PCR-SSCP法检测人肝癌组织中nm23-H1基因突变的研究

目的观察nm23-H1基因在人原发性肝细胞癌中的突变情况，探讨nm23-H1基因突变与肝癌发生、发展度转移的关系。方法采用PCR-SSCP方法对16例肝癌组织、12例癌旁组织和4例正常肝组织的nm23-H1基因的第1、2、4外显子进行突变检测。结果共有5例发生nm23-H1基因突变。在1例癌旁组织中检测到nm23-H1基因第2外显子纯合缺失；在1例肝癌组织中第1外显子、1例第2外显子，癌旁组织中2例第2外显子检测到基因突变。结论nm23-H1基因突变在肝癌组织中发生率低，nm23-H1基因突变可能与肝癌进展度转移有关。     

子宫内膜异位症中nm23-H1基因表达的意义

目的：探讨nm23-H1基因在子宫内膜异位症发生中的作用。方法：采用免疫组织化学SP法检测25例子宫内膜异位症和22例正常子宫内膜组织中nm23-H1的蛋白表达情况；采用RT-PCR检测研究33例子宫内膜异位症的异位子宫内膜组织和30例正常子宫内膜组织中nm23-H1基因的mRNA表达情况。 结果：25例子宫内膜异位症组中，nm23-H1的蛋白表达缺失为3例、弱阳性12例、强阳性10例，对照组中nm23-H1的蛋白表达缺失为0例、弱阳性3例、强阳性19例，两组差异显著（P<0.01）。33例子宫内膜异位症组中nm23-H1 mRNA表达缺失为4例、弱表达15例、强表达14例，30例对照组中，nm23-H1的mRNA表达缺失为1例、弱表达6例、强表达23例，子宫内膜异位症组明显低于正常对照组（P<0.05）。 结论：nm23-H1在子宫内膜异位症的发病过程中可能起重要的作用，进一步明确nm23-H1基因在子宫内膜异位症发生中的作用及其作用机制，对了解子宫内膜异位症的发病机制、临床诊断和治疗可能有一定意义。     

乳腺癌组织中uPA、uPAR及nm23-H1的表达

目的　观察乳腺癌组织中uPA、uPAR、nm2 3 H1的表达并探讨与腋窝淋巴结转移的关系。方法　用免疫组化EnVi sion两步法检测 6 9例乳腺癌组织中uPA、uPAR和nm2 3 H1表达的分布情况 ,观察其与肿瘤的分化程度以及与腋窝淋巴结转移的关系。结果　 (1)uPA阳性表达定位于癌细胞胞质 ;uPAR和nm2 3 H1阳性表达定位于癌细胞胞膜及胞质 ,多数癌旁乳腺上皮细胞呈nm2 3 H1阳性表达 ;高分化乳腺癌 (Ⅰ级 )uPA和uPAR表达阳性率 (30 0 %和 2 5 0 %)低于中低分化乳腺癌 (Ⅱ、Ⅲ级 ) (分别为 6 8 1%、72 7%和 70 0 %、74 1%) (P <0 0 5 ) ;nm2 3 H1表达阳性率在乳腺癌组织不同分化程度间差异无显著性 (P >0 0 5 ) ;(2 )腋窝淋巴结有转移者uPA和uPAR的表达阳性率 (73 2 %和 75 6 %)高于无淋巴结转移者 (35 7%和35 7%) (P <0 0 5 ) ;有腋窝淋巴结转移者nm2 3 H1的表达阳性率 (2 4 4 %)显著低于无淋巴结转移者 (5 0 0 %) (P <0 0 5 ) ;uPA、uPAR和nm2 3 H1的表达与淋巴结转移的个数均无关 ;(3)uPA阳性表达的癌组织其nm2 3 H1表达阳性率 (15 0 %)低于uPA阴性表达的癌组织 (6 2 1%) (P <0 0 5 )。结论　uPA和uPAR的高表达与乳腺癌腋窝淋巴结转移密切相关 ;uPA、uPAR和nm2 3 H1可以作为乳腺癌侵袭与淋巴结转移的     

