转化生长因子-β与葡萄膜黑色素瘤

转化生长因子-β(TGF-β)是一种具有多种生物学功能的多肽类生长因子。TGF-β在肿瘤的发生过程中起着双向调节作用,同时TGF-β对细胞的增殖与分化、细胞外基质的产生、血管的生成及机体免疫系统均起着重要作用。近年研究TGF-β的异常过表达以及信号传导通路的异常与葡萄膜黑色素瘤生长和侵袭转移能力密切相关。就TGF-β的结构、功能以及它在葡萄膜黑色素瘤发生过程中的作用进行综述。     

葡萄膜黑色素瘤染色体异常及其相关基因研究进展

葡萄膜黑色素瘤存在多种染色体异常及基因表达异常。3号染色体缺失和8号染色体获得与葡萄膜黑色素瘤的转移和预后密切相关，可作为其分型和预后评判的指标；6号染色体的异常与葡萄膜黑色素瘤不同表型的侵袭能力有关。Cyclin D1、MTS1、MDM2、Bcl-2等基因表达异常与葡萄膜黑色素瘤的发生有关，DDEF1、NBS1基因的表达与葡萄膜黑色素瘤的侵袭能力和转移密切相关。     

重视对葡萄膜黑色素瘤侵袭转移机制的研究

葡萄膜黑色素瘤（UM）是成人眼内最常见的恶性肿瘤。肿瘤组织浸润侵袭性强,易经血行途径发生转移,转移后临床预后较差。UM转移存在多种途径,目前认为与染色体异常、基因表达异常、细胞因子及信号转导通路调控异常等有关。通过对UM临床病理学特征及其侵袭转移机制进行研究,寻求检测UM早期转移的标志物及可能的治疗靶点,进行有效地监测和干预,对于提高患者生存率以及UM的防治水平具有重要意义。     

血清黑色素瘤活性抑制蛋白与葡萄膜黑色素瘤

目的检测葡萄膜黑色素瘤患者血清黑色素瘤活性抑制蛋白(MIA)的水平，并探讨其在葡萄膜黑色素瘤诊断和转移监测中的价值。方法应用酶联免疫吸附法（ELISA）分别检测不同组织病理分型的葡萄膜黑色素瘤（27例）、葡萄膜黑色素细胞瘤（6例）、其他眼部肿瘤患者（7例）以及正常成人（16人）外周血清中MIA的浓度。结果正常成人(16人)和睫状体无色素上皮腺瘤(4例)、视网膜母细胞瘤(2例)、视网膜血管瘤(1例)患者血清MIA浓度显著低于葡萄膜黑色素瘤患者；不伴巩膜浸润和远处转移的葡萄膜黑色素瘤患者血清MIA浓度明显低于伴有巩膜浸润和远处转移的患者，但与葡萄膜黑色素细胞瘤患者比较无明显差异；在不伴巩膜浸润和远处转移的葡萄膜黑色素瘤组，梭形细胞型患者血清MIA浓度与混合型和上皮细胞型患者相比无明显差异。结论血清MIA水平可能是临床诊断葡萄膜黑素瘤的一个较好指标，并且可用于肿瘤转移的监测。(中华眼底病杂志,2005,21:153-155)     

葡萄膜黑色素瘤的治疗现状

葡萄膜黑色素瘤（uveal melanoma，UM）是起源于葡萄膜黑色素细胞的眼内恶性肿瘤。由于其生存率很大程度上取决于原发肿瘤的大小，因此早发现、早治疗非常重要。目前，UM尚无有效的辅助治疗方法。但分子生物学方面的新发现，如GNAQ和GNA11突变和涉及下游信号传导通路MAPK、PI3K/Akt和Hippo为代表的多种治疗，则有助于对UM发病机制的理解。还有研究开辟了新的生物标志物范围，以提高对UM转移的检测。此外，许多研究和临床试验也正在进行中。     

