血管生成抑制因子1对胶质母细胞瘤的治疗作用及其机制的实验研究

目的探讨脑组织特异性血管生成抑制因子1(BAI1)对胶质母细胞瘤的治疗作用及其作用机制。方法采用COS-TPC法进行BAI1-腺病毒载体的构建,病毒重组子的成功构建和对肿瘤细胞的转染通过RT-PCR来验证。采用立体定向的方法将胶质母细胞瘤细胞U87MG接种于裸鼠脑内,待肿瘤形成后向瘤内注射病毒重组子AdeBAI1(AdeBAI1组)或AdeLacZ(Mock)(AdeMock组),两组均为6只裸鼠。观察裸鼠的存活时间。用AdeBAI1或AdeMock转染3个胶质母细胞瘤细胞系SW1783、U87MG和U373MG,48h后收集细胞,用MTT方法进行活细胞计数。用Trizol试剂提取总RNA。用RT-PCR方法研究BAI1及其他血管生成相关因子的mRNA的表达。结果BAI1mRNA的表达仅见于AdeBAI1转染的细胞中。颅内胶质母细胞瘤治疗结果显示,AdeBAI1组平均生成期为(26·0±4·6)d,AdeMock治疗组为(17·3±2·3)d,两组比较P<0·05。AdeBAI1组转染后细胞计数为(2·12±0·18)×105、AdeMock组为(4·23±0·18)×105,两组比较P<0·05。提示胶质母细胞瘤细胞的增殖可被BAI1抑制。AdeBAI1转染后血管生成相关因子Angiostatin和VEGF的表达降低,而VEGF-B和TSP1的表达升高。结论肿瘤内注射AdeBAI1可以抑制胶质母细胞瘤的生长。BAI1的表达升高可以影响其他血管生成相关因子的表达。BAI1的表达升高可以抑制肿瘤细胞的增殖作用。BAI1的抗肿瘤作用可能来自于抑制血管生成以及抑制肿瘤细胞的增殖两个方面。     

免疫微环境在胶质母细胞瘤中的研究进展

摘要: 胶质母细胞瘤是中枢神经系统恶性程度最高的胶质瘤,其标准治疗手段是最大程度的肿瘤切除、放疗和替莫唑胺辅助化疗,中位生存期仅为14个月。免疫疗法开启多种癌症治疗的新篇章,在胶质母细胞瘤中却未能带来生存获益。胶质母细胞瘤具有高度异质性和复杂的免疫抑制性微环境,肿瘤细胞与非肿瘤细胞相互作用,促进肿瘤的生长、侵袭、耐药。因此,剖析胶质母细胞瘤免疫微环境各组成成分,了解免疫细胞与肿瘤细胞之间的信号通路,有利于理解免疫治疗失败的原因,也为后续研究提供依据。     

丹皮酚对人胶质母细胞瘤细胞中血管内皮生长因子表达的影响

目的 研究丹皮酚(paeonol,Pae)对人胶质母细胞瘤细胞U251中血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响.方法 以实时定量PCR法检测VEGF转录水平,以Western blot检测VEGF表达水平.结果 ①Pae可呈剂量依赖性下调U251细胞中VEGF的转录水平,当Pae作用浓度分别为47、94、188、376、752和1 504 μmol/L时,对U251细胞VEGF mRNA的抑制率分别为29.6％、43.94％、73.78％、79.49％、83.11％和88.06％;②Pae对U251细胞VEGF转录的抑制作用随着时间的延长而逐渐增强,在188 μmol/L浓度下,当Pae作用时间分别为4、12和24 h时,对U251细胞VEGF mRNA的抑制率分别为67.13％、71.33％和82.04％;③Pae可显著降低U251细胞VEGF蛋白的表达,以47、188、376、1 504 μmol/L浓度的Pae分别作用U251细胞24 h后,VEGF蛋白的表达水平量明显降低,并呈剂量依赖性减弱,以上浓度Pae对U251细胞VEGF蛋白表达的抑制率分别为33.09％、73.93％、81.23％和87.85％.结论 Pae可显著降低人胶质母细胞瘤细胞中VEGF的转录及表达,有可能通过下调VEGF表达而干预人胶质母细胞瘤细胞的血管新生及化疗敏感性.     