肿瘤转移抑制基因nm23-H1 mRNA在乳腺癌中的转录表达

目的探讨nm23-H1基因mRNA在乳腺癌组织中的表达及其与临床的关系.方法采用半定量RT-PCR方法,检测30例乳腺癌组织nm23-H1的表达.结果①淋巴结阳性的原发灶组织nm23-H1 mRNA表达明显低于淋巴结阴性的原发灶组织,Ⅲ期乳腺癌组织nm23-H1 mRNA水平较Ⅰ、Ⅱ期的明显低.②多因素分析发现淋巴结转移与nm23-H1mRNA的表达有显著性相关.结论 nm23-H1基因mRNA表达强度与淋巴结转移呈负相关.在乳腺癌转移过程中,nm23-H1 mRNA起重要的作用.     

nm23-H1、c-erbB-2基因表达与结直肠癌转移的相关性

目的：探讨ｎｍ２３－Ｈ１、ｃ－ｅｒｂＢ－２基因表达与结直肠癌转移的关系。方法：采用免疫组化ＳＡＢＣ法，检测了８５例结直肠癌中ｎｍ２３－Ｈ１、ｃ－ｅｒｂＢ－２基因的蛋白产物。结果：ＤｕｋｅｓＤ期结直肠癌中ｎｍ２３－Ｈ１基因表达显著低于ＤｕｋｅｓＡ、Ｂ、Ｃ各期（Ｐ＜０．０１），ｃ－ｅｒｂＢ－２基因表达则显著高于ＤｕｋｅｓＡ、Ｂ、Ｃ各期（Ｐ＜０．０１），两基因在ＤｕｋｅｓＡ、Ｂ、Ｃ各期间的阳性表达差异无显著性（Ｐ＞０．０５）。结论：ｎｍ２３－Ｈ１与结直肠癌的肝转移呈负相关，ｃ－ｅｒｂＢ－２呈正相关，但两基因与淋巴结转移均无显著相关；两基因在蛋白水平上存在负相关性     

PCR－SSCP检测胃癌中nm23－H_1的基因突变

提取２０例胃癌及相应正常胃组织ＤＮＡ为样品，ＰＣＲ－ＳＳＣＰ方法快速筛查出４例胃癌组织中存在ｎｍ２３－Ｈ_１基因突变。此结果支持ｎｍ２３－Ｈ_１基因与肿瘤转移抑制密切相关。     

中国人肺鳞癌nm23H1基因遗传不稳定性的研究

目的 研究nm23H1基因5'端非转录区微卫星位点的微卫星不稳定(MSI)和杂合性缺失(LOH)对肺鳞癌(SLC)nm23H1蛋白表达的影响,以探讨nm23H1基因遗传不稳定性与肺鳞癌进展的关系,为肺鳞癌临床治疗和预后提供实验依据.方法 采用新鲜组织标本抽提DNA,应用荧光PCR-单链构象多态性(FPCR-SSCP)方法、免疫组织化学染色法,Leica-Qwin计算机图像分析等方法,进行nm23H1基因遗传不稳定性的研究.结果 在能提供信息(杂合子)的44例肺鳞癌患者中,MSI、LOH检出率和nm23H1蛋白阳性表达率分别为11.36%、25.00%和50.00%.在TNM Ⅰ、Ⅱ、Ⅲ期中,MSI的检出率分别为8.70%、10.00%和18.18%,而LOH分别为30.43%、20.00%和18.18%;MSI和LOH在淋巴结转移组的检出率为10.00%和20.00%,而无淋巴结转移组则为12.50%和29.17%.然而,以上各组在统计学上均无显著差异(P＞0.05).本研究中所有MSI(共5例)全部发生在分化良好(G1为2例和G2为3例)的存活患者(1年生存率)中,但MSI的检出率与肺鳞癌分化程度及患者生存状态并无统计学上的相关性(P＞0.05).nm23H1蛋白阳性表达率在TNM Ⅰ期(65.22%)、无淋巴结转移组(66.67%)显著高于TNM Ⅲ期(18.18%)、淋巴结转移组(30.00%)(P＜0.05).另外,统计学分析表明,MSI和LOH的检出率与nm23H1蛋白表达无关(P＞0.05).计算机图像定量分析显示,在各临床病理参数影响下,各组nm23H1蛋白的表达强度也没有差异(P＞0.05).结论 nm23H1蛋白可能对抑制肺鳞癌淋巴结转移有重要作用.nm23H1基因5'端非转录区微卫星的MSI和LOH对nm23H1蛋白的表达无影响,也未发现其与肺鳞癌发生、发展具有相关性.     

