCs间接共培养模拟肿瘤微环境下MSCs的生长,对照组为星型胶质细胞与MSCs间接共培养模拟正常微环境下MSCs的生长,空白对照组MSCs单独培养;QPCR,免疫荧光检测各组的P53和MDM2的基因和蛋白的表达;ELISA检测各组HGF、IL-6、BFGF的表达;免疫荧光检测对应升高的细胞因子作用于MSCs后的STAT3蛋白的表达。结果 实验组的MSCs形态肿瘤化;MSCs的MDM2 mRNA是对照组的1.56±0.21(P<0.05),MDM2蛋白表达率比对照组升高90±7%(P<0.01);MSCs的P53的mRNA表达是对照组的0.55±0.10, 而P53突变蛋白表达率比对照组升高87±5%(P<0.01);HGF、IL-6水平比对照组中达水平升高(P<0.05);过量IL-6处理组的MSCs的STAT3激活蛋白表达率比正常量IL-6处理组明显升高(P<0.01)。结论 IL-6参与MSCs在肿瘤微环境下的肿瘤转化的发生。     

C6胶质瘤细胞体外诱导神经干细胞的迁移

目的：体外观察 C6胶质瘤细胞的培养上清是否有诱导神经干细胞迁移的作用。方法：实验组取处于指数生长期的 C6胶质瘤细胞或星形胶质细胞的无血清培养上清加入细胞培养室的下室,并加无血清培养基;培养室的上室加处于对数生长期的神经干细胞球悬液,培养箱中共培养,光镜下计数迁移的细胞球数。结果：C6胶质瘤细胞的上清引起神经干细胞球迁移的数目明显多于星形胶质细胞的上清和新鲜无血清培养基。结论：C6胶质瘤细胞的培养上清中存在诱导神经干细胞球迁移的物质,这将为研究诱导神经干细胞迁移的物质提供很大的便利。     

颈内动脉移植骨髓间充质干细胞向C6胶质瘤的趋化迁移

目的探讨经颈内动脉移植的骨髓间充质干细胞(BMSC)向C6胶质瘤的趋化迁移能力及小剂量S缓激肽对其影响。方法密度梯度离心法分离BMSCs,体外培养、传代纯化。利用Transwell侵袭小室建立体外趋化迁移模型。立体定向方法建立大鼠C6胶质瘤模型。用BrdU标记BMSCs,经荷瘤侧颈内动脉灌注,观察其向C6胶质瘤组织的趋化能力以及在使用小剂量缓激肽后对其的影响。结果通过密度梯度离心法传至第3代获得了纯化的MSCs。体外模型中BMSCs可通过聚碳酸酯膜向下室内的C6细胞迁移。经颈内动脉灌注后BMSCs可以存活,并表现出了向脑胶质瘤趋化迁移的特性。分布区域主要位于肿瘤内部,在肿瘤与正常脑组织的交界位置亦有分布。使用小剂量缓激肽可以明显增加BMSCs通过血肿瘤屏障的数量。结论BMSCs具有通过血肿瘤屏障向C6胶质瘤趋化迁移的能力,经颈内动脉灌注是其有效的移植途径,小剂量缓激肽选择性开放血肿瘤屏障有助于BMSCs趋化迁移进入C6胶质瘤组织。     

间充质干细胞对神经胶质瘤趋化能力研究

本文探讨了间充质干细胞(MSCs)对胶质瘤组织的趋化能力及分布规律。从新生儿脐血中分离出MSCs加以培养,用流式细胞仪检测表面抗原,同时做体外诱导分化实验,证明间充质干细胞性质。立体定向接种C6大鼠胶质瘤细胞,建立胶质瘤模型。将 5 -溴脱氧尿核苷(5- BrdU)标记的人间充质干细胞借助立体定向仪打入胶质瘤模型鼠预定靶区,分为原位组、同侧半球组和同侧脑室组。数日后处死大鼠,灌注固定取脑,制成石蜡切片。以苏木素伊红 (H.E. )染色确定胶质瘤组织的范围;免疫组织化学染色显示MSCs,观察其在胶质瘤组织和周围正常脑组织中的分布规律。结果显示原位组、同侧半球组和同侧脑室组均可见MSCs在胶质瘤组织中广泛分布,而在周围正常脑组织中很少见。特别是在胶质瘤组织与正常组织交界处,这一定向分布更为明显。这一结果表明人间充质干细胞对胶质瘤组织有明显趋化性,能从远处向胶质瘤组织迁移并定位于其中,提示MSCs可作为一种潜在的细胞载体用于神经胶质瘤的靶向治疗。     

