肾细胞癌MN／CA9蛋白的表达及其生物学意义

目的：研究ＭＮ／ＣＡ９蛋白的表达和肾细胞癌的关系，探讨ＭＮ／ＣＡ９蛋白在肾细胞癌诊断研究中的应用。方法：应用免疫组化和免疫印迹的方法，对正常肾组织、良性肾损害及肾细胞癌组织的ＭＮ／ＣＡ９蛋白的表达进行了分析。结果：正常人肾组织和良性肾损害均为ＭＮ／ＣＡ９阴性，而在３０例肾癌中却有２８例为ＭＮ／ＣＡ９阳性，尤其是２６例富糖原型ＲＣＣ均呈现ＭＮ／ＣＡ９强阳性。结论：ＭＮ／ＣＡ９蛋白可作为肾细胞癌尤其是     

肾细胞癌MN/CA9蛋白的表达及其生物学意义

目的 :研究MN/CA9蛋白的表达和肾细胞癌的关系 ,探讨MN/CA9蛋白在肾细胞癌诊断研究中的应用。方法 :应用免疫组化和免疫印迹的方法 ,对正常肾组织、良性肾损害和肾细胞癌组织的MN/CA9蛋白的表达进行了分析。结果 :正常人肾组织和良性肾损害均为MN/CA9阴性 ,而在 30例肾癌中却有 2 8例为MN/CA9阳性 ,尤其是 2 6例富糖原型RCC均呈现MN/CA9强阳性。结论 :MN/CA9蛋白可作为肾细胞癌尤其是富糖原型肾细胞癌的一种标志物     

子宫颈鳞状细胞癌细胞外间质改变的免疫荧光抗体法研究

目的：通过对细胞外间质（ＥＣＭ）成分进行检测,探讨间质重构与子宫颈鳞状细胞癌侵袭性生长及播散的关系。方法：应用双重免疫荧光染色技术和激光共聚焦显微镜观察,对正常人及不同分化程度的子宫颈鳞状细胞癌患者的子宫颈石蜡包埋组织切片纤维结合素（ＦＮ）、层粘连蛋白（ＬＮ）及Ⅰ、Ⅲ、Ⅳ型胶原蛋白的表达进行检测。结果：正常子宫颈组织中,ＥＣＭ内可见结构致密的Ⅰ、Ⅲ型胶原和细条索状的ＦＮ蛋白表达,Ⅳ型胶原和ＬＮ蛋白     

增殖细胞核抗原在肝细胞癌、肝硬化和慢性肝炎中的表达

为了观察增殖细胞核抗原在肝细胞癌（ＨＣＣ）、肝硬化（ＬＣ）和慢性活动性肝炎（ＣＡＨ）中的表达，应用微波内免疫组化技术，对５５例ＨＣＣ切除标本和５７例肝穿刺活检标本进行了增殖细胞核抗原（ＰＣＮＡ）单克隆抗体的标记。结果发现，ＨＣＣ切除标本中ＰＣＮＡ阳性率７０．９％（３９／５５），ＰＣＮＡ标记指数与ＨＣＣ分级呈正相关，与癌旁组织相比差异显著，在肝穿刺活检标本中，ＰＣＮＡ阳性率：ＨＣＣ为６９．３％（９／１３）；ＬＣ为２３．８％（５／２１），ＣＡＨ为８．７％（２／２３）。本研究中共检出肝细胞不典型增生２７例，标记指数近似于ＨＣＣⅠ级，其中小细胞性不典型增生和大细胞性不典型增生的标记指数无差异。     

