丙酮酸乙酯对人脑胶质母细胞瘤U251细胞和HMGB1的抑制作用

目的探讨丙酮酸乙酯(EP)对人脑胶质母细胞瘤U251细胞的作用及可能的机制。方法不同浓度EP处理U251细胞后,分别用细胞计数试剂盒(CCK-8)和平板克隆的方法检测EP对U251增殖及克隆形成能力的影响,并采用Hoechst 33258染色和膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)染色流式细胞术观察细胞凋亡的变化,蛋白印迹法分析高迁移率族蛋白B1(HMGB1)表达水平的变化。结果 EP(10、15、20 mmol/L)分别作用8 h、16 h、24 h后均能抑制U251细胞的增殖,且存在显著的剂量-时间效应关系。与对照组相比,EP处理后U251细胞克隆形成能力减弱、细胞凋亡率升高。不同浓度组EP作用U251细胞24 h后HMGB1的蛋白表达水平随药物浓度的增加而降低。结论 EP能明显抑制人脑胶质母细胞瘤U251增殖,并诱导其凋亡,其机制可能与下调HMGB1的表达水平有关。     

Interleukin 1-β- and tumor necrosis factor-α-mediated regulation of ICAM-1 gene expression in astrocytes requires protein kinase C activity

Several adhesion molecules including intracellular adhesion molecule-1 (ICAM-1) are expressed by astrocytes, the predominant glial cell of the central nervous system (CNS). Such molecules are important in the trafficking of leukocytes to sites of inflammation, and in lymphocyte activation. ICAM-1 is constitutively expressed by neonatal rat astrocytes, and expression is enhanced by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), with IL-1β and TNF-α being the strongest inducers. In this study, we have examined the nature of the second messengers involved in ICAM-1 gene expression induced by the cytokines IL-1β and TNF-α. Our results indicate that stimuli related to protein kinase C (PKC) such as the phorbol ester phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 increase ICAM-1 mRNA expression, whereas cyclic nucleotide analogs and PKA agonists have no effect. Pharmacologic inhibitors of PKC such as H7, H8, and calphostin C inhibit ICAM-1 gene expression inducible by IL-1β and TNF-α. Prolonged treatment of astrocytes with PMA results in a time-dependent downregulating of the PKC isoforms α, δ, and ?, and a concomitant diminution of ICAM-1 mRNA expression induced by IL-1β, TNF-α, and PMA itself at specific time points post-PMA treatment. These data, collectively, demonstrate a role for various PKC isoforms in IL-1β and TNF-α enhancement of ICAM-1 gene expression in rat astrocytes. © 1995 Wiley-Liss, Inc.     

人参皂甙Rh2对人胶质瘤细胞U87MG凋亡的影响

目的 观察人参皂甙Rh2对胶质瘤细胞凋亡的影响并初步探讨其可能机制。方法 将培养的人胶质瘤细胞U87MG随机分为人参皂甙Rh2组、人参皂甙Rh2+尼莫地平组和对照组。人参皂甙Rh2组在常规培养细胞时加入20 μg/ml的人参皂甙Rh2，人参皂甙Rh2+尼莫地平组在人参皂甙Rh2组培养细胞时加入浓度为10 μmol/L的尼莫地平。利用流式细胞仪检测U87MG细胞凋亡，利用激光共聚焦显微镜和流式细胞仪检测U87MG细胞内钙离子浓度。结果 与对照组相比，人参皂甙Rh2促进U87MG细胞凋亡（P<0.05），且增加细胞内游离钙离子浓度（P<0.05）；尼莫地平显著减少人参皂甙Rh2引起的U87MG细胞凋亡（P<0.05）。结论 人参皂甙Rh2可以通过增加细胞内游离钙离子浓度促进U87MG细胞凋亡。     

Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: Evidence for a nuclear energy shuttle