葡萄膜黑色素瘤转移相关基因的表达及意义

目的：探讨肿瘤转移相关基因在葡萄膜黑色瘤中的表达及与浸润能力和病理分型的关系。方法：采用免疫组化方法定理检测96例葡萄膜黑色素瘤体标本中EGFR和nm23蛋白的表达，结果：肿瘤的浸润能力与EGFR的表达呈正相关，与nm23的表达则呈负相关，类上皮细胞型，混合细胞型，梭形细胞型EGFR的表达率依次降低，nm23的表达率依次增高，结论：EGFR和nm23是评价葡萄膜黑色素瘤转移潜能的有效指标。     

葡萄膜黑色素瘤动物模型的建立

葡萄膜黑色素瘤是成年人最常见的原发性眼内恶性肿瘤,在过去的数十年里,研究葡萄膜黑色素瘤所面对的主要挑战是建立合适的动物模型.最常见的葡萄膜黑色素瘤动物模型可分为动物的自发性肿瘤、诱发肿瘤和移植性肿瘤.开始应用人葡萄膜黑色素瘤细胞系和肿瘤组织碎片建立模型是原发的和转移的实验性动物模型的一大进步.葡萄膜黑色素瘤动物模型常被用于原发和转移葡萄膜黑色素瘤的预后因素及治疗手段等方面的研究.尽管动物模型与人类的疾病有一定差异,但它们确实为研究人葡萄膜黑色素瘤的发生、发展、转移预后及治疗手段等方面提供了有力的工具.本文对近年来葡萄膜黑色素瘤动物模型研究做一综述.     

葡萄膜黑色素瘤的肿瘤免疫研究进展

葡萄膜黑色素瘤是成年人眼内原发性恶性肿瘤中最常见的一种，具有高度的侵袭性和转移性。多数肿瘤由T细胞介导的特异性免疫应答起主要的抗肿瘤免疫作用，而葡萄膜黑色素瘤具有特殊的免疫学特性，在其发生、发展过程中由NK细胞发挥主要的免疫监视作用，且MHC-Ⅰ类分子的下降与预后改善呈正相关。该肿瘤细胞可通过多种方式如MHC-Ⅰ类分子表达异常和分泌免疫抑制因子如TGF-8、IL-10、MIF等逃避NK细胞的杀伤。对其进行免疫治疗时，应侧重于激活NK细胞的活性以提高患者的免疫应答，杀伤肿瘤细胞及阻止肿瘤转移。目前常用的免疫疗法有：将细胞因子基因注射到肿瘤局部，建立抗肿瘤免疫反应的微环境；促进黑色素瘤细胞的凋亡，加强外周血单核细胞对肿瘤细胞淋巴因子的激活杀伤效应。     

葡萄膜黑色素瘤染色体异常及检测方法

葡萄膜黑色素瘤是成年人最常见的眼内原发性恶性肿瘤,细胞遗传学的研究显示葡萄膜黑色素瘤细胞存在特征性的染色体异常,并与肿瘤的预后相关.这些特征性的染色体异常提示可能存在癌基因与抑癌基因,这有助于阐明肿瘤发生发展的机制.可用于检测葡萄膜黑色素瘤染色体异常的方法很多,随着科学技术的发展,检测手段也不断更新.本文对目前发现的葡...     

P38 MAPK在角膜创伤愈合过程中的作用

丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联是细胞内重要的信号传导系统。细胞通过这一信号传导系统将细胞外信号传递到细胞核内,介导细胞产生反应,调节细胞生长、增殖、分化和凋亡等生理过程及创伤愈合和炎症反应等病理过程。近年来发现一类新的MAPK通路—p38MAPK信号通路,对其结构和功能以及在角膜创伤愈合修复和炎症反应中的作用已有了进一步的了解,在信号通路水平调控p38MAPK的表达和活性,可能成为临床角膜创伤治疗的新途径。     

Involvement of PI3K/Akt signaling pathway in hepatocyte growth factor-induced migration of uveal melanoma cells