肝细胞癌nm23－H1蛋白表达的研究

应用免疫组化检测８８例肝细胞癌（ＨＣＣ）中ｎｍ２３－Ｈ１蛋白的表达。癌旁肝组织强阳性表达，５１例肝癌组织阳性表达（５８％）。阳性产物主要定位于肿瘤细胞胞浆。ｎｍ２３－Ｈ１蛋白表达与ＨＣＣ肿瘤体积，组织分型及Ｅｄｍｏｎｄｓｏｎ分级无关，而与肝内或肝外转移显著负相关。结果表明ｎｍ２３－Ｈ１在抑制ＨＣＣ肝内或肝外转移中起着重要作用，有可能成为评价ＨＣＣ病人预后的一项新指标。     

siRNA抑制nm23-H1基因表达对鼻咽癌6-10B细胞生物学行为的影响

目的：采用RNA干扰技术下调nm23-H1基因在鼻咽癌6-10B细胞中的表达,探讨nm23-H1表达下调对6-10B细胞生物学行为的影响。方法：采用脂质体法将nm23-H1基因siRNA(nm23-H1 siRNA)瞬间转染6-10B细胞,Western 印迹检测转染细胞中nm23-H1蛋白的表达水平,然后利用MTT法、流式细胞术和Transwell小室实验分别检测转染6-10B细胞的增殖、细胞周期和体外迁移侵袭等生物学行为的变化;测序检测6-10B细胞nm23-H1基因有无S120G 点突变。结果： nm23-H1 siRNA有效地下调nm23-H1基因的表达, nm23-H1 siRNA转染6-10B细胞的增殖能力增强,S期细胞增多,体外迁移和侵袭能力增强（P<0.05）。6-10B细胞nm23-H1 基因无S120G 点突变。结论：nm23-H1基因具有抑制人鼻咽癌细胞6-10B增殖和体外迁移侵袭的作用。     

乳腺癌病人nm23蛋白的检测与癌转移的关系研究

ｎｍ２３基因是一个与癌转移相关的肿瘤抑制基因，位于１７号染色体着丝点附近，其表达产物为１７ＫＤ的核苷二磷酸激酶。近年来报告，当癌发生转移时，病人的ｎｍ２３蛋白水平下降或检测不出ｎｍ２３蛋白。本文应用斑点ＥＬＩＳＡ法检测了３１例临床病理和Ｘ线诊断为乳腺癌的病人ｎｍ２３基因的表达水平。检查结果发现：３１例乳腺癌中，２０例阳性，１１例未测到ｎｍ２３基因的表达，经本法检查与临床病理和Ｘ线诊断比较，斑眯ＥＬ     

环孢素A对人滋养细胞nm23-H1表达的调控作用

目的:探讨环孢素A（CsA）对人滋养细胞系Bewo的23号非转移性基因（nometastatic gene23）H1型即nm23-H1表达的调控作用，为治疗滋养细胞疾病提供新的依据。方法:将Bewo细胞分为对照组（加溶媒）、环孢素组加入终浓度由10-2 μmol/L-10 μmol/L环孢素A。分别于48 h后用RT-PCR检测nm23-H1基因mRNA表达水平，于72 h后用In cell Western分析nm23-H1蛋白表达水平。结果:与对照组相比，nm23-H1的表达水平随环孢素A浓度10-2 μmol/L-1 μmol/L呈现降低趋势，其中1.0 μmol/L环孢素A nm23-H1 mRNA和蛋白水平均明显降低（P＜0.05， P＜0.01）；当浓度升高至10 μmol/L时，nm23-H1则呈升高趋势。结论:低浓度环孢素A可通过降调节nm23-H1的表达，继而改善滋养细胞的侵袭力。     

