1H‐MRSI pattern perturbation in a mouse glioma: the effects of acute hyperglycemia and moderate hypothermia

MR spectroscopic Imaging (MRSI), with PRESS localization, is used here to monitor the effects of acute hyperglycemia in the spectral pattern of 11 mice bearing GL261 gliomas at normothermia (36.5–37.5°C) and at hypothermia (28.5–29.5°C). These in vivo studies were complemented by ex vivo high resolution magic angle spinning (HR‐MAS) analysis of GL261 tumor samples from 6 animals sacrificed by focused microwave irradiation, and blood glucose measurements in 12 control mice. Apparent glucose levels, monitored by in vivo MRSI in brain tumors during acute hyperglycemia, rose to an average of 1.6‐fold during hypothermia (p < 0.05), while no significant changes were detected at normothermia, or in control experiments performed at euglycemia, or in normal/peritumoral brain regions. Ex vivo analysis of glioma‐bearing mouse brains at hypothermia revealed higher glucose increases in distinct regions during the acute hyperglycemic challenge (up to 6.6‐fold at the tumor center), in agreement with maximal in vivo blood glucose changes (5‐fold). Phantom studies on taurine plus glucose containing solutions explained the differences between in vivo and ex vivo measurements. Our results also indicate brain tumor heterogeneity in the four animal tumors investigated in response to a defined metabolic challenge. Copyright © 2009 John Wiley & Sons, Ltd.     

SH3GL2基因在人脑胶质瘤中表达的研究

目的研究SH3GL2基因的表达改变与人脑胶质瘤之间的相关性。方法选择42例人脑胶质瘤和26例正常人脑组织标本,应用荧光定量PCR和Western blot方法检测标本中SH3GL2基因mRNA和蛋白的表达情况。结果与正常脑组织标本相比,SH3GL2基因在人脑胶质瘤组织标本中的mRNA和蛋白的表达存在明显下调,结果具有统计学差异(0.05)。结论SH3GL2基因的表达改变与人脑胶质瘤相关,是一个新的候选的肿瘤抑制基因。P     

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。     

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。     

Significance of perivascular tumour cells defined by CD109 expression in progression of glioma

In the progression of glioma, tumour cells often exploit the perivascular microenvironment to promote their survival and resistance to conventional therapies. Some of these cells are considered to be brain tumour stem cells (BTSCs); however, the molecular nature of perivascular tumour cells has not been specifically clarified because of the complexity of glioma. Here, we identified CD109, a glycosylphosphatidylinositol‐anchored protein and regulator of multiple signalling pathways, as a critical regulator of the progression of lower‐grade glioma (World Health Organization grade II/III) by clinicopathological and whole‐genome sequencing analysis of tissues from human glioma. The importance of CD109‐positive perivascular tumour cells was confirmed not only in human lower‐grade glioma tissues but also in a mouse model that recapitulated human glioma. Intriguingly, BTSCs isolated from mouse glioma expressed high levels of CD109. CD109‐positive BTSCs exerted a proliferative effect on differentiated glioma cells treated with temozolomide. These data reveal the significance of tumour cells that populate perivascular regions during glioma progression, and indicate that CD109 is a potential therapeutic target for the disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Migratory activity of human glioma cell lines in vitro assessed by continuous single cell observation

A new migration assay, the time-lapse individual cell migration assay (TIM-assay), was developed, which allows the observation of cells over 24 h under controlled conditions. Using this technique, the migratory behavior of 8 human glioblastoma cell lines in vitro was studied. Special features are simultaneous documentation of migratory parameters of individual cells, i.e., migration velocities and migration paths of individual cells. Migration velocity for cell populations of the same cell line ranged from 0 to 24 μm/h. The migration paths were examined for being directional. Two thirds of all cells showed directional migration. Migration paths were further classified according to visual judgements for being linear, oscillating or mixed. The migration index had a mean of 91%. The presented TIM-assay allows the assessment of several new parameters, that may be useful to identify subgroups of gliomas with different biological characteristics. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vivo characterization of several rodent glioma models by 1H MRS