PURPOSE: Uveal melanoma is the most common primary intraocular malignancy in adult humans. Unlike cutaneous melanoma, uveal melanoma disseminates preferentially to the liver through the hematogenous system. To date, the mechanism underlying this metastatic homing is largely unknown. This study investigated the effect of hepatocyte growth factor (HGF)-triggered signaling pathways to identify the role of HGF and its downstream effectors in inducing the migration of uveal melanoma cells. METHODS: Migration of uveal melanoma cells was measured by in vitro wound healing and transwell migration assays. The expression and translocation of c-Met were detected using indirect immunofluorescence. The activation of extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways was analyzed using specific antibodies against phospho-ERK1/2 and phospho-Akt. The impact of HGF treatment on the expression of cell adhesion molecules was measured using Western blotting. RESULTS: HGF was found to enhance cell migration, and that HGF-induced migration depends on PI3K/Akt pathway. The activation of PI3K/Akt pathway induced by the HGF/c-Met axis is involved in the downregulation of cell adhesion molecules E-cadherin and beta-catenin, contributing to the attenuation of cell-cell adhesion and promoting the enhanced motility and migration of uveal melanoma cells. On HGF stimulation, receptor c-Met is translocated to the nucleus in a ligand-dependent manner, suggesting that c-Met may modulate the expression of genes involved in melanoma cell migration. CONCLUSIONS: Data from this study directly linked the central PI3K/Akt pathway to uveal melanoma migration and pointed to new avenues for therapeutic intervention in hepatic metastasis.     

Pyrophosphorolysis detects B-RAF mutations in primary uveal melanoma

PURPOSE: Mutations in the genes that control cell proliferation in cutaneous melanoma are generally uncommon in uveal melanoma. Despite the absence of known activating mutations, the RAF-MEK-ERK, or mitogen-activated protein kinase (MAPK), pathway is usually activated in uveal melanoma. An assay with increased potential to identify mutations is now available, and this study was therefore conducted to reanalyze uveal melanoma cell lines and primary tumors for this mutation. METHODS: Eleven uveal melanoma cell lines and 45 primary uveal melanomas were analyzed for mutations in exon 15 of the B-RAF gene by using pyrophosphorolysis-activated polymerization (PAP). Mutations were validated by sequencing of the PAP product. RESULTS: B-RAF mutations were detected in cell lines OCM-1 and -3 (V600E) and in six primary uveal melanomas. The V600K mutation was detected in one primary uveal melanoma, for which the V600E assay turned out to be sensitive as well. Direct sequencing of the exon 15 PCR product did not reveal the mutations found with the PAP-assay, indicating a low frequency of the mutant allele in primary samples. CONCLUSIONS: Because of the very sensitive PAP technology, B-RAF mutations were found in cell lines and primary uveal melanomas, which suggests that they may occasionally play a role in the activation of the MAPK pathway in uveal melanoma and indicates a higher prevalence of B-RAF mutations in uveal melanoma than was reported earlier. However, the relative scarcity of the B-RAF mutation excludes an elemental role for this mutation in uveal melanoma.     

Pharmacological drug screening to inhibit uveal melanoma metastatic cells either via EGF-R, MAPK, mTOR or PI3K

AIM: To screen five potential pharmacological substances specifically targeting EGF-R, MAPK, mTOR, or PI3K for their antiproliferative effects, possible impact on cell viability, as well as cell death rates on three different uveal melanoma metastasis cell lines in vitro. METHODS: Three different uveal melanoma metastasis cell lines (OMM2.5, OMM2.3, and OMM1), that originated from human hepatic and subcutaneous metastasis, were exposed to inhibitors of different targets: erlotinib (EGF-R), everolimus (mTOR), selumetinib (MAPK), trametinib (MAPK) or the alkylphosphocholine erufosine (PI3K). Cell viability was assessed with a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) dye reduction assay after 24h of treatment. Antiproliferative effects were evaluated separately after a 72-hour incubation of the cells with the pharmacological substance. Subsequently, the IC50 was calculated. Tumor cell death was investigated using a double stain apoptosis detection assay. RESULTS: Selumetinib, trametinib, and erufosine significantly decreased cell viability of all OMM cell lines (P<0.04). In addition, selumetinib and trametinib showed a significant inhibition of cell proliferation (P<0.05). Everolimus and erlotinib solely inhibited cell proliferation at the used concentrations (P<0.05). Besides an increase of necrotic cells after erufosine treatment (P<0.001), no changes in the number of dead cells for the other substances were observed. CONCLUSION: The preliminary drug screening demonstrates five new candidates, successfully targeting the canonical MAPK/ERK and PI3K/AKT/mTOR pathways in uveal melanoma metastasis cells in vitro. Hence, these findings provide an experimental basis to explore future single or combined therapy strategies for metastatic uveal melanoma.     

