卵巢癌高频转移细胞模型中nm23-H1基因表达的相关性研究

目的 筛选高频转移卵巢恶性肿瘤细胞，研究不同转移潜能的细胞和nm23的相关性。方法 通过反复动物接种和体外培养，观察动物肺转移状况，筛选高频转移细胞株，比较原发肿瘤和转移肿瘤的特征，并应用Northera-blot方法测定各类肿瘤细胞nm23 mRNA表达水平。结果 8株卵巢恶性肿瘤细胞中4株有较高转移潜能。多次培养接种可筛选出高频转移细胞亚群。测定各类细胞nm23 mRNA表达水平与肿瘤转移特性呈负相关。结论 由基因分子水平决定的肿瘤转移趋势在不同肿瘤种类及细胞亚群中有明显差异；卵巢癌中nm23 mRNA和蛋白的表达与其转移能力的降低有密切关系，可作为判定卵巢癌预后的敏感指标。     

nm23-H1基因表达与卵巢癌转移的相关性

背景与目的：转移是卵巢癌治疗失败及患者死亡的首要原因。然而,目前对卵巢癌转移潜能的分子机制知之不多。本研究旨在筛选高频转移卵巢恶性肿瘤细胞,分析卵巢癌高频转移细胞模型中nm23．H1基因表达与肿瘤转移特性的相关性,为系统实验研究和临床实践提供依据。方法：通过反复动物接种和体外培养,观察动物肺转移状况,筛选高频转移细胞株,比较原发肿瘤和转移肿瘤的特征,并应用Northem blot和Westem blot方法测定各类肿瘤细胞nm23 mRNA和蛋白表达水平。结果：8株卵巢恶性肿瘤细胞中,4株有较高转移潜能,多次培养接种可筛选出高频转移细胞亚群。各类细胞nm23mRNA和蛋白表达水平与肿瘤转移特性呈负相关(r=0．96,P=0．0001)。结论：由基因分子水平决定的肿瘤转移趋势在不同肿瘤种类及细胞亚群中有明显差异;卵巢癌中nm23 mRNA和蛋白的表达与其转移能力的降低有密切关系,可作为判定卵巢癌预后的敏感指标。     

卵巢浆液性囊腺癌中的nm23H1基因mRNA及其突变的研究

目的 探讨卵巢浆液性囊腺癌原发灶与相关淋巴结组织nm23H1mRNA表达及基因突变。方法 应用RT－PCR和SSCP技术对20例卵巢浆液性囊癌原发灶与相关淋巴结组织进行检测分析。结果 nm23H1mRNA表达水平在无淋巴结转移的卵巢癌原发灶组明显高于有淋巴结转移的原发灶组（P＜0．01）。所测组织标本未发现突变。结论 提示nm23H1mRNA表达水平可作为预测该肿瘤发生淋巴结转移的指标。卵巢浆液性囊腺癌基因改变可能不以突变为主。     

