MN/CAⅨ基因在肾癌组织中表达的实验研究

MN/CAⅨ基因在肾癌中有特异性的表达 ,是一种肿瘤相关抗原 (TAA) ,在肿瘤诊断以及生物治疗方面具有潜在的应用前景[1] 。我们采用RT PCR方法检测肾癌组织和正常肾组织中MN/CAⅨmRNA表达 ,探讨MN/CAⅨ基因表达作为肾癌肿瘤标记物的可能性 ,现报告如下。对象与方法　 2 0 0 2～ 2 0 0 3年我科肾癌根治手术标本 2 5例。男 16例 ,女 9例。年龄 2 3～ 6 5岁 ,平均 5 1岁。其中透明细胞癌 17例 ,颗粒细胞癌 8例。正常肾组织 2 1例 ,分别取自肾切除和T1肾癌原发灶远处正常肾组织。男 14例 ,女 7例 ,年龄 2 3～ 6 5岁 ,平均 4 9岁。GeneA…     

CAIX/MN/G250 mRNA在肾癌根治术前后表达的检测

目的探讨肾癌相关蛋白CAIX／MN／G250mRNA在肾细胞癌（renal cell carcinoma,RCC）手术前后的表达水平及临床意义。方法应用RT—PCR技术检测57例RCC患者癌组织及术前外周血中CAIX／MN／G250mRNA的表达情况,并选择15例癌旁肾组织、15例正常肾组织及40例健康人外周血作对照,对术前外周血CAIX／MN／G250mRNA阳性RCC患者术后随访4周,并对其外周血CAIX／MN／G250mRNA水平进行半定量检测。结果57例RCC患者,其CAIX／MN／G250mRNA阳性率在外周血中为59．6％（34／57）,癌组织中为78．9％（45／57）,显著高于对照组（P〈0．01）。在肾透明细胞癌患者的外周血及癌组织中,CAIX／MN／G250mRNA的阳性率分别为76．2％（32／42）和95．2％（40／42）,均显著高于相应对照组（P〈0．01）。RCC患者外周血及癌组织中CAIX／MN／G250mRNA阳性率均随临床分期的增加而增加（P〈0．01）。34例外周血阳性患者CAIX／MN／G250mRNA水平术前2h、术后7、14、21和28d分别为0．54±0．13、0．56±0．15、0．34±0．13、0．29±0．18和0．22±0．11。RCC患者外周血CAIX／MN／G250mRNA水平在肾癌根治术后随时间的推移呈下降趋势（P均〈0．05）。结论使用RT—PCR技术检测CAIX／MN／G250mRNA对肾癌尤其是透明细胞癌有较好的特异性和敏感性。外周血CAIX／MN／G250mRNA检测可作为肾癌微转移的检测指标用于RCC的诊断、疗效评价及预后判断。     

肾肿瘤和良性肾病组织βhCGmRNA表达的研究

目的：探讨βhCGmRNA在肾细胞癌（RCC）和良性肾脏疾病组织中的表达情况。方法：采用RT-PCR并结合限制性内切酶法检测44例RCC和24例良性肾脏疾病组织βhCG基因及其亚型的表达。结果：RCC的βhCGmRNA阳性表达率为52%，晚期和低分化RCC的阳性表达率较高，但无统计学意义。良性肾脏疾病组织的βhCGmRNA阳性表达率为54%，包括3例多囊肾（3/6），7例肾萎缩（7/13），2例嗜酸性细胞瘤，1例肾盂肾炎，β7基因是RCC和良性肾脏疾病组织中最常见的βhCGmRNA亚型。结论：βhCG基因可能在恶性肾肿瘤和良性肾脏疾病中发挥作用，其病理生理和临床意义值得进一步研究。     

肾癌患者外周血中碳酸酐酶9 mRNA表达的临床意义

目的：探讨肾细胞癌患者外周血中碳酸酐酶9（MN）mRNA表达的临床意义。方法：应用RT－PCR技术，对30例肾细胞癌患者及30例非肾细胞癌患者外周血中肾癌细胞标志物MN mRNA进行检测。结果：非肾癌组外周血中均不表达MN mRNA。肾癌患者外周血MN mRNA阳性表达13例（43％），其中肾癌转移8例，局限性肾癌5例；肾透明细胞癌12例，混合型癌1例。外周血MN mRNA表达与肾癌的临床分期无相关性（P＞0．05）；而肾透明细胞癌阳性率（12／22）明显高于其他组织类型（P＜0．05）。结论：RT－PCR技术可较敏感地检测到血液中肾癌细胞，有助于早期诊断肾癌微小转移。MN可被视为透明细胞癌特异的相关蛋白。     