ＤＮＡ结合抑制因子１在非小细胞肺癌组织中的表达

目的　探讨ＤＮＡ结合抑制因子１（Ｉｄ１）在非小细胞肺癌（ＮＳＣＬＣ）发生、发展中的作用及与ＮＳＣＬＣ临床病理特征之间的联系。方法　选取３０例ＮＳＣＬＣ癌组织、癌旁组织和正常肺组织以及１０例肺良性病变组织和正常肺组织,采用ＲＴ-ＰＣＲ和免疫组化ＳＰ法测定其Ｉｄ１ｍＲＮＡ和Ｉｄ１蛋白的表达情况。结果　（１）Ｉｄ１ｍＲＮＡ、Ｉｄ１蛋白在ＮＳＣＬＣ癌组织中的阳性表达率为９６.６７％（２９／３０）、９３.３３％（２８／３０）,在癌旁组织中的阳性表达率为１３.３３％（４／３０）、１０％（３／３０）,在ＮＳＣＬＣ正常肺组织、肺良性病变组织及其正常肺组织中均未见表达。（２）ＮＳＣＬＣ癌组织Ｉｄ１ｍＲＮＡ和Ｉｄ１蛋白的表达强度随癌细胞分化程度的降低而增强。（３）ＮＳＣＬＣ患者Ｉｄ１ｍＲＮＡ和Ｉｄ１蛋白的表达与病理类型、瘤体大小、淋巴结转移及预后无明显相关性。结论　Ｉｄ１因子可能是鉴别ＮＳＣＬＣ与肺良性病变的重要分子标记物之一,且其表达水平与ＮＳＣＬＣ癌细胞分化程度呈负相关。     

ＨＤＡＣ１和ＤＮＭＴ１蛋白在乳腺癌中的表达及临床意义

目的　探讨乳腺癌组织中组蛋白脱乙酰基酶１（ＨＤＡＣ１）和ＤＮＡ甲基转移酶１（ＤＮＭＴ１）的表达及临床意义。方法　应用组织芯片和免疫组织化学ＳＰ法检测１０５例乳腺癌组织和３７例癌旁组织中ＨＤＡＣ１和ＤＮＭＴ１蛋白的表达情况并分析其与ＥＲ、ＰＲ之间的关系。结果　（１）ＨＤＡＣ１蛋白在１０５例乳腺癌中的阳性表达率为６４.７6％,在癌旁组织中的阳性表达率为３５.１４％,两组差异有统计学意义（Ｐ＜０.０１）;ＤＮＭＴ１蛋白在乳腺癌中的阳性表达率为５５.２４％,在癌旁组织中的阳性表达率为２４.３２％,两组差异有统计学意义（Ｐ＜０.０１）;（２）ＨＤＡＣ１和ＤＮＭＴ１蛋白的表达与患者年龄、瘤体大小、组织学类型、组织学分级、腋窝淋巴结转移、临床分期均无相关性（Ｐ＞０.０５）;（３）乳腺癌组织中ＨＤＡＣ１和ＤＮＭＴ１蛋白在ＥＲ或ＰＲ阴性乳腺癌组织中的表达分别高于其在ＥＲ或ＰＲ阳性乳腺癌组织中的表达（Ｐ＜０.０１）。ＨＤＡＣ１与ＤＮＭＴ１蛋白表达具有一定的相关性。结论　乳腺癌组织中ＨＤＡＣ１和ＤＮＭＴ１蛋白过表达与肿瘤发生发展密切相关。     

脑肿瘤中Pten／MMAC1／Tep1蛋白的表达

目的：探讨Ｐｔｅｎ／ＭＭＡＣ１／Ｔｅｐ１蛋白在不同分化程度的脑肿瘤组织中的表达。方法：应用免疫组化技术检测３０例脑胶质细胞瘤和１５例脑膜瘤Ｐｔｅｎ／ＭＭＡＣ１／Ｔｅｐ１蛋白表达。结果：脑胶质细胞瘤组织中Ｐｔｅｎ／ＭＭＡＣ１／Ｔｅｐ１蛋白表达阳性率为５３．３３％,脑膜瘤组织中Ｐｔｅｎ／ＭＭＡＣ１／ｅｐ１蛋白表达阳性率为９３．３３％,两者差异有显著性（Ｐ〈０．０１）。结论Ｐｔｅｎ／ＭＭＡＣ１／Ｔｅｐ１     