To test the hypothesis that embryonic brain cells utilize a creatine phosphate energy shuttle, we examined the pattern of creatine kinase (CK) isoform expression and localization in the fetal rat brain. Moderate levels of CK activity are present at embryonic day 14 (7 U/mg protein) and decrease slightly until 3 days postpartum followed by a rapid, fourfold up- regulation to adult levels by 1 month (18 U/mg protein). In parallel with changes in enzyme activity, there is a biphasic and coordinate pattern of expression of brain-type CK (BCK) and ubiquitous mitochondrial CK (uMtCK) determined by nondenaturing electrophoresis and immunoblot analysis. The localization of CK isoforms was examined by immunocytochemistry, and, during the fetal period, BCK and uMtCK immunoreactivity was detected throughout the central and peripheral nervous system, especially in neuroepithelial regions of the cerebral vesicles and spinal cord. In large cells within the olfactory neuroepithelium and ventral spinal cord, differential compartmentation of CK isoforms was evident, with BCK localized primarily in cell nuclei, whereas uMtCK immunoreactivity was present in the cell body (but not within nuclei). In olfactory bulb neuroepithelium, both isoforms were expressed in the middle zone of the germinal layer associated with DNA synthesis. In embryonic skeletal and cardiac muscle, which also express BCK, the same compartmentation of BCK was seen, with BCK localized primarily in the cell nucleus of cardiac and skeletal myoblasts. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and uMtCK isoforms in the fetal brain that, in some cells, provides the anatomic basis for a nuclear energy shuttle. © 1995 Wiley-Liss, Inc.     

Effect of substance P and protein kinase inhibitors on β-amyloid peptide-induced proliferation of cultured brain cells

The present study investigated the effect of substance P (SP) and protein kinase inhibitors (H7 and HA1004) on β-amyloid peptide-induced proliferation of neonatal rat brain cells in primary cultures. The β-amyloid peptide_1–28 (designated as βAP28), at nanomolar concentrations (10⁻⁹ M), significantly (P 0.05) increased the proliferation of brain cells (presumably non-neuronal) as measured by [³H]thymidine uptake into DNA (mitogenesis). The effect was dependent on time of cultured, concentration of βAP28, and presence of fetal calf serum. The supplementation of SP into cell cultures at time zero reversed the proliferative response of βAP28. Moreover, the βAP28-induced proliferation was inhibited by protein kinase inhibitor H7, but not by HA1004. Since H7 is a selective protein kinase C (PKC) inhibitor and SP action involves PKC, we conclude that βAP28 induces normal brain cell proliferation through PKC pathway of cell signaling.     

Dissociated expression of mitochondrial and cytosolic creatine kinases in the human brain: a new perspective on the role of creatine in brain energy metabolism

The phosphocreatine/creatine kinase (PCr/CK) system in the brain is defined by the expression of two CK isozymes: the cytosolic brain-type CK (BCK) and the ubiquitous mitochondrial CK (uMtCK). The system plays an important role in supporting cellular energy metabolism by buffering adenosine triphosphate (ATP) consumption and improving the flux of high-energy phosphoryls around the cell. This system is well defined in muscle tissue, but there have been few detailed studies of this system in the brain, especially in humans. Creatine is known to be important for neurologic function, and its loss from the brain during development can lead to mental retardation. This study provides the first detailed immunohistochemical study of the expression pattern of BCK and uMtCK in the human brain. A strikingly dissociated pattern of expression was found: uMtCK was found to be ubiquitously and exclusively expressed in neuronal populations, whereas BCK was dominantly expressed in astrocytes, with a low and selective expression in neurons. This pattern indicates that the two CK isozymes are not widely coexpressed in the human brain, but rather are selectively expressed depending on the cell type. These results suggest that the brain cells may use only certain properties of the PCr/CK system depending on their energetic requirements.     

Histone deacetylase inhibitor, FK228, induces apoptosis and suppresses cell proliferation of human glioblastoma cells in vitro and in vivo

We investigated the effects of FK228 on cell proliferation and apoptosis against human glioblastoma (GM) T98G, U251MG, and U87MG cells. Upon exposure to FK228, cell proliferation was inhibited, and apoptosis detected by the cleavage of CPP32 was induced. FK228 increased the expression levels of p21 (WAF-1) and of pro-apoptotic Bad protein in all GM cells. Furthermore, FK228 treatment also reduced the anti-apoptotic protein Bcl-xL in all GM cells and anti-apoptotic Bcl-2 in U87MG cells, thereby shifting the cellular equilibrium from life to death. An increased accumulation of histone H4 was detected in the p21 (WAF-1) promoter and the structural gene (exon 2) and the Bad structural gene (exon 2 and 3) upon treatment with FK228, as assessed by chromatin immunoprecipitation (ChIP) assay. Thus, the results indicated that an increased expression of p21 (WAF1) and Bad due to FK228 is regulated, at least in part, by the degree of acetylation of the gene-associated histone. We also found that FK228 inhibits cellular invasiveness and decreases MMP-2 activity. In addition, the growth of transplanted human GM m-3 cells into the subcutaneous tissue of hereditary athymic mice was significantly inhibited, and apoptosis was induced with FK228 treatment. The results suggested that FK228 might be useful in the treatment of human GM, although further studies will be needed.     