肿瘤-睾丸抗原基因在肾透明细胞癌中的表达

目的 研究6种肿瘤.睾丸抗原(CT)基因在肾透明细胞癌中的表达及其临床意义.方法 采用逆转录-聚合酶链反应(RT-PCR)技术检测42例肾透明细胞癌患者癌组织(新鲜标本,T1期16例,12期12例,T3期10例,T4期4例;G1 10例,G2 18例,G3 14例)及其中14例患者癌旁组织的cTAGE-1、cTAGE-2、MAGE-A1、MAGE-A3、MAGE-A12及NY-ESO-1基因mRNA的表达.结果 42例肾透明细胞癌组织中100%(42/42)至少表达6种CT基因中一种,14例癌旁组织表达均阴性.肾透明细胞癌组织中MAGE-A12表达最高,其次为MAGE-A3,MAGE-A1,cTAGE-1,cTAGE-2及NY-ESO-1,分别为71%(30/42),69%(29/42),67%(28/42),64%(27/42),60%(25/42)及48%(20/42).肿瘤不同分期、不同分级之间6种CT基因表达的差异均无统计学意义(Pearson x2检验法,P＞0.05).结论 CT基因在肾透明细胞癌中有较高表达,可望成为肾透明细胞癌特异性免疫治疗的靶基因.     

Fibulin-1基因表达与肾细胞癌的关系研究

目的 检测肾细胞癌组织中Fibulin-1基因表达的改变,探讨其相关的临床意义.方法 选取2014年1月至2016年5月本院收治的肾细胞癌患者手术切除种癌组织83例,以及相应的癌旁正常非瘤肾脏组织.SYBR Green 荧光定量-PCR检测癌组织和非癌组织Fibulin-1基因表达量.结果 肾细胞癌肿瘤组织中Fibulin-1基因表达量为(0.176±0.028),显著低于对照非肿瘤肾组织的(0.384±0.052,P<0.01).肾细胞癌肿瘤组织中Fibulin-1基因表达量在不同性别、不同年龄和组织病理类型间差异无统计学意义(P>0.05).有淋巴结转移肿瘤组织Fibulin-1基因表达量为(0.152±0.022),显著低于无淋巴结转移肿瘤组织(0.204±0.035,P<0.05).中、低分化肿瘤组织Fibulin-1基因表达量为(0.160±0.021),显著低于高分化肿瘤组织(0.207±0.036,P<0.05).Ⅲ、Ⅳ期肿瘤组织中的Fibulin-1基因表达量为(0.148±0.019),显著低于Ⅰ、Ⅱ期肿瘤组织(0.199±0.034,P<0.05).结论 肾细胞癌患者fibulin-1基因表达下调,fibulin-1基因表达下调与肾细胞癌有无淋巴结转移、分化程度以及临床分期密切相关.     