高尔基体α-甘露糖苷酶Ⅱ、E-cadherin和α-catenin在卵巢上皮肿瘤组织和不同转移能力的卵巢癌细胞中的表达

目的:检测高尔基体α-甘露糖苷酶Ⅱ(Golgiα-mannosidaseⅡ,GMⅡ)、E-cadherin和α-catenin在不同卵巢上皮肿瘤组织和不同转移能力的卵巢癌细胞中的表达。方法:运用免疫组织化学法检测46例卵巢上皮恶性肿瘤(其中有大网膜或淋巴结转移者27例)、15例卵巢上皮交界性肿瘤及12例卵巢上皮良性肿瘤组织中GMⅡ、E-cadherin和α-catenin的表达情况。体外培养卵巢癌A2780、SKOV-3和HO-8910细胞,分别应用RT-PCR、免疫荧光和蛋白质印迹法检测GMⅡ、E-cadherin和α-catenin mRNA和蛋白的表达情况。结果:在卵巢上皮恶性肿瘤中GMⅡ的阳性表达率(87%)和E-cadherin、α-catenin的异常表达率(80%和72%)明显高于卵巢上皮交界性肿瘤(60%、20%和40%)和卵巢上皮良性肿瘤(42%、0%和17%),且有转移患者肿瘤组织中的阳性表达率和异常表达率明显高于无转移者(P<0.05)。A2780、SKOV-3和HO-8910细胞中GMⅡ荧光表达强度逐渐增强,E-cadherin和α-catenin荧光表达强度逐渐减弱。在A2780、SKOV-3、HO-8910中,GMⅡmRNA和蛋白表达水平逐渐增加,而E-cadherin和α-catenin mRNA及蛋白表达水平则逐渐减弱,各组间差异有统计学意义(P<0.05)。结论:GMⅡ、E-cadherin和α-catenin可能在卵巢癌的恶性进展中发挥重要作用,GMⅡ有望成为卵巢癌预后的标志和治疗靶点。     

新基因nm23-H1B在卵巢上皮性肿瘤中的表达研究

目的研究人类新基因nm23-H1B与卵巢肿瘤的关系。方法收集2003年4月~2004年2月手术切除各种卵巢肿瘤标本48例,正常卵巢组织8例。采用RT-PCR、Northern杂交、原位杂交检测nm23-H1BmRNA的表达。结果用RT-PCR在所有的标本中均检测到nm23-H1B基因mRNA的表达,其在正常卵巢组织中表达较低,而在所有的卵巢肿瘤中表达均出现明显上调,在早期卵巢癌中的表达高于晚期。Northern杂交显示nm23-H1B基因在正常卵巢组织的表达量较低,交界性肿瘤中的表达则与正常组织中相似,而在卵巢癌组织中表达明显增高,在早期卵巢癌中在分化好的组织中(Ⅰ级)表达量明显高于分化较差(Ⅱ、Ⅲ级)者。原位杂交显示nm23-H1B基因在正常卵巢组织和交界性肿瘤的表达量极低,而在卵巢癌组织中表达明显增高,mRNA表达产物主要位于细胞质内;早期卵巢癌中的mRNA表达量高于晚期卵巢癌。结论nm23-H1B基因与卵巢癌有明显的相关性。     

抑癌基因p16和转移抑制基因nm 23在卵巢上皮性肿瘤中的表达

目的:探讨抑癌基因p16及转移抑制基因nm23与卵巢上皮性肿瘤病理特点及生物学行为的关系。方法:免疫组化SP法检测30例正常卵巢,50例卵巢上皮性良性肿瘤及50例卵巢癌组织中P16蛋白和nm23蛋白,并分析其阳性表达与肿瘤的关系。结果:nm23蛋白在卵巢癌组织中的阳性率(70.0%)明显高于正常卵巢(43.3%)及良性肿瘤组(44.0%)(P<0.05);在浆液性癌(87.5%)和粘液性癌(47.8%)中的阳性差率也有显著差异(P<0.05);特别是在有淋巴结转移(39.1%)和无淋巴结转移(92.6%)的中检出率相非常显著(P<0.01),而与卵巢癌的临床分期及病理分级无明显相关性。p16蛋白在卵巢癌中的检出率(26.0%)明显低于正常卵巢(80.0%)和良性上皮性肿瘤(52.0%)(P<0.01),在晚期肿瘤中的表达率(20.5%)也远低于早期肿瘤(63.6%),而且在高度恶性组织中表达水平(11.1%)与中低度恶性组织的表达水平(43.5%)也有显著差异(P<0.05)。结论:nm23基因的表达与卵巢癌的淋巴结转移等生物学行为有关,p16基因可作为临床判断预后的重要指标之一。nm23、p16基因与卵巢上皮性肿瘤的发生、发展密切相关。     

