【作者】 吴桐；

【导师】 孙丰源；

【作者基本信息】 天津医科大学 ， 眼科学， 2010， 博士

【摘要】 [目的]葡萄膜黑色素瘤是一种成人最常见的原发性眼内恶性肿瘤,主要来源于葡萄膜黑色素细胞,因其血运丰富,主要经血行转移,而且特异性转移到肝脏,转移是主要的致死因素,尤其葡萄膜黑色素瘤的转移往往具有隐匿性,即在肿瘤早期就容易出现临床影像学无法诊断发现的微转移灶。因而目前对葡萄膜黑色素瘤研究的热点是确立转移潜能的预后标志,寻找可以早期准确客观预判葡萄膜黑色素瘤患者预后的生物标记物。legumain是一个新发现的半胱氨酸蛋白酶,其特点：在肿瘤组织中特异性表达,正常组织中无明显表达,与肿瘤免疫关系密切,而且与肿瘤生成,侵袭,转移,扩散密切相关。本研究旨在明确legumain在葡萄膜黑色素瘤中的表达,探讨legumain对葡萄膜黑色素瘤患者预后的影响,及其与影响葡萄膜黑色素瘤患者预后各因素(病理类型,肿瘤体积,转移情况,视神经受累,侵润程度)的关系。[方法]本研究首先选取两株葡萄膜黑色素瘤细胞株Mum-2B和Mum-2C,进行细胞培养传代,提取RNA和蛋白质,进行RT-PCR和Western-blot检测,同时利用两株细胞系进行细胞免疫荧光的染色,从而在基因和蛋白水平明确legumain在葡萄膜黑色素瘤细胞系的表达。在这个实验基础上,进而收集82例葡萄膜黑色素瘤患者的临床及预后资料,同时将这些患者保存的石蜡标本制作组织切片,进行legumain免疫组织化学染色,统计legumain的表达情况,运用Kaplan-meier单因素生存分析的原理与方法进行统计分析,将legumain的表达情况作为待检验的预后因素,与患者预后结果相联系,并运用Log-rank、Brelow、Tarone-Ware三种方法进行显著性检验,明确legumain对葡萄膜黑色素瘤患者预后的影响,同时对82例葡萄膜黑色素瘤的临床预后资料进行Kaplan-meier单因素生存分析,并运用Log-rank、Brelow、Tarone-Ware三种方法进行显著性检验,筛选出与葡萄膜黑色素瘤患者预后相关的因素,然后将legumain免疫组化结果分别与葡萄膜黑色素瘤患者预后相关的各因素进行相关分析,明确legumain与影响葡萄膜黑色素瘤预后各相关因素的关系。[结果]在细胞实验中,通过RT-PCR的检测,表明在基因水平上两株葡萄膜黑色素瘤细胞系legumain为高表达,从细胞免疫荧光也可见legumain在两株葡萄膜黑色素瘤细胞系细胞膜呈现为绿色荧光的高表达,利用western-blot技术对两株细胞系进行legumain的蛋白检测,可见legumain在两株细胞系中均为高表达,且在Mum-2B中表达较高。对82例葡萄膜黑色素瘤患者的石蜡病理切片进行免疫组化染色,结果显示在葡萄膜黑色素瘤的三种基本病理类型(上皮细胞型,梭形细胞型,混合细胞型)中legumain均有阳性(高表达)和阴性(低表达)的表达。通过Kaplan-meier单因素生存分析和Log-rank、Brelow、Tarone-Ware三种方法进行显著性检验,发现葡萄膜黑色素瘤患者性别的不同对患者预后无明显影响(P>0.05),其余因素legumain,病理类型,肿瘤体积,转移情况,视神经受累,侵润程度均对葡萄膜黑色素瘤患者的预后有显著性的影响(P<0.05)。经过双变量的相关分析,legumain与影响葡萄膜黑色素瘤患者预后的各因素(病理类型,肿瘤体积,转移情况,侵袭视神经,侵润程度)均呈正相关(P<0.01),而且与肿瘤侵润程度关系密切(相关系数大于0.5)。[结论]本研究通过对葡萄膜黑色素瘤细胞系和石蜡包埋病理标本进行legumain表达情况的检测,发现legumain在葡萄膜黑色素瘤中有明显的高表达,然后结合患者临床及预后资料进行葡萄膜黑色素瘤的预后研究和相关分析,明确legumain与葡萄膜黑色素瘤预后及影响预后各因素的关系,发现legumain不但与葡萄膜黑色素瘤患者预后有关,而且与影响葡萄膜黑色素瘤患者预后的各因素有一定的相关性,从而说明legumain不仅是判断葡萄膜黑色素瘤患者预后的一个重要指标,而且在葡萄膜黑色素瘤的发生发展过程中可能起着重要作用,legumain也有可能成为葡萄膜黑色素瘤治疗的一个新的靶点。更多还原