胶质母细胞瘤干细胞研究进展及其在胶质瘤治疗中的作用

胶质母细胞瘤是中枢神经系统肿瘤中最常见的恶性肿瘤,患者生存期仅1年左右.胶质母细胞瘤干细胞(GSC)是最早分离确认的实体瘤干细胞之一,在分子标记和生物学功能等方面与正常干细胞相似,具有自我更新、分化为多种细胞,以及少量细胞就可在免疫缺陷鼠成瘤的能力.GSC是肿瘤发生、发展、复发及转移的关键因素,具有易在体外长期培养并维持其遗传学、基因表达谱等特征,在肿瘤干细胞研究中具有重要示范意义.因此本文对GSC的研究进展及其在胶质瘤治疗中的作用等进行综述.     

miR-584对人胶质母细胞瘤侵袭和迁移能力的影响

目的探讨miR-584对人胶质母细胞瘤U251细胞侵袭和迁移能力的影响。方法将化学合成的miR-584-3p在脂质体Lipofectamine 2000的介导下,瞬时转染人胶质母细胞瘤U251,细胞分为空白对照组(无转染,其他条件相同)、mimics NC组(转染miR-584-3p mimics阴性对照序列)、miR-584-3p mimics组(转染miR-584-3p mimics)、inhibitor NC组(转染miR-584-3p inhibitor阴性对照序列)和miR-584-3p inhibitor组(转染miR-584-3p inhibitor)。Real-time PCR法检测细胞中miR-584-3p的表达水平;CCK-8法检测细胞增殖能力;划痕实验和Transw ell小室实验检测细胞的迁移和侵袭能力。结果与空白对照组和mimics NC组相比,miR-584-3p mimics组的miR-584-3p表达上调约100倍(P<0.05),细胞增殖能力无明显差异(P>0.05),细胞侵袭和迁移能力明显下降(P<0.05);与空白对照组和inhibitor NC组相比,miR-584-3p inhibitor组的miR-584-3p表达下调至空白对照组的5%(P<0.05),细胞增殖能力无明显差异(P>0.05),细胞侵袭和迁移能力显著增强(P<0.05)。结论 miR-584-3p mimics转染抑制了U251细胞的侵袭和迁移能力;miR-584-3p inhibitor转染能增强U251细胞的侵袭和迁移能力。     

雷帕霉素对人胶质母细胞瘤细胞增殖及凋亡的影响

目的:探讨雷帕霉素对人胶质母细胞瘤细胞增殖及凋亡的影响。方法:细胞培养技术培养人胶质母细胞瘤细胞系A172,将其分为对照组与实验组。MTT检测两组细胞增殖;细胞免疫化学检测磷酸化核糖体S6激酶(p-P70S6K)1蛋白表达;TUNEL检测A172凋亡。结果:MTT表明,实验组细胞存活增殖与对照组比差异有统计学意义(P<0.05);实验组p-P70S6K1与对照组比差异有统计学意义(P<0.05);雷帕霉素可诱导A172凋亡(P<0.05)。结论:雷帕霉素能有效抑制人胶质母细胞瘤A172增殖,也可诱导其凋亡。雷帕霉素可能通过m TOR/P70S6K1信号通路发挥抗肿瘤作用。     

AS2O3对胶质母细胞瘤细胞增殖的影响

目的探讨三氧化二砷（As2O3）及其联合地塞米松（DEX）对胶质母细胞瘤细胞的增殖抑制作用。方法实验分为空白对照组、As2O3组、DEX组、As2O3-4-DEX组，经倒置显微镜观察细胞形态、细胞计数、MTT法、流式细胞计数等方法研究As2O3及其联合DEX作用对胶质母细胞瘤细胞的增殖抑制作用。结果As2O3能够且呈浓度和时间的依赖性抑制BT-325细胞的增殖；DEX具有明显的协同效应。结论As2O3及其与DEX联合用药可能成为治疗胶质母细胞瘤的一条新途径。     

替莫唑胺对胶质母细胞瘤化疗的临床观察

目的探讨胶质母细胞瘤应用替莫唑胺化疗的疗效。方法回顾性分析接受替莫唑胺化疗的31例胶质母细胞瘤患者的临床疗效。结果所有患者均接受超过3个周期的替莫唑胺治疗,6个月有效率29.0%,无进展生存率64.5%。仅1例出现Ⅲ度骨髓抑制。结论胶质母细胞瘤手术和放射治疗后可以应用替莫唑胺化疗。     

