眼眶横纹肌肉瘤N—ras癌基因点突变及rasp21,p53蛋白的 …

研究眼眶横纹肌肉瘤（ｒｈａｂｄｏｍｙｏｓａｒｃｏｍａ，ＲＭＳ）Ｎ－ｒａｓ癌基因突变ｒａｓｐ２１、ｐ５３蛋白的异常表达，探讨基因突变在眼眶ＲＭＳ发病中的作用。方法应用特异性高突变区位不同的突变型寡核苷酸探针及突型变型ｒａｓｐ２１、ｐ５３单克隆抗体，对２１眼眶ＲＭＳ组织进行ＤＮＡ斑点杂交和免疫组织化学分析。结果４例眼眶ＲＭＳ细胞发生Ｎ－ｒａｓ癌基因１２密码子第２个碱基突变，５例眼眶ＲＭＳ组织中发生６１     

遗传性视网膜变性视细胞凋亡与p53基因表达

目的 研究遗传性视网膜变性视细胞凋亡的基因调控。方法 对出生后第１３－６０天的遗传性神经网膜变性动物模型（ＲＣＳ）大鼠视网膜,进行末端脱氧核糖核苷酸连接酶介导 ｄＵＴＰ末端标记（ＴＵＮＥＬ）凋恨细胞检测、ｐ５３蛋白免疫组织化学染色、ｐ５３ｍＲＮＡ原位杂交和ＲＴ－ＰＣＲ。结果 ＲＣＳ大鼠视网膜视细胞自生后第２５天出现凋亡,第３０－３５天达高峰;视细胞凋亡初期,妈隆后第２８－３０天,视细胞内Ｐ５３蛋白     

RDS／peripherin表达载体的构建和在COS—1细胞的表达

目的 构建大鼠ＲＤＳ／ｐｅｒｉｐｈｅｒｉｎ真核表达载体ｐＡＬＴＥＲ－ＲＤＳ,并观察在ＣＯＳ－１细胞中的表达,方法 将全长度的ＲＤＳ／ｐｅｒｉｐｈｅｒｉｎｃＤＮＡ（１．５ｋｂ）插入到真核表达载体ｐＡＬＴＥＲ－ＭＡＸ中,构建了ＲＤＳ真核表达载体ｐＡＬＴＥＲ－ＲＤＳ,采用电穿孔技术将其转染ＣＯＳ－１细胞,转染４８ｈ后,采用ＲＴ－ＰＣＲ检测ＲＤＳ／ｐｅｒｉｈｐｅｒｉｎｃＤＮＡ的表达,结果 采用ＲＴ－ＰＣＲ     

8-Br-cAMP可上调人视网膜母细胞瘤HX O-Rb44细胞抑癌基因的表达

目的 探讨８－Ｂｒ－ｃＡＭＰ对人网膜母细胞瘤ＨＸＯ－Ｒｂ４４细胞抑癌基因表达的效应，方法 应用ＲＮＡ斑点印迹技术检测细胞ｐ１６、ｐ２１ｗａｆｌ、ｗｐ５３、ｍｐ５３和Ｒｂ的ｍＲＮＡ、应用免疫组化斑点印迷技术检测细胞Ｐ１６、Ｐ２１ｗａｆｌ、ＰＲｂ、ｃｄｋ２、ｃｄｋ４和ＰＣＮＡ蛋白质表达的免疫反应（ＩＲ）。结果 在斑点印迹标本上，人ＨＸＯ－Ｂｂ４４细胞ｐ１６、ｐ２１ｗａｆｌ、ｗｐ５３及Ｒｂ的ｍＲＮＡ信号     

眼部鳞状细胞癌p53基因突变的研究

目的 探讨眼部鳞状细胞癌发生的分子生物学机制。方法 采用聚合酶链反应－限制性片段长度多态性（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ－ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍｓ,ＰＣＲ－ＲＦＬＰ）方法,对６３例患者的送检标本（包括３６例浸润性鳞状细胞癌７例原位癌,２０例乳头状瘤）,中ｐ５３基因２４８位点的突变进行检测,结果 ２２．２％（８／３６）的浸润性鳞状     