Autotaxin在人卵巢癌基因及蛋白水平表达的研究及意义

目的:探讨自分泌运动因子(Autotaxin,ATX)mRNA在人卵巢癌中的表达及其与卵巢癌临床病理特征之间的关系。方法:应用半定量逆转录聚合酶链反应(RT-PCR)、Western blot研究8例正常卵巢组织、18例良性卵巢上皮性肿瘤(以下简称良性卵巢肿瘤)、50例卵巢上皮性癌(以下简称卵巢癌)组织、23例大网膜转移灶中ATX mRNA及蛋白的表达。结果:8例正常卵巢组织、18例良性卵巢肿瘤、50例卵巢癌组织、23例大网膜转移灶中均有ATX mRNA表达;但ATX基因表达值在癌组织和大网膜转移灶中显著高于良性卵巢肿瘤和正常卵巢组织(P<0.001),分别为(89.31±10.15)%、(88.91±11.05)%、(40.18±16.71)%、(35.29±13.82)%。Western blot显示ATX蛋白在卵巢癌细胞中表达,在相对分子质量55×103、60×103大小的位置可见清楚的显色条带。ATX mRNA在卵巢癌细胞亦可见明显扩增条带。高表达ATX基因与卵巢癌手术分期和病理分化程度有关,而与年龄、病理类型等无关。结论:卵巢癌细胞中ATX分子在核酸与蛋白水平均有表达,两者结果一致;ATX基因过表达可能与卵巢癌的进展、转移有关。     

卵巢上皮性肿瘤组织Nectin-4表达临床意义的探讨

目的:探讨卵巢上皮性癌(卵巢癌)组织中Nectin-4 mRNA和蛋白的表达及其与临床病理参数之间的关系。方法:采用逆转录聚合酶链式反应(RT-PCR)技术,检测30例卵巢癌、25例卵巢上皮性肿瘤和30例正常卵巢组织中Nectin-4 mRNA的表达情况;采用免疫组织化学法检测Nectin-4蛋白的表达情况,并分析其与卵巢癌临床病理参数的关系。结果:卵巢癌组Nectin-4 mRNA表达水平为0.71±0.12,卵巢良性肿瘤组为0.32±0.09,正常卵巢组为0.30±0.09,卵巢癌组的mRNA表达量显著高于后两组,差异有统计学意义,P<0.01。卵巢癌组Nectin-4蛋白表达水平为3.35±0.52,卵巢良性上皮性肿瘤组1.36±0.41,正常卵巢组1.15±0.44,卵巢癌组织的Nectin-4蛋白表达量明显高于卵巢良性上皮性肿瘤和正常卵巢组织,差异有统计学意义,P<0.01。卵巢癌组织中Nectin-4 mRNA和蛋白表达水平,在不同年龄、不同病理类型间比较,差异均无统计学意义,P>0.05;而在不同病理分化程度、手术分期及有无淋巴结转移间比较,差异均有统计学意义,P<0.05。结论:Nectin-4 mRNA和蛋白在卵巢癌组织中均呈高表达,而在卵巢良性肿瘤组织和正常卵巢组织中仅有微量表达。在卵巢癌组织中,其表达程度与病理分化程度、手术病理分期及淋巴结转移有密切关系。     

卵巢癌细胞PCDGF的表达与意义

目的研究人卵巢癌细胞PCDGF的表达,探讨其与卵巢癌恶性表型的关系.方法应用半定量RT-PCR和Western blot分别检测3株卵巢癌细胞中PCDGF mRNA和蛋白表达水平.单层细胞增殖实验测定3株卵巢癌细胞的增殖能力;Boyden小室体外侵袭实验测定不同细胞株的侵袭能力.结果 3株卵巢癌细胞PCDGF转录和翻译水平呈同步变化(P<0.05),均为SW626最高,A2780稍低,Skov-3最低.而其增殖、侵袭能力亦为SW626最强, A2780次之, Skov-3最弱.结论 3株卵巢癌细胞PCDGF mRNA和蛋白表达水平与其增殖、侵袭能力呈正相关(前者r1=0.97, r2=0.89,后者r1=0.85,r2=0.84),提示PCDGF在卵巢癌发生演进中可能作为独立因素起多种作用,有望成为治疗的新靶点.     