Osteopontin expression and serum levels in metastatic uveal melanoma: a pilot study

PURPOSE: This was a pilot study conducted to examine the expression of osteopontin in uveal melanoma and to determine whether serum osteopontin can be used in detecting metastatic uveal melanoma. METHODS: Osteopontin mRNA was measured in three uveal melanoma cell lines of various invasive potential by real-time PCR. Tissue sections of primary and metastatic uveal melanomas were stained for osteopontin. Serum osteopontin levels were measured by ELISA assays in 15 patients with metastatic uveal melanoma and in 37 patients who were disease-free for at least 10 years after treatment of the primary tumor. Paired serum samples drawn from eight patients before and after development of metastasis were analyzed. RESULTS: By real-time PCR, highly invasive primary and metastatic uveal melanoma cells expressed 6- and 250-fold excess osteopontin mRNA, respectively, compared with poorly invasive primary uveal melanoma cells. Tissue sections of primary uveal melanomas lacking looping vasculogenic mimicry patterns either did not stain for osteopontin or exhibited weak, diffuse staining. In primary melanomas containing looping vasculogenic mimicry patterns, strong osteopontin staining was detected in the tumor periphery where patterns were located. Diffuse strong expression of osteopontin was detected in eight samples of uveal melanomas metastatic to the liver. Serum osteopontin levels were significantly higher in patients with metastatic uveal melanoma than in patients who had been disease free for at least 10 years after treatment (P = 0.0001) or in age-matched control subjects. Serum osteopontin levels were significantly higher (P = 0.008) after metastasis than before the detection of metastasis in eight patients. When a cutoff of 10 ng/mL was used, the sensitivity and specificity of serum osteopontin in detecting metastatic melanoma was 87.5%, and the area under the receiver operator characteristic curve was 96%. CONCLUSIONS: Osteopontin is expressed diffusely in tissue sections of hepatic metastases from uveal melanoma, and increased serum osteopontin levels correlate with melanoma metastasis to the liver with high specificity and sensitivity.     

Using the direct-injection model of early uveal melanoma hepatic metastasis to identify TPS as a potentially useful serum biomarker

PURPOSE: To develop a method to screen for serum biomarkers of early hepatic metastasis from uveal melanoma. METHODS: Cytokeratin 18 (TPS) was identified from gene expression profiles as protein generated by highly invasive uveal melanoma cells. Sera were collected from two groups of 15 SCID mice 2 weeks after injection of either tissue culture medium or MUM2B human metastatic uveal melanoma cells into the mouse liver. Serum TPS levels were assayed in 53 healthy human controls, 64 uveal melanoma patients who were disease free for at least 10 years, and 37 patients with metastatic uveal melanoma. RESULTS: After 2 weeks, small hepatic nodules (0.1-2.8 mm; mean, 0.80 mm) developed in 11 of 15 mice injected with MUM2B cells. Serum TPS levels in media-injected mice (84.7 U/L) were substantially lower than levels in MUM2B-injected mice (601 mug/L). TPS levels were significantly higher (P < 0.0001) in patients with metastatic uveal melanoma (139.63 +/- 22.20) than in healthy controls (54.23 +/- 0.01) or in patients free of disease (69.29 +/- 9.76). Significant differences were found between TPS levels before and after the development of hepatic metastases (P < 0.01), and serum TPS levels became elevated in four patients at least 6 months before the detection of hepatic metastases by abdominal ultrasonography. CONCLUSIONS: The direct-injection model of uveal melanoma in the mouse liver may be used to screen for potential serum biomarkers of metastatic uveal melanoma.     