The assessment of metabolites by (1)H MRS can provide information regarding glioma growth, and may be able to distinguish between different glioma models. Rat C6, 9 L/LacZ, F98 and RG2, and mouse GL261, cells were intracerebrally implanted into the respective rodents, and human U87 MG cells were implanted into athymic rats. Ethyl-nitrosourea induction was also used. Glioma metabolites [e.g. total choline (tCho), total creatine (tCr), N-acetylaspartate (NAA), lactate (Lac), glutamine (Gln), glutamate (Glu), aspartate (Asp), guanosine (Gua), mobile lipids and macromolecules (MMs)] were assessed from (1)H MRS using point-resolved spectroscopy (PRESS) [TE = 24 ms; TR = 2500 ms; variable pulse power and optimized relaxation delay (VAPOR) water suppression; 27-μL and 8-μL voxels in rats and mice, respectively] at 7 T. Alterations in metabolites (Totally Automatic Robust Quantitation in NMR, TARQUIN) in tumors were characterized by increases in lipids (Lip1.3: 8.8-54.5 mM for C6 and GL261) and decreases in NAA (1.3-2.0 mM for RG2, GL261 and C6) and tCr (0.8-4.0 mM for F98, RG2, GL261 and C6) in some models. F98, RG2, GL261 and C6 models all showed significantly decreased (p < 0.05) tCr, and RG2, GL261 and C6 models all exhibited significantly decreased (p < 0.05) NAA. The RG2 model showed significantly decreased (p < 0.05) Gln and Glu, the C6 model significantly decreased (p < 0.05) Asp, and the F98 and U87 models significantly decreased (p < 0.05) Gua, compared with controls. The GL261 model showed the greatest alterations in metabolites. (1)H MRS was able to differentiate the metabolic profiles in many of the seven rodent glioma models assessed. These models are considered to resemble certain characteristics of human glioblastomas, and this study may be helpful in selecting appropriate models.     

Foam cells generated by a combination of hyperglycemia and hyperlipemia in rats

Diabetes is a major risk factor for atherosclerosis, as well as hyperlipemia. Investigators have suggested that denatured lipoprotein in hyperglycemia transforms macrophages into foam cells, which correlates with the development or progression of atherosclerosis. In the present study, we examined the generation of foam cells in rats caused by a combination of hyperglycemia and hyperlipemia. Streptozotocin-induced diabetic male Wister rats were fed a high cholesterol diet (HCD) containing 1% cholesterol and 0.5% cholic acid to maintain a hyperglycemic and hyperlipemic state. Animals fed the HCD for 8 weeks or longer showed a high incidence of foam cell accumulation in the renal glomerulus, intima of aortic arch, splenic red pulp and marginal zone, liver sinusoid and intestinal lamina propria. The foam cells exhibited positive staining for antimonocyte/macrophage antibody and lipids in all these tissues. Anti-rat apolipoprotein B (apo B) antibody revealed that positive staining existed only in the cytoplasm of glomerular foam cells. These results suggest that the origin of these foam cells can be attributed to lipid-laden macrophages. The generation of foam cells in the hyperglycemia-hyperlipidemia supervening rat model presented in the present study might be a useful tool for investigations of the pathogenesis of foam cells.     

