多形性脑胶质母细胞瘤化学及基因治疗的研究进展

多形性脑胶质母细胞瘤是最常见的颅内肿瘤之一且预后差。即使积极采用手术,放疗及化疗的联合治疗方法,中位生存期小于1年。传统的恶性胶质瘤的化学治疗主要是应用细胞周期性或非周期性细胞毒性药物,直接杀死肿瘤细胞。随着人们对胶质瘤细胞生物学行为的深入研究,近年来新的细胞毒性药物(如替莫唑胺)不断开发出来;替莫唑胺及亚硝基脲类药物耐药性得到了部分解决以及非细胞毒性药物如酪氨酸激酶抑制剂及细胞间信号传导通路抑制剂在临床中的应用。此外,在导入自杀基因、修复或导入抑癌基因、肿瘤免疫基因治疗、血管生成抑制基因治疗、对流增强传送运输系统等基因治疗策略方面亦展开了广泛的临床研究。使得化学治疗及基因治疗成为GBM手术及放疗的有益补充。     

甲基转移样酶1在胶质母细胞瘤中高表达并与促胶质母细胞瘤生长有关

目的 探讨甲基转移样酶1(methyltransferase-like 1,METTL1)在人胶质母细胞瘤中的表达及其临床意义.方法 通过免疫荧光双标方法观察正常人脑组织中METTL1蛋白的表达定位.采用TCGA数据库分析比较METTL1 mRNA在胶质母细胞瘤及正常人脑组织中的表达差异.选取60例人胶质母细胞瘤及10...     

分泌蛋白YKL-40增强肿瘤细胞的上皮间质样转化

目的:分泌糖蛋白YKL-40在多种晚期肿瘤病人的血液中显著升高,提示YKL-40蛋白的血浓度是肿瘤恶变的生物标志物。本课题研究YKL-40重组蛋白和过表达YKL-40肿瘤细胞对肿瘤细胞的上皮间质样转化的作用。方法:构建YKL-40过表达的纤维状乳腺癌细胞系MDA-MB-231和非纤维状结肠癌细胞系HCT-116,观察细胞形态学变化,收集细胞和细胞培养液用于Western Blot(WB)检测YKL-40和上皮间质转化标记蛋白Vimentin和N-cadherin。观察重组蛋白YKL-40对原代MDA-MB-231细胞在无血清条件下的细胞存活影响;另外,用细胞存活试剂盒检测YKL-40过表达HCT-116细胞在无血清的培养液中细胞存活情况。最后,用细胞侵袭试验检测YKL-40过表达MDA-MB-231细胞的侵袭力,并用WB和Zymography来测定细胞分泌MMP9蛋白的表达和酶活性。结果:YKL-40过表达增强MDA-MB-231细胞的形态向上皮间质样转化,并显著提高Vimentin、N-cadherin蛋白的表达,但对HCT-116细胞无法诱导上皮间质样转化。在无血清培养基培养条件下,YKL-40可以增强两种细胞的存活能力,并且YKL-40过表达的MDA-MB-231细胞增强了细胞的侵袭能力,促进了MMP9蛋白表达和蛋白活性。结论:YKL-40可以增加肿瘤细胞的存活力,增强纤维状细胞向上皮间质样转化;并且,YKL-40增加MMP9蛋白表达和活性,增强细胞侵袭力。YKL-40是间质样肿瘤细胞EMT的增强子,此发现为抑制肿瘤恶变提供新靶点。     

肿瘤细胞和骨微环境在骨转移中的作用

随着肿瘤治疗水平的提高,肿瘤患者的生存期显著延长,转移性骨肿瘤的发生率呈增长趋势.骨转移引起的剧烈的临床症状和其较长的潜伏期,以及缺乏有效的治疗方法,极大降低了患者的生活质量.本文主要综述了骨转移相关的细胞特征及骨微环境在骨转移中的作用,并分析了影响骨转移形成的相关分子因素,为骨转移的定向分子治疗提供进一步的理论依据.     