ＴＲＡＩＬ联合蛋白酶抑制剂ＭＧ１３２诱导肺癌Ａ５４９细胞凋亡的实验研究

目的　观察ＴＲＡＩＬ对肺癌Ａ５４９细胞的体外生长抑制及诱导凋亡作用,并探讨ＴＲＡＩＬ与蛋白酶抑制剂ＭＧ１３２联合用药诱导细胞凋亡的作用及其机制。方法　采用ＭＴＴ法检测不同浓度ＴＲＡＩＬ对肺癌Ａ５４９细胞增殖的影响;应用流式细胞仪技术检测ＴＲＡＩＬ、蛋白酶抑制剂ＭＧ１３２及其两药联合干预２４ｈ对肺癌Ａ５４９细胞凋亡率的影响;采用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测药物干预前后细胞凋亡相关蛋白Ｃａｓｐａｓｅ-８、Ｃａｓｐａｓｅ-９以及核转录因子ＮＦ-κＢ的表达情况。结果　ＭＴＴ结果显示不同浓度ＴＲＡＩＬ对肺癌Ａ５４９细胞的体外抑制作用与剂量效应正相关,２４ｈＩＣ５０为９６.３２２μｇ／ｍｌ,４８ｈＩＣ５０为４３.５９μｇ／ｍｌ;流式细胞术显示ＴＲＡＩＬ与ＭＧ１３２联合用药后肺癌Ａ５４９细胞凋亡率明显增高（平均６７.６５％）,与ＴＲＡＩＬ（平均２３.５５％）、ＭＧ１３２（平均２５.９１％）单药组比较,有显著性差异（Ｐ＝０.０１６）;Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ结果显示,与ＴＲＡＩＬ单药组比较,两药联合后细胞凋亡相关蛋白Ｃａｓｐａｓｅ-８、Ｃａｓｐａｓｅ-９表达明显增强（Ｐ＝０.００６）,抑制细胞凋亡因子ＮＦ-κＢ表达则明显减弱（Ｐ＝０.０４）。结论　ＴＲＡＩＬ可抑制Ａ５４９细胞的增殖,呈浓度依赖关系;ＴＲＡＩＬ与蛋白酶抑制剂ＭＧ１３２联合应用可以明显提高细胞凋亡率,可能与蛋白酶抑制剂ＭＧ１３２降低ＮＦ-κＢ的活性,增强细胞对ＴＲＡＩＬ诱导凋亡的敏感性,促进凋亡相关蛋白Ｃａｓｐａｓｅ-８及Ｃａｓｐａｓｅ-９的表达有关。     

增殖细胞核怕在肝细胞癌,肝硬化和慢性肝炎中的表达

为了观察增殖细胞核抗原在肝细胞癌（ＨＣＣ）、肝硬化（ＬＣ）和慢性活动性肝炎（ＣＡＨ）Ｋ 的表达，应用微波内免疫组化技术，对５５例ＨＣＣ切除标本和５７例肝穿刺活检标本进行了增殖细胞核抗原（ＰＣＮＡ）单克隆抗体的标记。结果发现，ＨＣＣ切除标本中ＰＣＮＡ阳性率７０．９％（３９／５５），ＰＣＮＡ标记指数与ＨＣＣ分级呈正相关，与癌旁组织相比差异显著，在肝穿刺活检标本中，ＰＣＮＡ阳性率：ＨＣＣ为６９．３％（９     

肿瘤坏死因子-α促进乳腺癌细胞ＭＣＦ-７迁徙的机制探讨

目的：探讨肿瘤坏死因子（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ-α,ＴＮＦ-α）诱导黏附分子ＣＤ４４表达的机制。方法：以０、２、２０、２００ｎｇ／ｍｌ浓度处理ＭＣＦ-７细胞,了解对ＭＣＦ-７细胞增殖以及迁徙的影响。Ｗｅｓｔｅｒｎ-ｂｌｏｔ方法检测ＭＡＰＫ（ＪＮＫ、Ｐ３８、ＥＲＫ）信号通路中蛋白磷酸化水平的变化;用ＭＡＰＫ抑制剂预处理后,检测ＣＤ４４表达水平。结果：ＴＮＦ-α可以改变ＣＤ４４亚型表达,并促进ＭＣＦ-７细胞的迁徙能力;ＴＮＦ-α可以激活ＪＮＫ信号转导通路,对Ｐ３８和ＥＲＫ信号通路无明显激活效应;用特异性的ＪＮＫ信号通路抑制剂可以阻断ＴＮＦ-α诱导的ＣＤ４４蛋白表达水平。结论：ＴＮＦ-α通过ＪＮＫ信号通路调节ＣＤ４４并促进ＭＣＦ-７细胞的迁徙能力。     