Proton MRS of early post-natal mouse brain modifications in vivo

NMR provides a non-invasive tool for the phenotypic characterisation of mouse models. The aim of the present study was to apply reliable in vivo MRS techniques for non-invasive investigations of brain development in normal and transgenic mice, by monitoring metabolite concentrations in different brain regions. The conditions of anaesthesia, immobilisation and respiratory monitoring were optimized to carry out in vivo MRS studies in young mice. All the experiments were performed in normal mice, at 9.4 T, applying a point-resolved spectroscopy (PRESS) sequence (TR = 2,000 ms; TE = 130 ms). We obtained reproducible in vivo (1)H NMR spectra of wild-type mouse brains as early as post-natal day 5, which allowed us to follow brain maturation variations from post-natal days 5 to 21. The survival rate of animals was between 66 and 90% at post-natal days 5 and 21, respectively. Developmental changes of metabolite concentrations were measured in three brain regions: the thalamus, a region rich in cell bodies, the olfactory bulb, rich in fibre tracts actively myelinated during brain maturation, and the cerebellum. The voxel size varied from 2 to 8 microL according to the size of the brain structure analysed. The absolute concentrations of the total creatine, taurine, total choline, N-acetylaspartate and of the glutamate/glutamine pool were determined from (1)H NMR spectra obtained in the different brain regions at post-natal day 5, 10, 15 and 21. Variations observed during brain development were in accordance with those previously reported in mice using ex vivo MRS studies, and also in rats and humans in vivo. Possibilities of longitudinal MRS analysis in maturing mice brains provide new perspectives to characterise better the tremendous number of transgenic mutant mice generated with the aim of decrypting the complexity of brain development and neurodegenerative diseases but also to follow the impact of environmental and therapeutic factors.     

高果糖大鼠血清TG、UA、血糖的动态变化规律及机制研究

目的： 动态观察高果糖模型大鼠血清甘油三酯（TG）、尿酸（UA）、 血糖（GLU）及全血3-磷酸甘油醛脱氢酶（GAPDH）活性和肝脏基因表达的变化规律,并探讨其病理机制。方法：随机将SD大鼠分为对照组、果糖组和非诺贝特治疗组。对照组予普通大鼠饲料;果糖组给予高果糖饲料［5］;菲诺贝特治疗组给予高果糖饲料,同时灌胃菲诺贝特100 mg·kg-1·d-1进行给药。对照组、果糖组灌胃等量蒸馏水。实验期间动态检测血清 TG、UA、GLU;改进正向反应法测定全血GAPDH活性;Quanti Gene（QG）分析法测肝GAPDH mRNA转录水平。 结果：与对照组比,果糖组7-28 d TG持续明显升高; 14-28 d UA 明显升高;GLU仅在28 d明显升高; GAPDH活性及基因表达7-28 d均明显降低。菲诺贝特治疗组仅在 7 d见明显降TG作用;而对UA 则有明显升高的作用;对 GLU在28 d有明显的降低作用;对GAPDH活性及基因表达仅在造模7 d时有明显升高作用;其余未见明显变化。 结论：①高果糖造模初期TG明显升高,随造模时间延长相继伴发UA及GLU 明显升高。②TG、UA、GLU升高可能与GAPDH活性及基因表达降低有关。③菲诺贝特在高TG时有明显降TG作用,对伴发高UA和高GLU时未见明显影响。④菲诺贝特降TG的作用机制可能与提高GAPDH 活性和基因表达有关。     

仙台病毒 Tianjin 株缺陷干扰颗粒体内外诱导大鼠脑胶质瘤细胞凋亡的研究

目的探讨仙台病毒Tianjin株缺损干扰颗粒（ defective interfering particles ,DI颗粒）体内外诱导大鼠脑胶质瘤细胞C6凋亡的作用。方法将不同滴度仙台病毒Tianjin株DI颗粒分别与大鼠脑胶质瘤细胞C6作用不同时间,以培养基作为阴性对照、完整病毒作为阳性对照,通过DNA片段琼脂糖凝胶电泳、TUNEL染色、AnnexinⅤ-FITC/PI标记流式细胞仪分析等检测细胞凋亡情况。建立大鼠皮下胶质瘤模型,通过测量肿瘤大小观察DI颗粒抑瘤作用,病理切片HE染色观察肿瘤组织病理变化,TUNEL法检测肿瘤组织细胞凋亡情况。结果 C6细胞在体外经DI颗粒诱导后, DNA片段琼脂糖凝胶电泳呈阶梯状;流式细胞仪及TUNEL检测显示,DI颗粒组与完整病毒组细胞凋亡率明显增高,且呈时间-剂量依赖型。动物实验结果显示,DI颗粒和完整病毒均可明显抑制肿瘤生长;肿瘤组织病理切片HE染色显示DI颗粒组和完整病毒组瘤结节内瘤细胞较少;TUNEL原位细胞凋亡检测显示DI颗粒组和完整病毒组凋亡细胞明显增加,以上结果与阴性对照组比较差异均有统计学意义（P＜0．01）。结论仙台病毒Tianjin株DI颗粒在体内外均能引起大鼠脑胶质瘤C6细胞凋亡,且呈时间-剂量依赖型,提示DI颗粒有辅助治疗脑胶质瘤的可能性。     