人源化YKL-40中和抗体（Rosazumab） 体外阻断血管再生

摘要 目的：血管再生是实体肿瘤的生长和恶性转移的一个必须病理过程,阻断这一过程能够有效阻止恶性肿瘤的发生发展。YKL-40是血管再生因子,能刺激肿瘤的血管再生。本文探讨了一个原创性人源化抗YKL-40单克隆抗体（Rosazumab, 命名为洛沙单抗）阻断YKL-40血管生成的体外功能。方法：Western Blot检测洛沙单抗对分泌和重组YKL-40蛋白的特异性结合;Western Blot及考马斯亮蓝染色检测该抗体的抗体特异性和纯度;Live/Dead染色试验检测抗体的细胞毒性。以人微血管内皮细胞（Human microvascular endothelial cells, HMVECs）为研究对象,引入重组YKL-40蛋白或脑胶质瘤细胞（Glioblastoma serum-differentiated cells, GSDCs）的条件培养基进行培养。Transwell试验检测该抗体对HMVECs迁移力的影响,并用Matrigel试验检测对微管形成的作用。结果：洛沙单抗可特异性结合YKL-40,考马斯亮蓝染色进一步证明其纯度及特异性。体外血管再生试验包括细胞迁移力和微管形成证明该抗体可以有效中和重组YKL-40蛋白及肿瘤细胞条件培养基中的YKL-40,抑制HMVECs的迁移和血管形成。结论：洛沙单抗是首创的人源化中和YKL-40的抗体,其特异性高,细胞毒性低,能显著抑制YKL-40血管再生功能,为下一步体内试验奠定基础。本研究可为YKL-40诱导的肿瘤血管生成及恶性肿瘤转移提供一种新的治疗手段。     

IL-6在胶质母细胞瘤的表达及作用机制研究

目的:探讨白介素6(IL-6)在胶质母细胞瘤(GBM)中的表达,并探讨其高表达的作用机制,以期阐明GBM发生发展潜在分子机制。方法:采用免疫组化检测表皮生长因子变体3 (EGFRv III)阳性和阴性GBM组织IL-6的相对表达。以恶性胶质瘤细胞U87MG为研究对象,构建表达EGFRv III的U87MG-EGFRvIII细胞,用IL-1β分别处理U87MG、U87MG-EGFRvIII细胞,ELISA检测IL-6分泌量。采用EGFR下游效应通路p38MAPK、MK2、MEK1/2、JNK抑制剂SB、sc-48、PD、SP预处理细胞1小时,IL-1β刺激细胞后,检测各组IL-6分泌量变化。将IL-1β处理后的U87MG-EGFRvIII细胞记为IL-1β组,以不做任何处理细胞记为Control组,用联合SB、sc-48处理的IL-1β细胞依次命名为IL-1β+SB和IL-1β+sc-48组,western blot检测p38MAPK-MK2通路蛋白和IL-6蛋白表达,qPCR检测IL-6 m RNA表达。结果:IL-6在EGFRv III阳性GBM组织中普遍高表达,在EGFRv III阴性GBM组织中普遍低表达。EGFRv III可在未受IL-1β刺激的恶性胶质瘤细胞中上调IL-6基础分泌,也可在IL-1β刺激情况下进一步促进IL-6分泌。在U87MG细胞中,所有通路抑制剂对IL-6分泌均无影响;在U87MG-EGFRvIII细胞中p38 MAPK-MK2通路抑制剂SB和sc-48明显抑制IL-1β诱导的IL-6分泌,而MEK1/2、JNK抑制剂PD和SP则无明显影响。IL-1β能够诱导p38MAPK-MK2通路激活,诱导细胞内IL-6表达增加,联合SB、sc-48处理细胞后,p38MAPK-MK2通路活性降低,细胞内IL-6表达降低。结论:癌基因EGFRv III能够上调恶性胶质瘤细胞中IL-6基础分泌,IL-1β可进一步刺激IL-6分泌,其机制可能与p38MAPK-MK2通路激活有关。     