肝再生磷酸转移酶-3的表达与上皮性卵巢癌转移相关性

目的检测肝再生磷酸酶-3（PRL-3）mRNA在上皮性卵巢癌及其转移灶中的表达,探讨其与上皮性卵巢癌发生、发展以及侵袭转移的生物学行为之间的关系。方法以22例正常卵巢组织为对照,通过半定量PCR测定84例上皮性卵巢癌原发灶及58例大网膜转移灶PRL-3 mRNA相对表达水平,并分析其表达水平与临床病理学指标的关系。结果 84例卵巢癌组织中PRL-3基因表达量显著高于正常卵巢组织。58例转移灶PRL-3基因表达量（2.24＋0.52）显著高于原发肿瘤（1.50＋0.47）（P〈0.05）。PRL-3基因表达水平与肿瘤远处转移有相关性（P〈0.05）,而与临床分期、肿瘤病理类型、分化程度及年龄均无关（P〉0.05）,与3年生存率有关（P〈0.01）。结论 PRL-3基因在上皮性卵巢癌的发生、发展和侵袭转移中起重要作用,PRL-3可能成为判断上皮性卵巢癌转移高危因素的一个生物标志物。     

Nm23 and breast cancer metastasis

Summary The majority of breast cancer patients succumb to metastatic disease. We summarize published and recent research concerning thenm23 gene in breast cancer metastasis. In a murine developmental study, nm23 expression increased with the functional differentiation of the mammary gland in nulliparous and pregnant animals. In human breast cancer, five studies have now demonstrated a significant association between reduced nm23 expression, at the RNA or protein levels, and aggressive tumor behavior. Nm23-negative tumor cells have been observed in comedo ductal carcinoma in situ lesions in two independent studies, indicating that decreases innm23 expression begin prior to actual histologically identifiable invasion. Transfection studies, in which humannm23-H1 cDNA was expressed in the metastatic human MDA-MB-435 breast carcinoma cell line, indicate thatnm23-H1 suppresses in vivo metastatic potential by 50-90%. Finally, our data in melanoma and breast carcinoma transfection systems suggest that the biochemical mechanism of nm23 suppressive activity is likely not due to its nucleoside diphosphate kinase activity, association with GAP proteins, or secretion from cells.Supported by an educational grant from CIBA-GEIGY Corp., at the San Antonio Breast Cancer Symposium, Dec. 8, 1992.     

Expression of the nm23-2/NDP kinase alpha gene in rat mammary and oral carcinoma cells of varying metastatic potential.

Reduced expression of the putative metastasis-suppressor gene, nm23-1, has previously been correlated with high tumour metastatic potential. The involvement of the related and proximally-located nm23-2 gene in the suppression of tumour metastasis, however, has not yet been tested. In this study, we compared nm23-2 RNA levels in cell lines derived from three independent rat mammary carcinomas. Northern blot analysis revealed no correlation between nm23-2 RNA levels and metastatic potential in parent or clonal cell lines derived from chemically-induced (MAT 13762, DMBA-8) or spontaneous (BC1) rat mammary carcinomas. Cloning and sequencing of an nm23-2 cDNA from metastatic BC1 cells demonstrated that the predicted coding sequence of nm23-2 RNA in these cells was not inactivated by mutation. Further analysis showed that the nm23-2 gene was not down-regulated in H-ras-transfected metastatic clones or other metastatic cell lines derived from a spontaneous rat paraoral squamous cell carcinoma, B10. The data do not suggest a correlation between nm23-2 gene expression and metastasis-suppression in these tumours.     