Caveolin-1和HDAC1蛋白在肾细胞癌中的表达及意义

目的探讨Caveolin-1和HDAC1蛋白在肾细胞癌(renal cell carcinoma,RCC)及正常肾组织中的表达及临床意义,并探讨其与肿瘤生物学行为之间的关系。方法采用免疫组织化学方法检测51例RCC、15例正常肾组织及26例癌旁组织中Caveolin-1和HDAC1蛋白的表达,并分析其与患者临床病理特征的关系。51例RCC中组织学分级:高分化13例,中分化24例,低分化14例;临床分期:Ⅰ期16例,Ⅱ期20例,Ⅲ期10例,Ⅳ期5例。结果 Caveolin-1在正常肾组织、癌旁组织和RCC中的阳性表达率分别为33%(5/15)、42%(11/26)、65%(33/51),逐渐升高(P<0.05)。在51例RCC中,Caveolin-1的阳性表达率临床分期Ⅲ期和Ⅳ期高于Ⅰ期和Ⅱ期,低分化高于中、高分化,有淋巴结转移组高于无淋巴结转移组。Caveolin-1表达与病理学分期、分级和淋巴结转移均相关(P值分别为0.001、0.038和0.006,P均<0.05),随着肿瘤分期、分级的升高,Caveolin-1表达增强。RCC中Caveolin-1表达与年龄、性别、肿瘤所在部位均无关(P>0.05)。HDAC1在正常肾组织、癌旁组织和RCC中的阳性表达率分别为13%(2/15)、19%(5/26)、75%(38/51),呈现显著升高的趋势(P<0.05)。在51例RCC中,HDAC1的阳性表达率临床分期Ⅲ期和Ⅳ期高于Ⅰ期和Ⅱ期,低分化高于中、高分化,有淋巴结转移组高于无淋巴结转移组,肾癌组织中HDAC1表达率随肾癌TNM分期的增高和病理分级的增加而升高。RCC中HDAC1表达与年龄、性别、肿瘤所在部位均无关(P>0.05)。RCC中Caveolin-1的表达与HDAC1表达呈正相关。结论 Caveolin-1和HDAC1在RCC中高表达,可能是RCC发生、发展及浸润转移的重要因素之一,联合检测对判断RCC预后有指导意义。     

间隙连接蛋白43在肾细胞癌中的表达及意义

目的:探讨间隙连接蛋白Cx43在肾细胞癌(RCC)中的表达及其与RCC生物学行为之间的关系.方法:应用S-P免疫组织化学法检测Cx43蛋白在41例RCC,12例癌旁肾组织及10例正常肾组织中的表达情况.结果:Cx43阳性染色主要定位在细胞膜和细胞质上.Cx43蛋白在RCC中的阳性表达率明显低于在癌旁肾及正常肾组织的水平(P＜ 0.01);在透明细胞癌、颗粒细胞癌、梭形细胞癌中,其阳性表达率比较差异无统计学意义(P＞ 0.05);随着临床分期的增高,其阳性率明显下降(P＜ 0.05),并与RCC转移呈负相关(Spearman 等级相关系数r=- 0.483, P＜ 0.01);与肿瘤大小无明显关系(P＞ 0.05).结论:Cx43对RCC发生和转移有明显抑制作用,其表达减弱或消失可能与RCC的发生和发展密切相关.     

肿瘤相关基因MN在肾肿瘤中的表达及临床意义

目的探讨肿瘤相关基因MN在鲁肿瘤中的表达及临床意义。方法应用免疫组织化学技术对61例肾肿瘤组织及9例正常肾组织中MN的表达进行研究。结果除1例嫌色细胞癌外,所有恶性肾肿瘤中MN均呈阳性表达,相反在所有良性肾肿瘤及正常肾组织中表达阴性。结论MN的表达与肾细胞癌密切相关,可作为肾细胞癌诊断和治疗过程中的一种具有重要价值的生物学标记物。     

Connexin32、VEGF在肾细胞癌中的表达及临床意义

目的 研究间隙连接蛋白32(connexin32,Cx32)及血管内皮生长因子(vascular endothelial growth factor,VEGF)在肾细胞癌(renal cell cancer,RCC)组织中的表达及其与肾细胞癌的相关性,探讨其在RCC发生和发展中的作用.方法 运用免疫组织化学方法 (SP法)检测44例RCC和10例正常肾脏组织中Cx32及VEGF的蛋白表达,观察其表达的定位和表达量的差异并比较它们之间的差异性及相关性.方法 (1)Cx32蛋白主要在肾癌细胞胞质中表达,而在正常肾组织中,主要表达在细胞膜上.它在RCC组中的阳性表达率为27.3%,对照组中阳性表达率为90%,差异具有显著性(p<0.05).(2)VEGF蛋白主要表达在肿瘤细胞膜,在胞质中也有表达.它在RCC组中的阳性表达率为79.5%,对照组中阳性表达率为20%,差异具有极显著性(p<0.01).(3)在不同临床分期及病理分级的肾细胞癌组织中Cx32与VEGF的表达均呈负相关(p<0.01).结论 肾细胞癌组织中Cx32表达水平的明显下调以及VEGF表达水平的显著上调,可能与肾细胞癌的发生、发展、转移有关,检测二者的表达有助于综合判断RCC的生物学行为.     