MsrB1对ONOO－诱导的人晶状体上皮细胞周期的影响

目的　探讨蛋氨酸亚砜还原酶Ｂ１（ｍｅｔｈｉｏｎｉｎｅｓｕｌｆｏｘｉｄｅｒｅｄｕｃｔａｓｅＢ１,ＭｓｒＢ１）基因沉默对过氧亚硝酸根（ＯＮＯＯ－）诱导的人晶状体上皮细胞（ｈｕｍａｎｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｌｓ,ＨＬＥＣ）细胞周期的影响和ＥＲＫ信号通路在其中的调节作用。方法　将培养的ＨＬＥＣ分为两组:正常组和ＭｓｒＢ１基因沉默组,分别用０μｍｏｌ·Ｌ－１、１００μｍｏｌ·Ｌ－１、２００μｍｏｌ·Ｌ－１和３００μｍｏｌ·Ｌ－１ＯＮＯＯ－处理２０ｍｉｎ后,继续培养１２ｈ。用ＲＴ-ＰＣＲ法检测ＭｓｒＢ１基因表达水平变化,流式细胞仪检测细胞周期水平变化,Ｗｅｓｔｅｒｎｂｌｏｔ法检测ＭｓｒＢ１基因沉默和ＯＮＯＯ－对ｐＥＲＫ表达水平的影响。结果　３００μｍｏｌ·Ｌ－１ＯＮＯＯ－处理ＨＬＥＣ会导致ＭｓｒＢ１表达水平显著下降（Ｐ＜０．０１）,当ＭｓｒＢ１基因沉默细胞经３００μｍｏｌ·Ｌ－１ＯＮＯＯ－处理后,ＭｓｒＢ１表达水平进一步显著下调（Ｐ＜０．０５）。经２００μｍｏｌ·Ｌ－１或３００μｍｏｌ·Ｌ－１ ＯＮＯＯ－处理,ＨＬＥＣ细胞周期分别阻滞在Ｇ２／Ｍ期和Ｓ期,当ＭｓｒＢ１基因沉默细胞经２００μｍｏｌ·Ｌ－１或３００μｍｏｌ·Ｌ－１ＯＮＯＯ－处理后,更多的细胞阻滞在Ｇ２／Ｍ期或Ｓ期（Ｐ＜０．０５）。ｐＥＲＫ检测结果表明,ＭｓｒＢ１基因沉默前后经ＯＮＯＯ－处理导致的细胞周期阻滞和ｐＥＲＫ表达水平显著下降有关（Ｐ＜０．０１）。结论　ＯＮＯＯ－可以下调ＨＬＥＣＭｓｒＢ１表达水平,ＭｓｒＢ１基因沉默细胞经ＯＮＯＯ－处理,ｐＥＲＫ表达水平显著下降,导致更多细胞阻滞在Ｇ２／Ｍ期和Ｓ期。     

大鼠晶体上皮细胞Cu—ZnSOD的分子克隆表达和活性鉴定

不同来源的超氧化物歧化酶（ＳＯＤ）的克隆已有报道，用ＲＴ－ＰＣＲ方法我们成功地从大鼠晶休下皮细胞中克隆出了ＣＵ－ＺｎＳＯＤｃＤＮＡ并利用ｐＭａｌ载 大肠杆菌中高产表达出ＭＢＰ－ＳＯＤ融合蛋白可占菌体总蛋白４０％左右，采用的蛋白纯化系统的亲和层析柱就是利用的ＭＢＰ融合蛋白可被快速纯化而设计的，实验证明此方法可快捷、高效地表达有生物学活性的ＭＢＰ－ＳＯＤ融合蛋白。     

眼眶横纹肌肉瘤rasp21,p53蛋白表达及其与预后的关系

目的 研究ｒａｓｐ２１，ｐ５３癌基因蛋白产物在眼眶横纹肌肉瘤中的表达及其与预后的关系。方法 对确诊的２１例眼眶ＲＭＳ用免疫组化ＡＢＣ法标记ｒａｓｐ２１，ｐ５３蛋白。结果 发现ｒａｓｐ２１，ｐ５３在眼眶ＲＭＳ的阳性率分别为４２．９％和７６．２％。随访的１３例患者中，ｐ５３阳性表达组１００％预后不好，ｒａｓｐ２１／ｐ５３阳性表达组８０％预后不好，ｒａｓｐ２１／ｐ５３阴性组２５％预后不好。     