Semiquantitative immunoblot analysis of nm23-H1 and -H2 isoforms in adenocarcinomas of the lung: prognostic significance

Total amounts of nm23 protein and relative levels of H1 and H2 isoforms were studied in 27 fresh-frozen samples of pulmonary adenocarcinoma and adjacent non-neoplastic tissues that were obtained at surgery. Semiquantitative immunoblotting with a monoclonal antibody (Pan-242) against nm23 protein demonstrated both isoforms, recognized as 20.5 kDa for H1 and 18.5 kDa for H2, to be present in all cases. Both H1 and H2 levels in neoplastic tissues were higher than in the corresponding non-neoplastic samples. Expression of H2 was usually greater than of H1. The H2/H1 ratio varied from 1.9 to 14.1 (mean value 5.2) in non-neoplastic tissues and 1.0-5.9 (mean value 2.5) in neoplastic tissues, although this ratio did not correlate with any prognostic factor like tumor size, nodal status or distant metastasis (TNM tumor stage). H1 and H2 levels were significantly lower (mean values 4.3 and 2.4) in well-differentiated than in moderately and poorly differentiated adenocarcinomas (8.3 and 3.0) (P < 0.03 and P < 0.05, respectively). These data indicate that H1 and H2 isoform levels correlate with histological differentiation, but not the metastatic potential or stage of pulmonary adenocarcinoma.     

Squamous cell lung carcinomas: the role of nm23-H1 gene

 This analysis of 32 pairs of human squamous cell lung carcinomas and normal matched control DNA demonstrates that loss of heterozygosity (LOH) is infrequent at the nm23-H1 locus, affecting only 2 of the 18 informative cases. Both LOH cases were in the tumor stage IIIA. One tumor was of poor and the other of moderate histological grade. These and an additional 34 tumor samples were also analyzed immunohistochemically for the presence of nm23-H1 protein. Of the 66 cases tested for the presence of nm23-H1 protein 54 were negative. Eight samples exhibited up to 35% positive cells (with weak immunostaining intensity) and four between 35% and 70% (moderate immunostaining intensity); no sample showed more than 70% positive cells. Noncancerous lung parts contained no nm23-H1 protein. nm23-H1 expression was independent of TNM stage, grade, tumor size, and patient’s survival. Two samples with LOH were negative for nm23-H1 protein. We therefore conclude that neither loss of heterozygosity of the nm23-H1 gene nor the intensity of specific protein expression are related to squamous cell lung carcinoma development and progression. Received: 27 January 1997 / Accepted: 9 May 1997     

nm23-H1 protein immunoreactivity in intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix

Differences In the Immunohlstochemlcal expression of the 17 kDa protein encoded by the human nm23-H1 gene were studied In premallgnant lesions and Invasive squamous cell carcinoma (SCC) (N = 8) of the cervix using routine streptavldln-blotln Immunohistochemlstry and a polyclonal antibody to the nm23-H1 protein. The premalignant lesions were kollocytic atypla due to wart virus Infection (N = 5), low-grade cervical intraepithelial neoplasia (CIN) (N = 7) and high-grade CIN (N = 7). The carcinomas were either moderately (N = 3) or poorly differentiated (N = 5). The non-neoplastlc controls were normal squamous epithelium from cases with uterine prolapse (N = 7) and normal squamous epithelium not affected by the Infective or neoplastic areas of some of the cases with wart virus Infection (N = 2) and carcinoma (N = 2). Moderate to strong cytoplasmic and, occasionally, nuclear Immunostainlng for the nm23-H1 protein was seen in all cells above the basal layer of the normal squamous epithelium. However, most of the cervical SCC show a relative reduction in nm23-H1 immunoreactivity (7/8 cases; 88%). This difference In nm23-H1 expression was statistically significant (P = 0.0006; Chi-squared test with continuity correction). All of the cases with wart virus Infection (N = 5; 100%) displayed moderately strong nm23-H1 immunostaining throughout the squamous epithelium except for the basal layer where no staining was observed. The cases that had low-grade squamous dysplasia of the cervix (CIN Ml) (N = 7; 100%) also displayed moderate to strong nm23-H1 Immunoreactivity In the epithelium except for the basal layer (CIN I) or the lower two-thirds of the epithelium (CIN II). nm23-H1 Immunoreactivity was either absent or was significantly reduced in all of the high-grade CIN (CIN III) cases (At = 7; 100%) in which only the non-dysplastJc superficial third of the squamous epithelium displayed nm23-H1 immunolabeling. The difference in nm23-H1 expression between low-grade and high-grade CIN cases was statistically significant (P = 0.0013; Chi-squared test with continuity correction). Similarly, the difference between low-grade CIN and SCC cases in the expression of nm23-H1 was also significant (P = 0.0041; Chi-squared test with continuity correction). However, no statistically significant difference in nm23-H1 immunoreactivity was found between cases of high-grade CIN and SCC. In conclusion, nm23-H1 protein immunoreactivity is reduced in high-grade CIN and cervical SCC but not in low-grade CIN. These findings suggest that reduced expression of the protein may be Important early in the sequential development of cervical squamous neoplasia.     