Expression of nm23-H1 in uveal melanoma

Uveal melanoma (UM) is the most common malignant intraocular tumor in adults. Despite the high accuracy of clinical diagnosis and advances in local treatment, more than 50% of UM patients develop metastasis within 10 years of initial diagnosis. NM23 is one of the human metastasis suppressor genes. Reduced nm23-H1 expression is correlated with high metastatic potential in many different cancers. The purpose of this study is to investigate the expression of nm23-H1 in UM and its potential value as a prognostic marker. Immunostaining of nm23-H1 was verified in five human UM cell lines with different metastatic potentials. The expression level of nm23-H1 mRNA was evaluated with one-step quantitative real-time PCR. The invasion ability of the cell lines was assessed before and after silencing nm23-H1 with small interference RNA. Thirty-two cases of paraffin-embedded specimens of human UM were immunostained with nm23-H1 monoclonal antibody. The immunostaining was evaluated in a semiquantitative fashion based on extent and intensity. The real-time PCR results of five human UM cell lines showed that expression of nm23-H1 was higher in cell lines with low metastatic potential compared with those with high metastatic potential (P<0.05). The invasive ability of the UM cell lines increased after silencing nm23-H1 expression with small interference RNA (P<0.05). The immunostaining of nm23-H1 was cytoplasmic in all cell lines and UM patients samples. The increased immunostaining intensity of nm23-H1 in patients' samples was associated with better survival rate (Kaplan-Meier test P=0.0097). The expression of nm23-H1 was not correlated with other prognostic factors. It can be concluded that nm23-H1 may be a prognostic marker to predict the survival rate of UM patients and it has the potential to identify high-risk patients.     

NM23, an example of a metastasis suppressor gene

Metastasis suppressor genes - unlike tumor suppressor genes - are defined by their capacity to control metastatic dissemination in vivo without affecting growth of the primary tumor. The first of these metastasis suppressor genes, NM23, was identified in 1988. Since then, expression of NM23 has been studied widely in human tumor cohorts, often with contradictory results. Not only is NM23 overexpressed in most human solid tumors when compared to healthy tissues, but also low expression of NM23 correlates with metastasis and poor clinical prognosis in the advanced stages of a number of epithelial cancer types, including melanoma, breast, colon, and liver carcinoma. This does not hold true, however, for other cancer types such as neuroblastoma and hematological malignancies, in which high NM23 expression correlates with more aggressive disease. Genetic alterations in the NM23 gene - loss of heterozygosity, spontaneous mutations and polymorphisms - are rarely found in tumors; thus, the metastatic potential of tumor cells is probably affected by NM23 protein levels. Three lines of evidence demonstrate the anti-metastatic activity of NM23: first, overexpression of NM23 in metastatic cell lines reduces their metastatic potential in xenograft models; second, the incidence of lung metastases is elevated in NM23 knockout mice prone to develop hepatocellular carcinoma, and, third, silencing NM23 by RNA interference confers a "metastatic phenotype" on non-invasive human epithelial liver and colon cancer cell lines. It appears that NM23 is crucial for inhibiting invasive migration, so acting at early stages of metastatic dissemination. The mechanistic basis of the metastasis suppressor function of NM23 and its regulated expression still remains obscure, however. Reactivation of expression of the endogenous NM23 gene in tumor cells, or stimulation of the pathways it controls, constitutes a promising avenue for anti-metastatic therapy.     

人肿瘤裸鼠移植瘤株模型转移抑制基因nm23-H1mRNA的表达

目的ｎｍ２３基因与肿瘤移植瘤株转移的关系。方法应用地高辛标记的ｎｍ２３┐Ｈ１反义ｃＲＮＡ探针原位杂交方法对人克隆化肝癌，人骨肉瘤，肾癌三种不同肿瘤的裸鼠移植瘤株模型（Ｈ┐ＣＳ，Ｍ┐ＯＳ，Ｍ┐ＲＣＣ）中瘤内ｎｍ２３┐Ｈ１进行检测。结果ｎｍ２３Ｈ１ｍＲＮＡ在三模型中均有不同程度快的表达，其中有较强转移能力的Ｈ┐ＣＳ，和潜伏期短、侵袭生长速度快的Ｍ┐ＲＣＣ杂交信号５０％为弱阳性，而Ｍ┐ＯＳ仅有２５％为弱阳性。结论ｎｍ２３的弱阳性表达可能和Ｈ┐ＣＳ生长速度及转移倾向有一定联系。     