Expression of MN/CA9 protein in Papanicolaou smears containing atypical glandular cells of undetermined significance is a diagnostic biomarker of cervical dysplasia and neoplasia

BACKGROUND: Despite the enormous impact that Papanicolaou (Pap) smear screening has had on the incidence of cervical carcinoma in developed countries, there is still an unacceptably high frequency of occurrence of this cancer. In part, this is due to human error associated with cytologic diagnoses of Pap smears. Also, the use of new sampling devices, such as the cytobrush, has increased the complexity of diagnosing benign and neoplastic cervical cytology. This is particularly apparent in the diagnosis of atypical glandular cells of undetermined significance (AGUS). Approximately 40% of AGUS diagnoses have a corresponding significant lesion at biopsy follow-up, and 60% do not. There is clearly a need for an adjunct to cytologic diagnosis that can readily identify AGUS smears that are diagnostic of significant lesions. The authors have identified the MN/CA9 antigen as a strong candidate for an adjunct biomarker. METHODS: A total of 245 Pap smears of all AGUS diagnostic categories with histologic confirmation were studied. The median age of the patients was 39 years. The Bethesda system classification (AGUS-favor reactive, AGUS-not otherwise specified, and AGUS-favor neoplastic) was used. All of the Pap smears were decolorized and immunostained with monoclonal antibody to MN/CA9 antigen by the immunoperoxidase technique. The results of MN/CA9 immunoreactivity were correlated with the histologic data in a semiblinded fashion. RESULTS: The follow-up biopsies showed that a high percentage (70%) of patients had low and high grade cervical intraepithelial neoplasia lesions, respectively (CIN I and CIN II or III). Clinically significant lesions-adenocarcinoma in situ/carcinoma (AIS/CA) and CIN II or III-were found in 50% of the cases. Among these, 11% were AIS/CA. In the three subcategories of AGUS diagnosis, the AGUS-not otherwise specified showed the broadest range of lesions in the follow-up biopsies. Three patterns of MN/CA9 immunoreactivity were observed in the Pap smears: 1) atypical cells, 2) normal endocervical cells only, and 3) all cells negative. All Pap smears that were MN/CA9 positive were histologically confirmed to be clinically significant lesions or CIN I; in addition, there were a very small number (n = 12) of cases of atypia. None of the benign lesions showed MN/CA9 expression in the corresponding Pap smears. Furthermore, the pattern of atypical cell immunostaining identified all cases with significant lesions (AIS/CA and CIN II or III) in the cervices. Conversely, the majority of CIN I cases (82%) and all cases of atypia showed positive immunostaining restricted to normal endocervical cells only. CONCLUSIONS: There is a clear association between MN/CA9 immunostaining of atypical cells and the presence of significant lesions in the cervix. Similarly, there is a clear association between lack of expression of MN/CA9 and the absence of cervical lesions. However, the screen does not allow discrimination between CIN I and atypia. The authors also found that, based on the combined patterns of morphology and immunostaining, they are able to discriminate between AIS and CIN II or III in AGUS Pap smear diagnoses. Thus, expression of the MN/CA9 antigen is indeed a discriminator of significant lesions in AGUS Pap smear diagnoses.     

The expression of G250/mn/CA9 antigen by flow cytometry: its possible implication for detection of micrometastatic renal cancer cells.