Osteopontin as a serologic marker for metastatic uveal melanoma: results of a pilot study

PURPOSE: To evaluate the protein osteopontin (OPN) as a potential new marker for screening and detection of metastatic uveal melanoma. DESIGN: Prospective, clinical study. METHODS: Twenty-eight plasma samples of 27 patients with uveal melanoma were analyzed, and the OPN plasma levels were quantified. Eight of these patients showed liver metastasis. As a control, we measured OPN levels in eight healthy, age-matched individuals. RESULTS: The median plasma concentration of OPN in patients with melanoma without metastasis was 46.78 ng/ml (range, 14.5 to 118.67 ng/ml). In contrast, increased median levels of OPN of 170.72 ng/ml (range, 87.37 to 375.54 ng/ml, P <.001) were seen in eight patients with proven metastatic disease. Healthy patients without uveal melanoma showed a median plasma concentration of OPN of 54.6 ng/ml (range, 38.23 to 71.21 ng/ml). CONCLUSION: The protein OPN seems to be a promising tumor marker for detecting metastatic disease in patients with uveal melanoma.     

不同病理类型的葡萄膜黑色素瘤中微小RNA差异表达谱分析

背景 目前研究证实微小RNA(miRNA)参与大多数人类肿瘤疾病的发生和发展,其作用类似于抑癌基因或癌基因.葡萄膜黑色素瘤(UM)是成人常见的眼部恶性肿瘤,其发生和转移机制仍未完全阐明.探讨UM组织中miRNA的差异表达情况有望为UM的靶向治疗提供依据. 目的 筛选不同病理类型的UM组织中特异性miRNA表达谱. 方法 收集于2013年3月至2015年10月在北京同仁医院手术局部切除并经常规组织病理学和免疫组织化学检测证实为梭型细胞型UM的标本4例和上皮细胞型UM标本4例,采用miRNA芯片分别检测2种UM组织中miRNA的表达,收集同期死于非肿瘤疾病的8个供体眼的正常葡萄膜组织作为对照,利用组间差异倍数筛选出差异≥2倍差异表达的miRNA;用在线软件预测差异表达miRNA的靶基因,采用生物信息学方法分析靶基因参与的信号功能通路.采用实时定量PCR法验证芯片检测结果.结果 收集的梭形细胞型和上皮细胞型UM标本经组织病理学检查均得到确诊,免疫组织化学检测梭形细胞型及上皮细胞型UM组织中HMB45、黑色素-A和S-100均呈阳性反应.与正常葡萄膜组织比较,在梭形细胞型UM组织中差异表达的miRNA有109个,其中29个上调,80个下调,上调的miRNA包括miR-146a-5p、miR-25-3p和miR-29b-1-5p,下调的miRNA包括miR-126-5 p、miR-183-5p和miR-96-5p;上皮细胞型UM中差异表达的miRNA有50个,其中23个上调,27个下调,上调的miRNA包括miR-155-5p、miR-210和miR-378 a-5p;下调的miRNA包括miR-199a-5p、miR-143-3p和miR-143-5p.在梭形细胞型和上皮细胞型UM组织中共同上调的miRNA为miR-132-3p、miR-21-5p、miR-34a-5p和miR-34b-5p,共同下调的miRNA为miR-125b-2-3p、miR-126-3p、miR-199a-3p和miR-214-3p.梭形细胞型和上皮细胞型UM组织中差异表达的miRNA所预测的靶基因分别参与癌症通路、丝裂原活化蛋白激酶(MAPK)信号通路、Wnt信号通路、细胞间黏附、胞吞作用、前列腺癌通路、结直肠癌通路和细胞黏附通路.结论 与正常葡萄膜组织相比,梭形细胞型UM和上皮细胞型UM组织中存在多种miRNA的差异表达,梭形细胞型UM和上皮细胞型UM组织之间也存在明显的miRNA差异表达,这些差异表达的miRNA可通过不同的信号转导通路参与调控UM的生物学行为.     