免疫毒素DTATEGF对人非小细胞肺癌脑转移瘤的影响及机制

目的：观察免疫毒素DTATEGF对体外培养的人NSCLC脑转移瘤细胞增殖、凋亡及其对肿瘤血管生成的影响。方法：MTT法检测不同浓度靶向毒素DTATEGF对体外培养的人NSCLC脑转移瘤PC9-BrM3细胞增殖的影响,流式细胞仪分析DTATEGF作用于PC9-BrM3细胞系48h后细胞凋亡和细胞周期变化。12只皮下种植肿瘤的裸小鼠分为2组：瘤床内分别注入DTATEGF或对照液2ug,隔天一次,共5次,测量肿瘤体积及其微血管密度（MVD）。结果：DTATEGF明显抑制PC9-BrM3细胞的体外增殖,呈剂量依赖关系,其诱导PC9-BrM3细胞凋亡,1pmol／L的DTATEGF作用PC9-BrM3细胞48h后细胞凋亡率为（64．0±0．5）％,对照组为（1．5±0．4）％,差异有统计学意义（P〈0．01）;细胞周期检测显示：DTATEGF处理组SubG0／G1期和s期细胞别为（32．0±1．5）％和（2．0±0．4）％,而空白对照组分别为（5．0±0．6）％和（11．4±0．8）％,差异均有统计学意义（p〈0．01）。动物实验显示DTATEGF处理组肿瘤体积较对照组生长缓慢,且DTATEGF处理组MVD为（15．6±4．6）／mm2,而空白对照组为（31．2±5．4）／mm2,差异均有统计学意义（P〈0．05）。结论：DTATEGF抑制PC9-BrM3增殖、诱导细胞凋亡,明显抑制裸小鼠皮下种植的人NSCLC脑转移瘤细胞的生长及其新生血管的形成。     

Genome-wide allelotype study of primary glioblastoma multiforme

Objective To investigate the molecular genetic pathogenesis of primary glioblastoma multiforme (GBM) and identify which chromosomes or chromosomal regions of the entire genome may harbor tumor suppressor genes (TSGs) associated with GBM.Methods A high-resolution allelotype study of 21 cases of primary GBM was performed by PCR-based loss of heterozygosity （LOH）analysis. Three hundred and eighty-two fluorescent dye-labeled microsatellite markers covering all 22 autosomes were applied. The mean genetic distance between two flanking markers was about 10 cM.Results LOH was observed on all 39 nonacrocentric autosomal arms examined in this study. The LOH frequencies of 10q, 10p, 9p, 17p and 13q were the highest (&gt;50%). Furthermore, high LOH frequencies were detected in the regions containing known TSGs including PTEN, DMBT1, p16, p15, p53 and RB; the LOH frequencies on 14q, 3q, 22q, 11p, 9q, 19q were also high (&gt;40.5%). Our study observed the following commonly deleted regions: 9p22-23, 10p12.2-14, 10q21.3, 13q12.1-14.1, 13q14.3-31, 17p11.2-12, 17p13, 3q25.2-26.2, 11p12-13, 14q13-31, 14q32.1, 14q11.1-13, 22q13.3, 4q35, 4q31.1-31.2, 6q27 and 6q21-23.3. Conclusions The molecular pathogenesis of GBM is very complicated and associated with a variety of genetic abnormalities on many chromosomal arms. The most closely related chromosomal arms to the pathogenesis of GBM are 10q, 10p, 9p, 17p and 13q. Besides the well-known TSGs including PTEN, DMBT1, p16, p15, p53 and RB, multiple unknown TSGs associated with GBM may be present on the commonly deleted regions detected in the present study.     

Prognostic factors influencing clinical outcomes of glioblastoma multiforme

Background Glioblastoma multiforme （GBM） is the most malignant kind of astrocytic tumors and is associated with a poor prognosis. In this retrospective study, we assessed the clinical, radiological, genetic molecular and treatment factors that influence clinical outcomes of patients with GBM. Methods A total of 116 patients with GBM who received surgery and radiation between January 2006 and December 2007 were included in this study. Kaplan-Meier survival analysis and Cox regression analysis were used to find the factors independently influencing patients＇ progression free survival （PFS） time and overall survival （OS） time. Results Age, preoperative Karnofsky Performance Scale （KPS） score, KPS score change at 2 weeks after operation, neurological deficit symptoms, tumor resection extent, maximal tumor diameter, involvement of eloquent cortex or deep structure, involvement of brain lobe, Ki-67 expression level and adjuvant chemotherapy were statistically significant factors （P 〈0.05） for both PFS and OS in the univariate analysis. Cox proportional hazards modeling revealed that age 〈50 years, preoperative KPS score 〉80, KPS score change after operation 〉0, involvement of single frontal lobe, non-eloquent area or deep structure involvement, low Ki-67 expression and adjuvant chemotherapy were independent favorable factors （P 〈0.05） for patients＇ clinical outcomes. Conclusions Age at diagnosis, preoperative KPS score, KPS score change at 2 weeks postoperation, involvement of brain lobe, involvement of eloquent cortex or deep structure, Ki-67 expression level and adjuvant chemotherapy correlate significantly with the prognosis of patients with GBM.     