Monoclonal antibody (mAb) G250 is a well characterized and specific mAb to renal cell carcinoma (RCC). The gene G250 was recently cloned and was proved to be homologous to MN/CA9. The G250/MN/CA9 antigen was recently explored as a potential marker for RCC. Flow cytometry (FCM) allows quantitative analysis of cells. The present study describes a flow cytometric method to detect this antigen in human cell lines and in malignant and normal renal tissues. Twelve human carcinoma cell lines (HeLa, Colo205, HT29, BxPC3, OVCAR3, SKOV3, ACHN, A704, CAKI-2, SKRC-59, SKRC-10, and SKRC-52), 10 specimens of normal peripheral blood mononuclear cells, and 38 malignant and 36 adjacent normal renal tissues were studied. The malignant and normal renal tissues were disaggregated mechanically into a single-cell suspension, stained by mAb G250, and analyzed by FCM. All 22 of the clear cell carcinomas, 6 of 8 mixed cell carcinomas, and 3 of 6 granular cell carcinomas were positive for G250/MN/CA9 antigen. SKRC-52 and SKRC-10 were strongly positive for G250/ MN/CA9. The G250/MN/CA9 antigen could also be detected in HeLa, SKOV3, HT29, and A704 cells. One chromophobic, one chromophilic cell carcinoma, the normal renal tissues, and normal peripheral blood mononuclear cells were considered as negative. Our results further confirmed that the G250/MN/CA9 antigen was an ideal marker for RCC, especially for clear cell carcinomas, and that this antigen was present in several types of malignant cells. FCM may serve as a fast tool of immunocytochemical detection of renal cancer cells. Flow cytometric detection of renal cancer cells by using mAb G250 should be further explored.     

Clinical significance for detection of circulating cancer cells in renal cell carcinoma

Renal cell carcinoma has a very poor prognosis since various therapeutic modalities other than radical operation are not effective. Early detection and treatment are of considerable importance to cure the patients; however, early diagnosis of renal cell carcinoma is not easy because no specific tumor marker, like PSA for prostate cancer, is available. MN/CA9 is considered to be one of the carbonic anhydrase isoenzymes, and is expressed in approximately 90% of renal cell carcinomas. Expression of MN/CA9 in normal tissues is very limited. Using optimal RT-PCR with specific primers, MN/CA9 positive cells were clearly detected in the blood. The sensitivity and specificity were found to be approximately 40% and 90% respectively. The detection of circulating renal cell carcinoma cells using RT-PCR for MN/CA9 mRNA is useful for diagnosis of the presence of renal cell carcinomas. This RT-PCR assay may also be able to provide information with which to predict the prognosis of the patients.     

Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma

MN/CA9 is a cancer-related gene, frequently activated in human renal cell carcinomas (RCCs). To reveal the activation mechanism, we investigated the relationship between methylation status of the MN/CA9 promoter region and gene expression using 13 human RCCs, and examined the effect of in vitro CpG methylation on the MN/CA9 promoter activity using a human RCC cell line (SK-RC-44), expressing MN/CA9. MN/CA9 expression was evaluated by RT-PCR and observed in 10 of 13 RCCs (77%). A total of 9 out of 10 MN/CA9 -positive RCCs (90%) contained clear cell components. Methylation status of 6 CpGs in the MN/CA9 promoter region was decided by using the bisulfite genomic sequencing protocol. Out of 13 RCCs 9 (69%) showed partial hypomethylation of the CpG at -74 bp, while the other 4 RCCs and 3 normal kidney tissue samples showed complete methylation. Hypomethylation of the CpG at -74 bp was strongly correlated with MN/CA9 expression. Luciferase assay revealed that the MN/CA9 promoter activity was strongly suppressed by methylation of the CpG at -74 bp. These findings suggest that hypomethylation of the CpG at -74 bp in the MN/CA9 promoter region might play an important role in this gene activation of human RCC.     

肿瘤异常糖链糖蛋白在肾癌中的诊断价值

目的:研究外周血肿瘤异常糖链糖蛋白（tumor abnormal protein,TAP）的表达对早期肾癌的诊断价值。方法:对45例肾癌患者（肾癌组）、32例肾脏良性病变患者（肾良性病变组）和42例体检健康人群（正常组）的外周血TAP值进行分析;绘制受试者工作特征（receiver operating characteristics,ROC）曲线评估TAP对肾癌的诊断效能;分析肾癌组中TAP阳性表达与患者临床病理特征的关系。结果:肾癌组中外周血TAP水平明显高于肾良性病变组和正常组。通过评估TAP对肾癌的诊断效能,发现TAP诊断肾癌的ROC曲线下面积为0.92(95%CI:0.82~0.95),其诊断肾癌的灵敏度和特异度分别为73%和91%。结论:肾癌患者外周血中TAP呈高表达,可作为肾癌早期诊断的肿瘤标志物。