胶质瘤U87细胞中AKT2基因表达对化疗药物替尼泊苷敏感性的影响

目的探讨脑胶质瘤U87细胞中AKT2基因表达对替尼泊苷（VM-26）敏感性的影响。方法体外将慢病毒介导的AKT2-shRNA表达载体转染胶质瘤U87细胞,作为实验组;转染GFP—shRNA的U87细胞作为阴性对照组,未转染的U87细胞作为空白对照组。应用RT—PCR、Westernblot检测3组U87细胞中AKT2mRNA和蛋白的表达变化。应用MTT法检测3组U87细胞对VM-26敏感性的变化。结果与阴性对照组和空白对照组比较,实验组转染后的U87细胞AKT2mRNA和蛋白的表达水平明显下降（P〈0．01）。实验组VM-26对U87细胞的半数抑制量（IC50）为（6．46±0．42）μg／ml,阴性对照组为（1．16±0．25）μg／ml,空白对照组为（1．15±0．22）μg／ml,实验组与两对照组间差异有统计学意义（P〈0．05）。结论AKT2-shRNA抑制AKT2基因表达,能增加人脑胶质瘤U87细胞株对化疗药物VM-26的敏感性。     

Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain

Cytosolic tyrosine kinases play a critical role both in neural development and in adult brain function and plasticity. Here we isolated a cDNA with high homology to human Ack1 and mouse Tnk2. This cDNA directs the expression of a 125-kD protein that can be autophosphorylated in tyrosines. Initially, this clone was named Pyk1 for proline-rich tyrosine kinase (Lev et al., 1995); however, since it corresponds to the mouse homolog of Ack1, here we called it Ack1/Pyk1. In this study we show that Ack1/Pyk1 mRNA and protein is highly expressed in the developing and adult brain. The highest levels of Ack1/Pyk1 expression were detected in the hippocampus, neocortex, and cerebellum. Electron microscopy studies showed that Ack1/Pyk1 protein is expressed in these regions both at dendritic spines and presynaptic axon terminals, indicating a role in synaptic function. Furthermore, we demonstrate that Ack1/Pyk1 mRNA levels are strongly upregulated by increased neural activity, produced by intraperitoneal kainate injections. During development, Ack1/Pyk1 was also expressed in the proliferative ventricular zones and in postmitotic maturing neurons. In neuronal cultures, Ack1/Pyk1 was detected in developing dendrites and axons, including dendritic tips and growth cones. Moreover, Ack1/Pyk1 colocalized with Cdc42 GTPase in neuronal cultures and coimmunoprecipitated with Cdc42 in HEK 293T cells. Altogether, our findings indicate that Ack1/Pyk1 tyrosine kinase may be involved both in adult synaptic function and plasticity and in brain development.     

TIMP-2对人脑胶质瘤U87体外侵袭力的抑制作用

目的 探讨TIMP-2对人脑胶质瘤细胞体外侵袭力的抑制作用。方法 用携带TIMP-2基因的重组腺病毒载体AdTIMP-2，体外转染人胶质瘤细胞系U87，用Western blotting检测目的蛋白的表达。通过Boyden小室法检测U87转染前后细胞体外侵袭力的变化，同时采用胶原酶谱法分析MMP-2、MMP-9活性改变。结果 D转染AdTIMP-2病毒后的U87细胞目的蛋白表达上调，转染后U87细胞的MMP-2、MMP-9明显下降，同时体外侵袭力受到明显抑制。结论 重组腺病毒载体介导的TIMP-2基因治疗，是一种有效的抗胶质瘤方法。     

胶质母细胞瘤肿瘤干细胞的分离培养与生物学特性研究

目的从人脑胶质母细胞瘤中分离培养脑肿瘤干细胞（brain tumorstem cells,BTSC）,并研究其生物学特性。方法利用无血清培养基和悬浮培养方法,从6例人胶质母细胞瘤标本中分离培养BTSC,并通过单克隆形成实验观察脑肿瘤干细胞球（brain tumor sphere,BTS）的形成过程。将BTSC接种于含血清培养基,观察其在体外的分化特点。将BTSC子代分化细胞更换培养条件,观察其在无血清培养基中的逆向分化现象。应用细胞免疫荧光染色对BTSC及其分化细胞进行鉴定。结果在人胶质母细胞瘤中成功分离出BTSC,其在无血清培养基中呈悬浮球状生长,具有很强的自我更新和增殖能力;免疫荧光显示其表达CD133。BTSC在含血清培养基中发生贴壁分化。分化后的子代细胞可表达CD133、神经元特异性烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）。BTSC子代分化细胞在无血清条件下培养,能够再次增殖形成BTS,呈逆向分化现象。结论人胶质母细胞瘤中存在BTSC,其具有自我更新、无限增殖、多向分化以及逆向分化等生物学特性。     