Expression pattern of Popdc2 during mouse embryogenesis and in the adult.

The Popdc2 gene is a member of the Popeye domain containing gene family encoding membrane proteins with prominent expression in striated and smooth muscle tissue. After introducing a LacZ reporter gene into the Popdc2 locus, expression was studied during embryonic development and postnatal life. At embryonic day (E) 7.5, expression was present in cardiac and extraembryonic mesoderm. At E10.5, expression was found in heart, somites, and mesothelial cells lining the coelom. At E12.5, expression was present in the coelomic mesothelium, pericardial and myocardial layer of the heart, skeletal muscle, bladder, gut, and umbilical vessels. Postnatal expression was found in cardiac and skeletal muscle and in the smooth muscle layer of colon, rectum, and bladder. In the stomach, Popdc2 was exclusively present in the pyloric epithelium. In conclusion, Popdc2 is expressed in various muscle and nonmuscle cell types during embryonic development and in postnatal life.     

探讨人胶质瘤细胞系U87细胞中反义miR-21诱导凋亡机制

目的 探讨下调miR-21诱导人胶质瘤细胞系U87细胞凋亡机制.方法 应用化学方法合成的反义miR-21寡聚核苷酸(anti-miR-21),通过瞬转法转染U87细胞,检测U87细胞凋亡、增殖、侵袭等变化,并结合生物信息学分析、Western印迹验证在U87细胞中miR-21和PTEN基因及caspase间关系.结果 体外转染反义miR-21寡聚核苷酸能明显抑制U87细胞生长,诱导其凋亡,降低侵袭能力;增加caspase-3表达活性,活化caspase-9表达,但不影响PTEN和caspase-8表达.结论 miR-21可能是人胶质瘤U87细胞的抗凋亡微RNA(microRNA,miRNA),反义miR-21可能通过easpase-9、3而不是PTEN诱导肿瘤细胞凋亡.     

PcG蛋白EZH2在全反式维甲酸诱导的小鼠iPS细胞系分化过程中的作用

目的探讨组蛋白甲基转移酶EZH2在小鼠iPS细胞系IP14D-1向生殖细胞分化过程中的作用。方法将小鼠iPS细胞系IP14D-1通过悬浮培养分化形成拟胚体(iEBs)作为向生殖细胞分化的启动步骤,1μmol/L全反式维甲酸(atRA)持续诱导iEBs 2、4和7 d,采用实时RT-PCR、荧光定量RT-PCR、免疫荧光检测atRA处理前后EZH2和生殖细胞分化标志性基因Stra8在iEBs中的表达。结果在atRA诱导IP14D-1来源的iEBs中,EZH2表达在诱导后2 d达到高峰,伴随atRA诱导,EZH2表达减弱;而生殖细胞分化标志性基因Stra8其变化特点与EZH2相反,无atRA诱导时EBs表达Stra8较弱,伴随atRA诱导,Stra8表达增强。EZH2和Stra8阳性信号分别定位于胞膜和胞质,其变化特点与PT-PCP结果相一致。结论 atRA诱导使EZH2低水平表达时期提前出现,伴随Stra8增强表达,启动了小鼠iPS细胞向生殖细胞的分化进程。     