胶质母细胞瘤外泌体的作用机制研究进展

胶质母细胞瘤(glioblastoma,GBM)因组织学异质性、侵袭能力强、术后复发快等问题,致使患者经手术治疗、化疗和放疗后的预后差,总体生存期短。GBM细胞来源外泌体(GBM cell-derived exosome,GBM-exo)能够通过其携带的细胞因子、miRNA、DNA和蛋白质等调节GBM细胞的增殖和迁移,通过血管生成蛋白和非编码RNA促进肿瘤血管生成,通过调节因子、蛋白质和药物靶向免疫检查点等介导肿瘤免疫逃逸,以及通过非编码RNA对抗GBM细胞的耐药性,有望成为个性化治疗GBM的重要靶标,且可以作为GBM的诊断和预后标志物。本文阐述了GBM-exo的制备方法和生物学特征,及其在GBM细胞增殖、血管生成、免疫逃逸和耐药性方面的作用和分子机制,为研发诊治GBM的新策略提供参考。     

多形胶质母细胞瘤细胞系端粒酶活性的测定

端粒缩短见于星形细胞瘤发展过程中,但其长度在胶质母细胞瘤／细胞系相对稳定,提示胶质瘤细胞内存在端粒修复机制的可能性．为证实此点,利用端粒重复片段扩增技术（ＴＲＡＰ）,对８株人／大鼠多形胶质母细胞系的蛋白提取液中端粒酶活性加以测定．结果显示：８例胶质瘤样本的反应液均可见端粒ＰＣＲ扩增片段;用无ＤＮａｓｅ的ＲＮａｓｅ事先处理蛋白提取液,可明显降低或消除ＰＣＲ产物的出现,说明ＴＲＡＰ反应中的ＰＣＲ扩增是在端粒酶的介导下进行而非ＤＮＡ污染或其它端粒修复因子所致．从而不但建立起检测人癌细胞内端粒酶活性的可靠方法,也为针对端粒酶的胶质母细胞瘤生物／药物治疗提供了实验依据．     

YKL-40 和NF-κ B 在子宫内膜异位症中的表达及相关性研究

目的:通过检测人类软骨蛋白39(YKL-40)和核转录因子KappaB(NF-κB)在子宫内膜异位症组织中的表达,探讨二者与子宫内膜异位症的关系及二者的相关性。方法:用免疫组化二步法检测40例子宫内膜异位症(EMS)患者的异位内膜和在位内膜及40例子宫肌瘤患者子宫内膜(对照组)YKL-40和NF-κB的表达,并对检测结果进行统计学分析。结果:YKL-40、NF-κB在子宫内膜异位症患者的异位和在位内膜及对照组内膜中的表达率分别为67.5%、62.5%、35%及88.5%、57.5%、32.5%,差异有统计学意义(P<0.01);在不同月经周期YKL-40表达无统计学意义;在位内膜和正常内膜NF-κB的表达分泌期高于增殖期,差异有统计学意义(P<0.05);YKL-40和NF-κB在三种内膜中的表达具有正相关性,相关系数分别是0.305,0.267和0.457(P<0.01)。结论:YKL-40和NF-κB在EMS发生发展中起重要的作用。     

Role of YKL-40 in the angiogenesis, radioresistance, and progression of glioblastoma