High-level expression of CD109 is frequently detected in lung squamous cell carcinomas

CD109 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein, which is a member of the alpha2-macroglobulin/C3, C4, C5 family of thioester-containing proteins. It has been reported that CD109 is expressed in a subset of hematopoietic cells, endothelial cells and several kinds of human tumors. Herein it is reported that the CD109 protein is preferentially expressed in lung squamous cell carcinomas compared with other types of lung carcinoma including adenocarcinomas, large cell carcinomas and small cell carcinomas. Immunohistochemical staining of surgically resected lung specimens using an anti-CD109 antibody detected CD109 expression in basal cells of bronchial and bronchiolar epithelia and myoepithelial cells of bronchial secretary glands, but not in bronchial and bronchiolar apical epithelial cells and alveolar epithelial cells. Furthermore, the CD109 immunoreactivity was observed in squamous cell carcinomas at a high frequency compared with other types of lung carcinoma. Although the detailed function of CD109 protein is unclear, these results suggest that CD109 expression may play a role in the development of lung squamous cell carcinoma.     

The Utility and Limitations of Neurosphere Assay,CD133 Immunophenotyping and Side Population Assay in Glioma Stem Cell Research

The newly proposed glioma stem cell (GSC) hypothesis may re‐model the way we diagnose and treat the tumor, which highlights the need for a complete knowledge on the genetic and epigenetic “blueprints” of GSCs. To identify the true “stemness” signatures, pure GSC populations are primarily needed. Reliable in vitro methods enriching for GSCs and thereby identifying the key stem‐like characteristics constitute the preliminary step forward. We discuss in this review the current widely used methods for enriching and isolating GSCs, namely neurosphere assay, CD133 Immunophenotyping and side population assay, and detail their limitations and potential pitfalls that could complicate interpretation of corresponding results.     

鼠神经干细胞对胶质瘤C6细胞侵袭能力的影响

目的探讨鼠神经干细胞对鼠胶质瘤C6细胞侵袭能力的影响及机制。方法取新生1～2天SD大鼠大脑皮层组织,于无血清培养基中分离培养神经干细胞并进行NES免疫细胞化学染色,并将神经干细胞和鼠胶质瘤C6细胞于体外共培养,利用2D、3D生长实验和Transwell实验分别检测与鼠神经干细胞共培养鼠胶质瘤C6细胞(实验组)和单独培养鼠胶质瘤C6细胞(对照组)的侵袭能力。结果 2D实验中C6细胞的对照组和实验组的平均直径分别为(18.53±1.32)μm和(13.44±1.45)μm,差异有统计学意义(P<0.01)。3D实验中C6细胞的对照组和实验组的平均直径分别为(20.34±1.25)μm和(15.52±1.43)μm,差异有统计学意义(P<0.01)。Transwell侵袭实验发现胶质瘤C6细胞组中对照组和实验组平均视野侵袭细胞为(36.45±1.36)个和(22.73±1.66)个,差异有统计学意义(P<0.01)。结论与神经干细胞共同培养后C6细胞的侵袭能力明显降低。     

Characterization of side-population cells in human normal endometrium

BACKGROUND: It has been proposed that the human endometrium may contain a population of adult stem cells that are responsible for its remarkable regenerative capability. Recently, a subset of stem cells or progenitor cells in adult tissue has been identified as side-population cells (SP cells) displaying low staining with Hoechst 33342 by fluorescence-activated cell sorter (FACS) analysis. In this study, we isolated SP cells from the human endometrium and analysed their properties. METHOD: Endometrial cells were obtained using enzymatic digestion from uterine hysterectomy for the treatment of uterine myoma and stained with Hoechst 33342 dye either alone or in combination with verapamil. The cells were then analysed using FACS. RESULTS: SP cells were present among normal human endometrial cells. Most SP cells were enriched in the CD9(-)CD13(-) fraction. These SP cells showed long-term repopulating properties and produced gland (CD9(+))- and stroma (CD13(+))-like cells. CD9(-)CD13(-) cells isolated from the endometrium also generated gland- or stroma-like cells. CONCLUSIONS: SP cells in the human endometrium can function as progenitor cells. This is the first report of the phenotype of SP cells from normal human endometrial cells.