人脑胶质母细胞瘤PDX模型的建立

目的：探讨患者来源的异种移植（patient derived xenograft,PDX）胶质母细胞瘤模型的建立方法,为临床和生物学研究提供重要的理论依据和技术支持。方法：收集胶质母细胞瘤组织,建立皮下肿瘤模型,待皮下肿瘤生长取出肿瘤,分散肿瘤细胞并种于小鼠颅内。HE染色比较原发肿瘤组织与PDX肿瘤形态学特点,免疫组织化学染色方法检测Ki?67的表达,比较肿瘤增殖能力。结果：成功建立人脑胶质母细胞PDX模型,原发肿瘤和PDX肿瘤形态学特点相似,PDX肿瘤保留了原发肿瘤的增殖能力。结论：通过建立PDX模型,可以将PDX作为胶质母细胞瘤患者个体化治疗的重要临床前研究工具。     

VM-26对人多形性胶质母细胞瘤BT-325细胞增殖的影响

目的　研究替尼泊甙 (VM -2 6)对人多形性胶质母细胞瘤BT -3 2 5细胞增殖的影响。方法　应用免疫细胞化学方法 ,观察BT -3 2 5细胞经不同剂量 (1μg/ml和 5μg/ml)VM -2 6作用后的不同时间 (48小时、72小时 )克隆形成率的变化以及PCNA、CyclinD1表达的改变。结果　克隆形成率实验显示VM -2 6作用后克隆形成率降低 ,与对照组比较差别显著 (P <0 .0 1) ;对照组PCNA和CyclinD1均为强阳性 ;经VM -2 6作用后 ,PCNA和CyclinD1表达均受到显著的抑制 ,尤以 5μg/ml、72小时组效果最佳 ,与对照组比较差别显著 (P <0 .0 1)。结论　VM -2 6可显著抑制BT -3 2 5细胞恶性增殖     

Relationship between magnetic resonance imaging and molecular pathology in patients with glioblastoma multiforme

Background  Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor in adults. Magnetic resonance imaging (MRI) is routinely used in the diagnosis, characterization and clinical management of GBM. The diagnosis and treatment of GBM is largely guided by histopathology and immunohistochemistry. This study aimed to identify the relationship between magnetic resonance features and molecular pathology of GBM.

Methods  MRI images of 43 glioblastoma patients were collected. Four imaging features, degree of edema, contrast tumor enhanced/T2 ratio, multiple lesions and tumor across the midline, were selected to identify their relationship with P53, Ki-67 and O⁶-methylguanine-DNA methltransferase (MGMT) expression in patients with GBM. The relationship between imaging features and molecular pathology was studied by chi-square test using the software SPSS 13.0.

Results  High expression of P53 was found correlated with low contrast tumor enhanced/T2 ratio, low expression of Ki-67 was correlated with multiple lesions and high expression of KI-67 may be related with tumor across the midline, low expression of MGMT was correlated with edema.

Conclusion  Some MRI features such as the degree of edema, contrast tumor enhanced/T2 ratio, multiple lesions and tumor acrossing the midline are correlated with P53, Ki-67 and MGMT of GBM.

     

A genome-wide allelotype study of primary and corresponding recurrent glioblastoma multiforme in one patient

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain tumor. Although comprehensive therapeutic measures are available, recurrence is very frequent and the prognosis of GBM remains dismal. To date, little is known about the molecular pathogenesis associated with GBM recurrence. According to Knudson ' s two-hit hypothesis of tumor suppressor gene (TSG) inactivation,1 deletion of a chromosomal region, as revealed by loss of heterozygosity (LOH), is often indicative of the presence of a potential TSG. Allelotype studies involving a comprehensive LOH analysis of the whole genome can provide more detailed and thorough information for detecting genetic anomalies than traditional LOH analysis. The present study is designed to conduct a genome-wide allelotype analysis of one patient ' s primary and corresponding recurrent GBM tumors in an effort to reveal molecular genetic alterations associated with the recurrence of this malignancy.     

A genome-wide allelotype study of primary and corresponding recurrent glioblastoma multiforme in one patient

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain tumor. Although comprehensive therapeutic measures are available, recurrence is very frequent and the prognosis of GBM remains dismal. To date, little is known about the molecular pathogenesis associated with GBM recurrence.     