Inhibition of ovarian cancer metastasis by adeno-associated virus-mediated gene transfer of nm23H1 in an orthotopic implantation model

Ovarian cancer is one of the most threatening malignant tumors in females due to the frequent occurrence of metastasis that precedes diagnosis. The present study explored the possibility of preventing ovarian cancer metastasis by promoting nm23H1 expression through adeno-associated virus (AAV)-mediated gene transfer. A cell line of high metastatic potential, SW626-M4, was derived by in vivo selection and used to establish an ovarian cancer metastasis model in the mouse. Liver metastasis and animal survival time were measured after transfer of a recombinant adeno-associated viral vector expressing nm23H1 (AAV-nm23H1) into the aforementioned model. Intraperitoneal injection of AAV-nm23H1 into this orthotopic implantation model of ovarian cancer resulted in (1) expression of the exogenous gene in more than 95% of tumor cells in situ in nude mice; (2) a 60% reduction in the number of animals developing liver metastases; and (3) a 35-day prolongation of median survival time compared with the untreated host group. In conclusion, the results support the feasibility of induction of nm23H1 expression through gene transfer as a therapeutic strategy for preventing metastases and prolonging host survival time, and indicate that AAV vectors deserve attention in the design of future gene therapy approaches to achieving long-term expression of curative genes in vivo.     

脂质体-c-erbB-2反义寡核苷酸转染对人卵巢癌裸鼠皮下移植瘤形成的抑制作用研究

目的　探讨经脂质体—硫代磷酸化修饰的c erbB 2反义寡脱氧核苷酸 (ASODN )作用后的卵巢上皮癌细胞株SKOV3 在裸鼠皮下的成瘤能力。方法　利用脂质体将经硫代磷酸化修饰的寡核苷酸导入SKOV3 细胞内 ,然后将这种被转染的细胞接种于裸鼠皮下 ,观察肿瘤体积的变化 ,并计算抑瘤率。结果　经脂质体 c erbB 2ASODN作用后的卵巢上皮癌细胞在裸鼠皮下成瘤能力降低 ,最大抑瘤率达 4 0 .0 %(P <0 .0 5 )。首次出现目检可见肿瘤的平均时间延长为 9.6天 (P <0 .0 5 )。结论　c erbB 2癌基因的反义调控能降低卵巢上皮癌细胞的成瘤能力 ,抑制肿瘤生长 ,在卵巢上皮癌的基因治疗中有一定价值。     

Search for metastasis suppressor genes

Cell fusion experiments have predicted the existence of cancer metastasis suppressor genes. The E1a gene of Adenovirus 2 has been demonstrated to suppress c-Ha-ras induction of experimental metastatic potential in rat embryo fibroblasts. Another approach to the identification of candidate metastasis suppressor genes has utilized differential or subtraction hybridizations to clone genes which are downregulated as cells become highly metastatic. To date, three such genes have been identified: nm23, WDNM1, and fibronectin. With regard to nm23, downregulation of nm23 RNA levels in high metastatic potential cells has been demonstrated in a wide variety of rodent metastasis systems, including K-1735 murine melanoma cell lines, nitrosomethylurea-induced rat mammary tumors, MMTV-induced mouse mammary tumors, and ras +/- E1a transfected rat embryo fibroblasts. Whether the expression of the nm23 gene, and other down-regulated genes in tumor metastasis, correlates with changes in metastatic potential, or actually has suppressive activity, will require transfection experiments.