Expression of the nm23 homologues nm23-H4, nm23-H6, and nm23-H7 in human gastric and colon cancer

Eight members of the nm23-gene family have been described. The involvement of nm23-H1 and nm23-H2 in tumour progression and metastasis, as well as in gene regulation and apoptosis, has been shown in numerous studies. Whether nm23-H4, -H6, and -H7 play a role in tumours is, however, largely unknown. This study describes data on the expression of these three nm23 homologues in human colon and gastric cancer by real-time RT-PCR and immunohistochemistry. Increased expression of these genes, most strikingly nm23-H4 and -H7, was observed in the majority of tumours analysed. No correlation with tumour stage according to the TNM classification was found. In contrast, by immunohistochemical analysis, nm23-H4 and -H6 overexpression correlated with the intestinal tumour type in gastric cancer tissues, whereas no increased immunoreactivity for the three nm23 proteins was noted in the diffuse type tumour specimens. These findings indicate that nm23-H6, and particularly nm23-H4 and -H7, may be involved in the development of colon and gastric carcinoma, the latter possibly in a type-specific manner. A contribution to tumour progression or metastasis could not, however, be proven. Elucidation of the specific mechanisms by which the nm23 homologues nm23-H4, -H6, and -H7 are involved in tumour development requires further studies.     

nm23-H1基因在人肺癌细胞L9981恶性表型逆转中的作用

nm23-H1基因是肿瘤转移抑制基因,转染野生型的nm23-H1基因可以逆转肺癌的恶性表型,我们拟建立转染野生型nm23-H1基因的人大细胞肺癌细胞株L9981-nm23-H1,并初步探讨nm23-H1基因在逆转L9981细胞株恶性表型中的作用。应用基因克隆技术构建质粒载体pLXSN-nm23-H1-EGFP,并建立L9981-nm23-H1细胞株。同时应用Westernblot检测L9981-nm23-H1细胞中nm23-H1蛋白表达。细胞培养及改良的Boyden小室法分别检测L9981-nm23-H1细胞株的体外生物学行为,动物实验检测L9981-nm23-H1细胞株在裸鼠体内的成瘤性及肺部转移能力。结果成功构建了逆转录病毒载体pLXSN-nm23-H1-EGFP;并建立了人大细胞肺癌细胞株L9981-nm23-H1;nm23-H1基因在细胞株L9981-nm23-H1中稳定、高效表达;L9981-nm23-H1细胞体外增殖能力,克隆形成力,体外侵袭力显著降(P<0.01);L9981-nm23-H1裸鼠体内的成瘤性及肺部转移能力显著低于L9981和L9981-pLXSN(P<0.01);nm23-H1基因的抑瘤率82.56%。本研究资料提示转染野生型nm23-H1基因可以逆转人大细胞肺癌细胞株L9981的恶性表型。