Relationship between MRI features and miRNA gene expression in patients with glioblastoma multiforme

BACKGROUND: Magnetic resonance imaging (MRI) is commonly utilized as the part of the diagnostic workup for the clinical diagnosis of glioblastoma multiforme (GBM), further guiding the clinical treatment of this aggressive cancer. Recent research has shown that micro RNA’s (miRNAs) may act as oncogenes, or in some cases, tumor suppressor genes that in turn may reflect the genotypic features of GBM. METHODS: In order to identify the relationship between the radiographic findings of MRI with those identified changes in miRNA gene expression of GBM, we reviewed the MRI images of GBM patients and compared them to the identified miRNA expression profiles utilizing microarray analysis of paired GBM tumor samples. We chose five MRI imaging features: 1. contrast tumor enhanced/necrosis ratio, 2. contrast tumor enhanced/T2 ratio, 3. multiple lesions, 4. hemorrhage and 5. necrotic volume. The relationship between these five imaging features and miRNA expression was studied using Significance Analysis of Microarrays (SAM) analysis. RESULTS: We found that the expression of miRNA’s hsa-miR-892b, hsa-miR-892a, hsa-miR-888 was inversely correlated with a enhanced/necrosis ratio ≥ 1. The miRNA’s hsa-miR-95, hsa-miR-498 and hsa-miR-1300 were associated with a contrast tumor enhanced/T2 ratio ≥ 1. The miRNA’s hsa-miR-612,hsa-miR-524-3 and hsa-miR-1282 were associated with multiple lesions identified on MRI and the expression of miR-221 was associated with hemorrhage in GBM. The expression of miR-let-7, including miR-let-7f, miR-let-7i, miR-let-7f-1*, were down-regulated in the hemorrhage group. The gene expression of of miRNA’s hsa-miR-140-5p, hsa-miR-30e and hsa-miR-301a was relatively low when compared with the larger necrotic volume group as identified by MRI. CONCLUSION: The miRNA gene expression profiles correlate with several select MRI features of patients with GBM. Further analysis of key imaging features of MRI with correlation with miRNA gene expression patterns may help to guide treatment decisions based upon these unique correlative profiles of GBM.     

Relationship between magnetic resonance imaging features and miRNA gene expression in patients with glioblastoma multiforme

Background Magnetic resonance imaging (MRI) is commonly utilized as part of the diagnostic workup for the clinical diagnosis of glioblastoma multiforme (GBM), further guiding the clinical treatment of this aggressive cancer. Recent research has shown that microRNAs (miRNAs) may act as oncogenes, or in some cases, tumor suppressor genes that in turn may reflect the genotypic features of GBM. This study aimed to investigate the relationship between MRI features and miRNA gene expression in patients with glioblastoma multiforme.

Methods In order to identify the relationship between the radiographic findings of MRI and those identified changes in miRNA gene expression of GBM, we reviewed the MRI images of GBM patients and compared them with the identified miRNA expression profiles utilizing microarray analysis of paired GBM tumor samples. We chose five MRI imaging features: (1) contrast tumor enhanced/necrosis ratio, (2) contrast tumor enhanced/T2 ratio, (3) multiple lesions, (4) hemorrhage, and (5) necrotic volume. The relationship between these five imaging features and miRNA expression was studied using significance analysis of microarrays analysis.

Results We found that the expression of miRNAs such as hsa-miR-892b, hsa-miR-892a, and hsa-miR-888 was inversely correlated with an enhanced/necrosis ratio ≥1. The miRNAs such as hsa-miR-95, hsa-miR-498, and hsa-miR-1300 were associated with a contrast tumor enhanced/T2 ratio ≥1. The miRNAs such as hsa-miR-612, hsa-miR-524-3, and hsa-miR-1282 were associated with multiple lesions identified on MRI and the expression of miR-221 was associated with hemorrhage by GBM. The expression of miR-let-7, including miR-let-7f, miR-let-7i, and miR-let-7f-1*, was downregulated in the hemorrhage group. The gene expression of miRNAs such as hsa-miR-140-5p, hsa-miR-30e, and hsa-miR-301a was relatively low when compared with the larger necrotic volume group as identified by MRI.

Conclusions The miRNA gene expression profiles correlate with several selected MRI features of patients with GBM. Further analysis of key imaging features of MRI with correlation with miRNA gene expression patterns may help to guide treatment decisions based on these unique correlative profiles of GBM.