糖尿病视网膜病变与红细胞变形能力和膜磷脂及收缩蛋白变化的关系

目的　探讨糖尿病视网膜病变（ｄｉａｂｅｔｉｃ ｒｅｔｉｎｏｐａｔｈｙ，ＤＲ）与红细胞变形能力（ｅｒｙｔｈｒｏｃｙｔｅ ｄｅｆｏｒｍ ａｂｉｌｉｔｙ，ＥＤ）、红细胞膜磷脂成分及红细胞膜收缩蛋白（ｓｐｅｃｔｒｉｎ，ＳＰ）变化的关系。　方法　１０８例Ⅱ型糖尿病患者根据视网膜病变的有无分为ＤＲ组（５５例）和非ＤＲ（ｎｏｎｄｉａｂｅｔｉｃ ｒｅｔｉｎｏｐａｔｈｙ，ＮＤＲ）组（５３例），检测其ＥＤ、红细胞膜磷脂成分和红细胞膜收缩蛋白二聚体（ｓｐｅｃｔｒｉｎ ｄｉｍｅｒ，ＳＰＤ）、四聚体（ｓｐｅｃｔｒｉｎ ｔｅｔｒａｍ ｅｒ，ＳＰＴ）相对含量的变化。并与正常对照组５３例的相同检测结果作对比。　结果　ＤＲ患者红细胞滤过指数（ｅｒｙｔｈｒｏｃｙｔｅ ｆｉｌｔｒａｔｉｏｎ ｉｎｄｅｘ，ＥＦＩ）、ＳＰＤ、ＳＰＤ／ＳＰＴ、神经鞘磷脂（ｓｐｉｎｇｏｍ ｙｌｉｎｅ，ＳＭ）／磷脂酰胆碱（ｐｈｏｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ，ＰＣ）明显增高，而ＳＰＴ、ＳＭ、ＰＣ、磷脂酰丝氨酸（ｐｈｏｐｈａｔｉｄｙｌｓｅｒｉｎｅ，ＰＳ）和磷脂酰乙醇胺（ｐｈａｔｉｄｙｌｔｈａｎｏｌａｍ ｉｎｅ，ＰＥ）明显降低，与对照组和ＮＤＲ 组比较有显著差异（Ｆ＝ ８．     

小儿激素敏感性肾病综合征辅助性T细胞亚群研究

目的 进一步探讨微小病变型肾病综合征（ＭＣＮＳ）的发病有否ＴＨ－１／ＴＨ－２反应失衡。方法 通过逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）和双抗体夹心ＥＬＩＳＡ法，检测１１激素敏感性肾病综合征患儿外周血单个核细胞（ＰＢＭＣ）淋巴因子基因表达和蛋白产生。结果 与正常对照组相比，ＮＳ患儿ＩＬ－１０、ＩＬ－４基因表达和蛋白产生明显增高，ＩＬ－２、ＩＬ－６降低，干扰素（ＩＦＮ）－γ和ＩＬ－５无明显差异。结论 激     

The effect of ultraviolet radiation on choroidal melanocytes and melanoma cell lines: cell survival and matrix metalloproteinase production

Background Ultraviolet radiation (UVR) can induce DNA damage and regulate the expression of factors important for tumour growth and metastasis, including matrix metalloproteinases (MMPs). Epidemiological studies suggest that chronic UVR exposure, especially during early adulthood, may be a risk factor in patients with choroidal melanoma. However, the effects of UV(R)-B on human choroidal melanocyte survival and growth are unknown. In this study, we investigated if UV(R)-B affected the in vitro survival, growth and MMP production of choroidal melanocytes and melanoma cells. Methods Cultures of primary choroidal melanocytes and melanoma cell lines (OCM-1 and OCM-8) were exposed to UV(R)-B (0–30 mJ/cm²). The cell morphology and growth were examined, and cell viability was assessed using an MTT assay. Gelatin zymography was used to assess the enzymatic activity for MMP-2 and -9 in conditioned media following UV(R)-B treatment. Results UV(R)-B ≥20 mJ/cm² was cytotoxic for choroidal melanocytes. Cytotoxic doses of 5 to 10 mJ/cm² were found for OCM-8 and OCM-1 melanoma cell lines. Low levels of UV(R)-B (2.5 and 3.5 mJ/cm²) significantly reduced melanoma cell viability after 48 h, although melanocyte viability was not affected by doses of UV(R)-B <10 mJ/cm². Conditioned media from melanoma cells and melanocytes displayed pro-MMP-2 activity independent of UV(R)-B. Control and UV(R)-B-treated OCM-1 cells secreted active MMP-2 up to 72 h. Pro-MMP-9 activity was seen from 36 h for control and UV(R)-B-treated OCM-1 and OCM-8 cells. Conclusions Melanocytes appeared more resistant to physiological doses of UV(R)-B than melanoma cells; the potential of melanocytes to initially survive DNA damage following UV(R)-B exposure may be relevant to the subsequent transformation of melanocytes to melanomas. Although UV(R)-B did not induce the production and/or activation of MMP-2 and -9 in melanocytes or melanoma cells, we are currently investigating whether DNA damage-response genes such as p53 and p21 can be regulated following UVR exposure, and whether they are important for choroidal melanoma development. This study was supported in part by grants from the Sydney Foundation for Medical Research (MCM) and the National Health and Medical Research Council, Australia (RMC).     

p53 Immunoreactivity,Ki-67 expression,and microcirculation patterns in melanoma of the iris,ciliary body,and choroid