【Abstract】 [objective] In adults, uveal melanoma is the most common malignant intraocular tumor, which originated from melanocytes. Because of its rich circulation of blood, uveal melanoma can disseminate hematogenously, which preferentially metastasizes to the liver, and metastasis is the main cause of death. Especially insidious potential is the main character of UM metastasis, so at the time of diagnosis, many patients may have subclinical metastasis, which underscores the critical need to identify prognostic markers indicative of uveal melanoma invasive metastatic potential. Legumain, an novel asparaginyl endopeptidase, is expressed in some humam solid carcinoma, whereas it can not been detected in normal tissures, whose overexpression has been shown to be combined with increased migration, invasion, and metastasis in some solid carcinaoma. In our study, to make sure the expression of legumain in uveal melanoma, and to investigate its prognostic value in the patients of uveal melanoma and its relationship with other prognostic factors (cell type, tumor size, metastasis, involvement of optic nerve, and extent of invasion) in uveal melanoma.[Methods] In our study, firstly, to detect legumain in two uveal melanoma cell lines by using RT-PCR and western-blot, after the culture of cell lines and extraction of RNA and protein from cell lines. Simultaneously, immunofluorescence staining can be used to show the expression of legumain in uveal melanoma cell lines. Secondly, Paraffin-embedded sections from 82 clinicopathologically well-characterized cases of primary uveal melanoma were immunohistochemically stained for legumain. Legumain expression was evaluated in terms of both the intensity and the extent of staining for each specimen. Thirdly, to investigate the relationship between the expression of legumain and prognostic outcome of uveal melanoma patients through Kaplan-Meier univariate analysis and Log-rank, Brelow, Tarone-Ware tests, and some other prognostic risk factors were to be determined after the prognostic risk factors wre preliminary srcreened respectively with Kaplan-Meier univariate analysis and Log-rank, Brelow, Tarone-Ware tests. Last but not least, we investigated the relationship between the expression of legumain and other prognostic risk factors through bivariate correlation analysis. [Results] In our experiment, the expression of legumain was detected in two uveal melanoma cell lines by using RT-PCR, western-blot and immunofluorescence staining, and especially the expression of legumain was more obvious in Mum-2B cell line by western-blot. Both positive and negative expression of legumain can be shown in the three main cell types of uveal melanoma (epitheloid, spindle, mixed cell) by immunohistochemically staining for paraffin-embedded sections from 82 cases of uveal melanoma. Through Kaplan-Meier univariate analysis and Log-rank, Brelow, Tarone-Ware tests, the expression of legumain, cell type, tumor size, metastasis, involvement of optic nerve, and extent of invasion (P<0.05) except sex (P>0.05) had not only great influence on the prognosis of uveal melanoma patients, but also the expression of legumain had positive correlations with other prognostic risk factors including cell type, tumor size, metastasis, involvement of optic nerve, and extent of invasion (P<0.01), especially with the extent of invasion (correlation coefficient> 0.5), by using bivariate correlation analysis.[Conclusion] In this study, by detecting legumain in the two cell lines of uveal melanomna and paraffin-embedded sections from 82 cases of uveal melanoma, it can be proved that legumain was expressed in uveal melanoma, and in combination with clinical data, through survival study and correlation analysis, it is to find that upregulation of legumain expression appears to be associated with poor prognosis in uveal melanoma, and also has positive correlations with other prognostic risk factors. Therefore, legumain is not only a valuable predictive marker for prognosis of uveal melanoma patients, but also it is involved in the pathgenesis and progress of uveal melanoma. In the near future, legumain may be the important target for effective treatment of uveal melanoma.更多还原