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。     

Expression pattern of NLRC5 in the postnatal mouse brain

Nucleotide oligomerization domain-like receptors (NLRs), belonging to a large family of pattern recognition receptors, participate in the host’s first line of defense against invading pathogens. Caspase recruitment domain containing 5 (NLRC5), the largest member in the NLR family, is demonstrated to be involved in the innate immune response and inflammatory diseases far and wide. Recent studies report that NLRC5 is associated with some central nervous system (CNS) diseases. Besides, NLRC5 is a mastery regulator for the expression of MHC class I both in the immune system and the CNS, while MHC class I is expressed and exerts its function in the brain. Therefore, it is necessary to investigate the expression pattern of NLRC5 in the developing and adult CNS. In our study, postnatal brain sections of C57BL/6 J mice are analyzed for the expression of NLRC5 protein by immunofluorescence. In the postnatal stages of developing telencephalon, NLRC5 exhibits a spatial and temporal expression pattern. NLRC5 is time-specifically expressed in subfields of hippocampus and different layers of prefrontal cortex. Moreover, it is shown that NLRC5 is highly cell type specific. It can be expressed in large quantities by neurons and microglia, but rarely expressed by astrocytes. Taken together, our research is important for further understanding the biological characteristics of NLRC5 and its function in the CNS.     

Feasibility of neurochemically profiling mouse embryonic brain and its development in utero using 1H MRS at 14.1 T

We aimed to evaluate the feasibility of neurochemical profiling of embryonic mouse brain developments in utero and to seek potential in vivo evidence of an energy shift in a mitochondrial pyruvate carrier 1 (MPC1) deficient mouse model. C57BL/6 embryonic mouse brains were studied in utero by anatomical MRI and short echo localized proton (¹H) MRS at 14.1 T. Two embryonic stages were studied, the energy shift (e.g., embryonic day 12.5–13, E12.5–13) and close to the birth (E17.5–18). In addition, embryonic brains devoid of MPC1 were studied at E12.5–13. The MRI provided sufficient anatomical contrasts for visualization of embryonic brain. Localized ¹H MRS offered abundant metabolites through the embryonic development from E12.5 and close to the birth, e.g., E17.5 and beyond. The abundant neurochemical information at E12.5 provided metabolic status and processes relating to cellular development at this stage, i.e., the energy shift from glycolysis to oxidative phosphorylation, evidenced by accumulation of lactate in E12.5–13 embryonic brain devoid of MPC1. The further evolution of the neurochemical profile of embryonic brains at E17.5–18 is consistent with cellular and metabolic processes towards the birth. Localized ¹H MRS study of embryonic brain development in utero is feasible, and longitudinal neurochemical profiling of embryonic brains offers valuable insight into early brain development.     

1H MRS characterization of neurochemical profiles in orthotopic mouse models of human brain tumors

Glioblastoma (GBM), the most common primary brain tumor, is resistant to currently available treatments. The development of mouse models of human GBM has provided a tool for studying mechanisms involved in tumor initiation and growth as well as a platform for preclinical investigation of new drugs. In this study we used ¹H MR spectroscopy to study the neurochemical profile of a human orthotopic tumor (HOT) mouse model of human GBM. The goal of this study was to evaluate differences in metabolite concentrations in the GBM HOT mice when compared with normal mouse brain in order to determine if MRS could reliably differentiate tumor from normal brain. A T_E =19 ms PRESS sequence at 9.4 T was used for measuring metabolite levels in 12 GBM mice and 8 healthy mice. Levels for 12 metabolites and for lipids/macromolecules at 0.9 ppm and at 1.3 ppm were reliably detected in all mouse spectra. The tumors had significantly lower concentrations of total creatine, GABA, glutamate, total N‐acetylaspartate, aspartate, lipids/macromolecules at 0.9 ppm, and lipids/macromolecules at 1.3 ppm than did the brains of normal mice. The concentrations of glycine and lactate, however, were significantly higher in tumors than in normal brain. Copyright © 2014 John Wiley & Sons, Ltd.