PURPOSE: The major role of p53 is to modulate cell proliferation, but recently, it has been found that p53 also modulates angiogenesis through several pathways. Because both cellular proliferation and microcirculation patterns are important prognostic markers in uveal melanoma, we tested the relationships between p53 expression, the expression of the cell proliferation marker Ki-67, and the presence of various microcirculation patterns in uveal melanoma. METHODS: Immunostaining of p53 and Ki-67 using the bp53.12 and the MIB-1 antibodies, respectively, were preformed in 98 uveal melanomas (18 melanomas confined to the iris, 30 ciliary body melanomas, and 50 choroidal melanomas). Percent of p53 positive cells, and the mean MIB-1 positive cell count per high power field were calculated in each section by two observers. Microcirculation patterns were assessed in adjacent PAS stained sections. RESULTS: p53 immunoreactivity was found in 14 of the 98 melanomas. High proliferative activity and epithelioid cell type were associated with p53 immunoreactivity. However, p53 immunoreactivity was not associated with any of the microcirculation patterns or with tumor location. CONCLUSIONS: The findings suggest that alterations in p53 expression are associated with the expression of the cellular proliferation marker, Ki-67, but are not associated with the presence of microcirculation patterns.     

Prospects for developing treatment of uveal melanoma from the position of modern carcinogenesis concepts

Immunohistochemical studies of regulator proteins p53, Bcl-2, Bax, Ki-67, and CD-95L showed that apoptosis suppression, allowing unlimited proliferation of tumor cells, plays an important role in the carcinogenesis of uveal melanoma. This fact dictates the need of including drugs arresting suppression or inducing apoptosis in therapeutic protocols for uveal melanoma. Prognostic significance of p53, Bcl-2, Bax, and CD-95L was demonstrated. Each of the peptides can be used as a prognostic marker.     

Expression and distribution of MMPs and TIMPs in human uveal melanoma

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas (n=18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0-3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was > or =Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).     

p21 ras和p53的表达与眼睑恶性肿瘤临床病理学的关系

目的探讨ras和p53基因突变与眼睑恶性肿瘤发生的关系及临床病理学意义.方法采用免疫组化SABC法检测了98例眼睑恶性肿瘤组织中p21ras和p53蛋白的表达.结果眼睑正常及轻度不典型增生上皮中未见p21ras和p53蛋白表达;而中、重度不典型增生、原位癌、浸润癌p21ras和p53蛋白阳性表达率逐渐增高,随着肿瘤分化程度的降低,p21ras和p53蛋白阳性表达率及阳性表达强度逐渐增高;SCC中p21ras及p53蛋白阳性表达强度与组织分化程度之间呈显著负相关,且差别有显著性(P＜0.05).结论p21ras和p53基因突变与眼睑恶性肿瘤发生、发展密切相关.     

基质金属蛋白酶家族及细胞外基质金属蛋白酶诱导因子与常见眼内恶性肿瘤的关系

基质金属蛋白酶家族包括基质金属蛋白酶及其抑制剂。基质金属蛋白酶是一组Zn2+依赖性蛋白水解酶,能降解细胞外基质,基质金属蛋白酶抑制剂则能抑制其活性。细胞外基质金属蛋白酶诱导因子广泛地存在于人类各种肿瘤细胞,刺激与肿瘤有关的成纤维细胞和肿瘤细胞自身的基质金属蛋白酶合成增加。三者常在眼部肿瘤中异常表达,与眼肿瘤的发展及浸润转移关系密切。本文就其与眼部常见恶性肿瘤视网膜母细胞瘤及脉络膜黑色素瘤的关系作一综述。     

c-myc, p53, and Bcl-2 expression and clinical outcome in uveal melanoma

AIMS: Overexpression of c-myc protein has independent prognostic significance in a variety of primary and metastatic cutaneous melanomas which suggests a possible role for this gene in melanomagenesis. We have therefore examined the importance of this oncogene in uveal melanoma and studied the coexpression of two other gene products, Bcl-2 and p53, which might contribute to its effect. METHODS: The percentage of cells positive for nuclear c-myc expression was estimated by flow cytometric analysis of nuclei extracted from paraffin blocks. The expression of Bcl-2 and p53 protein was assessed by immunohistochemistry. A total of 71 tumours were studied and the results compared with survival with a mean follow up period of 6 years. RESULTS: c-myc was expressed in > 50% of the cells by 70% of the tumours, and was independently associated with improved survival in a Cox multiple regression-model. Although Bcl-2 was expressed by the majority of the cells in 67% tumours, it was without effect on prognosis. None of the cases studied showed convincing positivity for p53. Analysis of coexpression showed that the best survival was seen in c-myc+/Bcl-2+ tumours and the worst in c-myc-/Bcl-2-tumours. CONCLUSION: The finding of improved rather than reduced survival in c-myc positive tumours is at variance with skin melanoma. There was no evidence to suggest that c-myc was modulated by upregulation of Bcl-2 or p53 inactivation/mutation. Although Bcl-2 is unlikely to have any effect on tumour growth or metastasis, it could contribute to the general lack of susceptibility to apoptosis in these tumours.