Glioblastoma is one of the most fatal cancers, characterized by a strong vascularized phenotype. YKL-40, a secreted glycoprotein, is overexpressed in patients with glioblastomas and has potential as a novel tumor biomarker. The molecular mechanisms of YKL-40 in glioblastoma development, however, are poorly understood. Here, we aimed to elucidate the role YKL-40 plays in the regulation of VEGF expression, tumor angiogenesis, and radioresistance. YKL-40 up-regulated VEGF expression in glioblastoma cell line U87, and both YKL-40 and VEGF synergistically promote endothelial cell angiogenesis. Interestingly, long term inhibition of VEGF up-regulated YKL-40. YKL-40 induced coordination of membrane receptor syndecan-1 and integrin αvβ5, and triggered a signaling cascade through FAK(397) to ERK-1 and ERK-2, leading to elevated VEGF and enhanced angiogenesis. In addition, γ-irradiation of U87 cells increased YKL-40 expression that protects cell death through AKT activation and also enhances endothelial cell angiogenesis. Blockade of YKL-40 activity or expression decreased tumor growth, angiogenesis, and metastasis in xenografted animals. Immunohistochemical analysis of human glioblastomas revealed a correlation between YKL-40, VEGF, and patient survival. These findings have shed light on the mechanisms by which YKL-40 promotes tumor angiogenesis and malignancy, and thus provide a therapeutic target for tumor treatment.     

Elevated serum YKL-40 levels in patients with Kawasaki disease

Context: YKL-40 is an inflammatory biomarker for endothelial dysfunction that may have a role in Kawasaki disease (KD).

Objectives: We investigated the association of serum YKL-40 levels with KD and established laboratory parameters for YKL-40 levels and other inflammatory markers.

Methods: YKL-40 levels and other inflammatory markers of 23 KD patients, 9 disease control patients and 11 age-matched healthy controls.

Results: YKL-40 concentration in the serum of KD patients significantly increased during the acute disease phase compared with those of disease controls and healthy controls.

Conclusions: Increased YKL-40 levels may provide a useful inflammatory marker for patients with KD.     

Glioblastoma stem cell-specific histamine secretion drives pro-angiogenic tumor microenvironment remodeling

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YKL-40 in patients with end-stage renal disease receiving haemodialysis

Purpose: This study aimed to determine serum YKL-40 in patients with end-stage renal disease (ESRD) on haemodialysis (HD) and to evaluate the prognostic value of serum YKL-40.

Methods: Patients >18?years on maintenance HD were included. Serum YKL-40 was measured using ELISA before and after a single HD treatment.

Results: A total of 306 patients were included. Median serum YKL-40 concentration was 238?µgL^?1 (IQR: 193–291?µgL^?1) before HD treatment and 198?µgL^?1 (IQR: 147–258?µgL^?1) after HD treatment, which corresponded to age-corrected 93th percentile in healthy subjects. All-cause mortality after 2.8?years was 35.9%. Patients with serum YKL-40 in the highest quartile compared with the lowest quartile had a univariate HR of 4.0 (95% CI: 2.2–7.3, p?p?=?0.01) in multivariate analysis. Time-dependent receiver operating characteristic curves showed that serum YKL-40 after HD treatment had significant higher area under the curves from 90?d (p?=?0.004) and throughout the rest of the follow-up period when compared to serum YKL-40 before HD treatment.

Conclusion: YKL-40 was highly elevated in patients with ESRD on HD, and dialysis reduced serum YKL-40 concentrations approximately one-sixth. YKL-40 measured after dialysis was independently associated with mortality in HD patients.     

The role of YKL-40 in the pathogenesis of autoimmune diseases: a comprehensive review

YKL-40, a chitinase-3-like protein 1 (CHI3L1) or human cartilage glycoprotein 39 (HC gp-39), is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells and vascular smooth muscle cells. Its biological function is not well elucidated, but it is speculated to have some connection with inflammatory reactions and autoimmune diseases. Although having important biological roles in autoimmunity, there were only attempts to elucidate relationships of YKL-40 with a single or couple of diseases in the literature. Therefore, in order to analyze the relationship between YKL-40 and the overall diseases, we reviewed 51 articles that discussed the association of YKL-40 with rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease. Several studies showed that YKL-40 could be assumed as a marker for disease diagnosis, prognosis, disease activity and severity. It is also shown to be involved in response to disease treatment. However, other studies showed controversial results particularly in the case of Behçet disease activity. Therefore, further studies are needed to elucidate the exact role of YKL-40 in autoimmunity and to investigate its potential in therapeutics.     

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.     