The role of MN/CA IX antigen in carcinogenesis and metastasis of renal cell carcinoma]

MN/CA IX is a carbonic anhydrase (CA) isoenzyme expressed in normal alimentary tract in a tissue-specific manner. This antigen is activated in the majority of renal cell carcinomas (RCC) but not in normal kidney tissues. Although the exact role of CA activity in carcinogenesis and metastasis has not been established, MN/CA9 has been suggested to be implicated in acidification of extracellular milieu surrounding the cancer cells and thus create a microenvironment conductive to tumor growth and spread. Mutations in the von Hippel-Lindau (VHL) gene cause the familial syndrome and are also found in the majority of sporadic RCC. Wild-type VHL was recently described to down-regulate MN/CA9 in RCC cell lines, and the molecular mechanism of MN/CA9 and VHL in renal carcinogenesis is of interest. To investigate the mechanism of MN/CA9 activation in RCC, we examined the methylation status of this gene in RCC cell lines and human tissue samples and found that hypomethylation in the promoter region may play an important role in the expression of MN/CA9. RT-PCR analysis of blood samples from RCC patients revealed the presence of circulating MN-positive cells in the blood. This antigen may be a potential therapeutic target as well as diagnostic marker for RCC. Therefore, we are currently investigating whether or not MN/CA IX peptide could be an appropriate molecule for use antigen specific immunotherapy on RCC patients.     

MN/CA IX antigen as a potential target for renal cell carcinoma

MN/CA IX is considered as a carbonic anhydrase isoenzyme expressed in the normal alimentary tract in a tissue-specific manner. This antigen is activated in the majority of renal cell carcinomas (RCC) but not in the normal kidney tissues. Our previous study revealed that increase of malignant potential is related to down-regulation of MN/CA9. To investigate the mechanism of MN activation in RCC, we examined the methylation status of this gene (MN/CA9) in RCC cell lines (SKRC-1, 6, 10, 12, 14, 44, 59). Moreover, we analyzed the circulating blood of patients for the presence of RCC cells by RT-PCR, to determine whether detection of circulating RCC cells could be useful as a biomarker. CpG methylation was investigated at 7 CpG sites in the MN/CA9 5' region. Clear mRNA signals were observed in 5 cell lines (SKRC-1, 6, 10, 44, 59), e.g., MN/CA9 positive. These 5 MN-positive cell lines showed hypomethylation in the 5' region. In contrast, all CpG sites were methylated in the remaining 2 lines and 3 normal kidney tissue samples. These results suggest that hypomethylation in the 5' region may play an important role in the expression of MN/CA9 in RCC. RT-PCR analysis of blood samples from RCC patients revealed the presence of circulating MN-positive cancer cells in the blood. Although a significant correlation with tumor stage and grade was not observed, the analysis of blood samples from patients with metastases resulted in a high detection rate of 82%. These findings suggest the usefulness of MN/CA IX as a potential diagnostic marker for detection of RCC.     

The use of MN/CA9 gene expression in identifying malignant solid renal tumors

OBJECTIVE: Small solid renal tumors are increasingly encountered. It is important to determine the malignancy of solid renal tumors for the choice of treatment. MN/CA9 is expressed in malignant renal cells but absent in normal cells. MN/CA9 is one of the most powerful gene markers available for RCC. The objective of this pilot study is to utilize MN/CA9 gene expression in FNA biopsy to determine the malignancy of imaging-indeterminate solid renal tumors. METHODS: A total of 35 patients with an imaging-indeterminate solid renal mass entered into this study. The molecular protocol consisted of a rapid column extraction of RNA and one-step RT-PCR for the detection of MN/CA9 gene expression. The preoperative molecular diagnosis was compared with postoperative pathology. RESULTS: There were 28 RCCs (19 clear cell carcinomas, 7 papillary carcinomas and 2 chromophobe carcinomas) and 7 benign tumors proved by postoperative pathology. The overall sensitivity and specificity for MN/CA9 were respectively 68% and 100%. MN/CA9 was positive in 16/19 (84%) FNA biopsies of clear cell RCCs. No false positive appeared for MN/CA9 gene expression. Moreover, MN/CA9 gene expression was positive in 8/13 (62%) of false negative or suspect cytology. CONCLUSION: Detection of MN/CA9 gene expression in FNA biopsy is possible. Its detection can be helpful in identifying the malignancy among renal tumors.     