骨桥蛋白在不同侵袭转移潜能葡萄膜黑色素瘤中的表达及意义

背景 骨桥蛋白(OPN)在多种转移性肿瘤组织中表达增高,但在葡萄膜黑色素瘤(UM)中的表达与临床病理特征及侵袭转移是否相关尚不清楚. 目的 研究UM组织及不同转移潜能人UM细胞系MUM-2B、C918和OCM-1A中OPN的表达情况,分析OPN的组织及细胞系表达水平与UM患者临床病理特征与转移预后之间的关系.方法 收集2004年1月至2007年12月在北京同仁医院行眼球摘除手术并已经病理证实为脉络膜黑色素瘤组织的标本共50例,应用免疫组织化学法检测石蜡标本中OPN的表达,分析其与临床资料的相关性.应用定量聚合酶链反应(Q-PCR)技术检测不同侵袭性转移潜能的人UM细胞系中OPN mRNA的表达情况.结果 50例UM组织中发生肝转移者13例,其中10例OPN表达阳性,未发生肝转移的37例中14例OPN表达阳性;上皮型与非上皮型脉络膜黑色素瘤OPN表达阳性者分别为11/15和13/35;肿瘤累及睫状体和未累及者OPN表达阳性者分别为20/30和4/20;上述指标的比较差异均有统计学意义(X2=5.888、5.510、10.470,P＜0.05).患者不同性别、年龄、眼别、肿瘤最大基底径以及是否侵犯巩膜导管间OPN表达阳性例数的比较差异均无统计学意义(P=0.536、0.256、0.802、0.848、0.555).转移潜能细胞系由高到低依次为MUM-2B、C918、OCM-1A,其OPN mRNA相对表达量分别为1.00±0.04、0.91±0.03、0.08±0.01,差异有统计学意义(F=33.135,P＜0.05),MUM-2B、C918中OPN mRNA的表达水平较OCM-1A中明显增高,差异均有统计学意义(P=0.00),而MUM-2B、C918中OPN mRNA的表达水平差异无统计学意义(P=0.804).结论 OPN与UM侵袭能力及转移潜能密切相关,发生转移的UM组织及高侵袭转移性细胞系中OPN表达水平明显升高,提示OPN可能作为预测UM侵袭能力、转移潜能以及患者预后的指标.     

PD-L1: PD-1 interaction contributes to the functional suppression of T-cell responses to human uveal melanoma cells in vitro

PURPOSE: To assess the expression of PD-L1 on human uveal melanomas and its potential to suppress T-cell function. METHODS: A panel of primary and metastatic uveal melanoma cell lines was evaluated for PD-L1 expression by RT-PCR and flow cytometric analysis. Uveal melanoma-containing eyes were examined for PD-L1 expression by immunohistochemistry. PD-L1 function was tested by coculturing IFN-gamma-pretreated uveal melanoma cells with activated Jurkat T cells for 48 hours and assessing T-cell production of IL-2 by ELISA. RESULTS: Five of the nine primary and one of the five metastatic uveal melanoma cell lines tested constitutively expressed PD-L1 protein at various levels. However, all primary and metastatic uveal melanoma cell lines upregulated PD-L1 expression after stimulation with IFN-gamma. Immunohistochemistry demonstrated that PD-L1 was not expressed by primary uveal melanomas in situ. IL-2 production by activated Jurkat T cells was decreased significantly when the cells were cocultured with IFN-gamma-pretreated uveal melanoma cells. More than 70% of IL-2 production was restored by addition of either anti-PD-L1 or anti-PD-1 antibody to the coculture assays (P < 0.01). CONCLUSIONS: Expression of PD-L1 by uveal melanoma cells regulates T-cell function by suppressing IL-2 production. The results imply that the presence of IFN-gamma in the tumor local microenvironment promotes upregulation of PD-L1 expression by uveal melanoma, which may, in part, promote immune escape by impairing T-cell function. The selective blockade of PD-L1 is a potential strategy in T-cell-based immunotherapy for uveal melanoma.