YKL-40和NF—KB在子宫内膜异位症中的表达及相关性研究

目的：通过检测人类软骨蛋白39（YKL-40）和核转录因子KappaB（NF—KB）在子宫内膜异位症组织中的表达,探讨二者与子宫内膜异位症的关系及二者的相关性。方法：用免疫组化二步法检测40例子宫内膜异位症（EMS）患者的异位内膜和在位内膜及40例子宫肌瘤患者子宫内膜（对照组）YKL-40和NF—KB的表达,并对检测结果进行统计学分析。结果：YKL-40、NF—KB在子宫内膜异位症患者的异位和在位内膜及对照组内膜中的表达率分别为67．5％、62．5％、35％及88．5％、57．5％、32．5％,差异有统计学意义（P〈0．01）;在不同月经周期YKL-40表达无统计学意义;在位内膜和正常内膜NF-KB的表达分泌期高于增殖期,差异有统计学意义（P〈0．05）;YKL-40和NF-KB在三种内膜中的表达具有正相关性,相关系数分别是0．305,0．267和0．457（P〈0．01）。结论：YKL-40和NF—KB在EMS发生发展中起重要的作用。     

Association between serum YKL-40 level and dysglycemia in cystic fibrosis

BackgroundYKL-40, a chitinase-like protein, is a biomarker for type 1 and type 2 diabetes prognosis. We hypothesized that YKL-40 protein levels are elevated in CF patients with dysglycemia.MethodsSeventeen healthy control subjects and 66 CF patients were prospectively recruited and subjected to an oral glucose tolerance test. In all participants, fasting serum YKL-40 was compared between control and CF patients and between normal glucose-tolerant patients (NG-CF) and CF patients with dysglycemia (DG-CF). A Botnia clamp procedure was performed on a subset of patients for each group to determine the impact of acute increases of either glucose or insulin on YKL-40 concentration.ResultsCF patients had higher serum YKL-40 values than the controls (113 [49;288] vs. 38 [30;50] ng/ml, p < 0.001). YKL-40 concentrations in CF patients were mainly increased in the DG-CF group, who had significantly higher values: 213 [93;383] vs. 67 [27;97] ng/ml in the NG-CF group, p < 0.001). No significant modulation of YKL-40 concentration was observed in serum of CF (NG or DG-CF) or non-CF patients, after acute exposure to glucose or insulin.ConclusionsHigher serum YKL-40 levels in CF patients are significantly associated with dysglycemia. The increase in YKL-40 is potentially associated with an inflammatory response resulting from chronic glucose intolerance or CF disease evolution.     

蛋白质翻译后修饰SUMOylation在肿瘤微环境中的功能作用

目前肿瘤仍然是人类生存中无法克服的一大难题,治疗方案多种多样,但还未找到一种行之有效的方法。随着越来越多的研究发现,人们把治疗肿瘤的目光投向了一种新的领域——肿瘤微环境（tumor microenvironment,TME）,指癌细胞周围各种基质细胞的动态和复杂的环境。TME是宿主免疫系统和肿瘤之间的关键交互区域,TME内的细胞相互影响并与癌细胞相互作用以影响癌细胞的侵袭、肿瘤的生长和转移,是治疗癌症的一个全新的方向。在TME复杂的环境中,蛋白质的翻译后修饰（post-translational modification,PTMs）被证明在TME中发挥着重要的作用。PTMs通过调节蛋白质的结构、空间定位和相互作用调控其功能。在PTMs中有一种可逆的翻译后修饰被称为SUMOylation,是通过小泛素样修饰物（small ubiquitin-like modifier,SUMO）靶向赖氨酸残基修饰的翻译后修饰,是细胞过程中普遍存在的调控机制。SUMOylation广泛参与致癌、DNA损伤反应、癌细胞增殖、转移和凋亡,在TME中发挥举足轻重的作用。本综述旨在总结蛋白质的SUMOylation动态修饰对多种免疫细胞的影响,从而探究其在TME中发挥的作用。