结直肠癌患者CEAmRNA表达与血清CEA及相关蛋白联合检测的临床意义

为探讨结直肠癌患者外周血癌胚抗原信使核糖核酸（CEAmRNA）的表达与血清癌胚抗原（CEA）联合CA19—9、CA242和CA724蛋白检测的临床意义,采用逆转录-聚合酶链反应（RT—PCR）和化学发光法,检测125例术前和96例术后结直肠癌患者及150名健康对照者外周血中CEAmRNA表达和血清CEA、CA19-9、CA242和CA724蛋白水平。结果结直肠癌患者血清CEA及相关蛋白联合检测的阳性率为79．1％,高于CEAmRNA（53．6％）和健康对照者（2．7％）,有显著性差异（P〈0．01）。两种方法阳性检出率与肿瘤Dukes分期、淋巴结及脏器转移明显相关。血清CEA及相关蛋白联合检测阳性率在分化程度低的患者组中为74．3％,明显高于高分化组（52．8％）,有显著性差异（P〈0．01）,而CEAmRNA表达与肿瘤细胞分化程度无关。术后随访中发现CEAmRNA和血清CEA及相关蛋白水平明显增高的患者,均发生肝、肺和盆腔等远处转移。结果表明,外周血CEAmRNA和血清CEA联合CA19—9、CA242和CA724的检测可作为判断结直肠癌恶性程度、转移及监测疗效的重要指标,血清CEA及相关蛋白联合检测的敏感度优于外周血CEAmRNA表达。     

检测不同生物学行为肾癌内LVD和MVD的临床意义

目的探讨淋巴管和微血管在不同生物学行为肾细胞癌（RCC）组织内的密度及其临床应用价值。方法40例肾细胞癌按其不同的生物学行为特点分为长期生存组和肿瘤转移组。肿瘤组织切片分别以针对血管内皮生长因子受体-3（VEGFR-3）和淋巴内皮透明质酸受-体1（LYVE-1）的单克隆抗体实施免疫组化染色,并对瘤组织内微血管和淋巴管进行计数。结果VEGFR-3非特异地表达于微血管和淋巴管内皮细胞浆,而LYVE-1仅特异地表达于淋巴管内皮细胞浆中。转移组瘤内MVD显著高于长期生存组（P〈0.05）。LYVE-1在转移组中仅有3例瘤内显示阳性。结论MVD可作为判断RCC患者预后的可靠指标。RCC缺乏瘤内淋巴管形成。     

Human kidney injury molecule-1 is a tissue and urinary tumor marker of renal cell carcinoma

Human kidney injury molecule-1 (hKIM-1) is a type 1 transmembrane protein that is not detectable in normal kidney tissue but is expressed at high levels in human and rodent kidneys with dedifferentiated proximal tubule epithelial cells after ischemic or toxic injury. Therefore, it was hypothesized that renal tumors express hKIM-1 and release this protein into the urine. Forty renal cell carcinoma (RCC) and 484 nonrenal tumors were analyzed by immunohistochemistry for expression of hKIM-1 (group 1). Urine samples before nephrectomy and nephrectomy tissue samples were collected from an additional 42 patients with renal tumors, from 30 normal control subjects, and also from 10 patients with prostate carcinoma (group 2). In five additional patients with RCC, urine was collected before and after nephrectomy (group 3). Tissue was examined for expression of hKIM-1, and cell-free urine supernatants were analyzed for hKIM-1 by ELISA. Urinary hKIM-1 was normalized to the urinary creatinine concentration (U(Cr)). Expression of hKIM-1 was present in 32 tissue sections (91%) of 35 clear cell RCC (group 1). In group 2, the normalized urinary hKIM-1 levels were significantly higher in patients with clear cell RCC (0.39 +/- 0.08 ng/mg U(Cr); n = 21), compared with levels in patients with prostate carcinoma (0.12 +/- 0.03 ng/mg U(Cr); P < 0.02; n = 10), or normal control subjects (0.05 +/- 0.01 ng/mg U(Cr); P < 0.005; n = 30). Tissue sections from 28 (82%) of 34 primary RCC stained positively for the expression of hKIM-1. In all patients with a detectable prenephrectomy urinary hKIM-1 level, there was either complete disappearance or marked reduction after nephrectomy (group 3). In conclusion, the cleaved ectodomain of hKIM-1 can be detected in the urine of patients with RCC and may serve as a new biomarker for early detection of RCC.