胰岛素样生长因子结合蛋白-2在U251细胞中对FAK磷酸化的促进作用

目的探讨外源性胰岛素样生长因子结合蛋白-2（insulin-like growth factor binding protein-2,IGFBP-2）在人胶质母细胞瘤细胞株U251细胞中,对黏着斑激酶（focal adhesion kinase,FAK）磷酸化的影响,从而提示IGFBP-2增强肿瘤细胞迁移、侵袭的机制。方法通过免疫荧光染色及蛋白印记试验检测IGFBP-2对FAK磷酸化的影响。结果在U251细胞中,500ng/mlIGFBP-2作用5分钟后,FAK磷酸化增强近一倍（P〈0.05）,作用30min后,FAK磷酸化增强近2倍（P〈0.01）。结论IGFBP-2可能通过FAK信号通路促进胶质瘤细胞迁移和侵袭。     

Biphasic changes in the levels of poly(ADP-ribose) polymerase-1 and caspase 3 in the immature brain following hypoxia–ischemia

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair-associated enzyme that has multiple roles in cell death. This study examined the involvement of PARP-1 in ischemic brain injury in the 7-day old rat, 0.5–48 h after unilateral carotid artery ligation and 2 h of 7.8% oxygen. This experimental paradigm produced a mild to moderate injury; 40–67% of animals in the ligated groups had histological evidence of neuronal death. Ipsilateral cortical injury was seen at all survival times, while mild contralateral cortical injury was seen only at the 1 h survival time. Hippocampal injury was delayed relative to the cortex and did not show a biphasic pattern. Immunohistochemical staining for PARP showed bilateral increased staining as early as 1 h post-hypoxia. PARP staining at early time periods was most intense in layer V of cortex, but did not demonstrate a pattern of cell clusters or columns. Ipsilateral PARP-1 levels quantified by western blotting showed a biphasic pattern of elevation with peaks at 0.5 and 12 h post-hypoxia. Contralateral PARP-1 levels were also elevated at 0.5 and 24 h. PARP activity as determined by immunoreactivity for poly(ADP-ribose) (PAR) was increased ipsilaterally at 0.5, 2 and 12 h survival times. Cortical caspase 3-activity was increased ipsilaterally at 6, 12, and 24 h and contralaterally at 0.5, 1, 2 and 6 h post-hypoxia.

There are three main findings in this study. First, changes in the distribution and amount of cell death correlate well with measured PARP-1 levels after hypoxia–ischemia, and both display biphasic characteristics. Second, there are significant early, transient morphological and biochemical changes in the contralateral cortex after neonatal hypoxia–ischemia due to unilateral permanent occlusion of a carotid artery followed by 2 h of systemic hypoxia. Third, variability in the responses of individual pups to hypoxia–ischemia suggests the presence of unidentified confounding factors.     

Differential expression of protein kinase C βI (PKCβI) but not PKCα and PKCβII in the suprachiasmatic nucleus of selected house mouse lines, and the relationship to arginine-vasopressin

The functional significance of the suprachiasmatic nucleus (SCN) in circadian rhythm control of mammals has been well documented. The role of protein phosphorylation mediated by protein kinase C (PKC), however, is not well known. We report the immunocytochemical localization of three Ca²⁺-dependent PKC isoforms (α, βI, βII) within the SCN of selected house mouse lines that differ in behavioral circadian rhythm parameters. Optical density measurements revealed that the adult mice selected for low levels of nest-building behavior (small nest-builders) had more than threefold higher PKCβI immunostaining in the SCN than the mice selected for high levels of nest-building behavior (big nest-builders). A similar twofold difference between the adult small and big nest-builders was observed for the number of PKCβI-containing cells in the SCN. The non-selected control lines were intermediate. Ten-day-old pups revealed similar differences in PKCβI immunostaining in the SCN between the small and big nest-builders. PKCα and PKCβII immunostaining in the SCN was not different among the lines. PKCβI immunostaining was not different among the selected lines in the lateroanterior hypothalamic nucleus (LA) and the cornu ammonis field 1 (CA1) of the dorsal hippocampus and confirms the specificity of the difference in PKCβI immunostaining in the SCN among the selected lines. The significance of these findings is discussed in the context of differences among the lines in arginine-vasopressin (AVP) and light-induced Fos expression in the SCN, behavioral phase-delay responses to 15-min light pulses in constant darkness, and measures of the strength of the circadian activity rhythm expressed.