肾癌抑制性消减杂交文库的构建及意义

目的　应用抑制性消减杂交技术构建人肾癌组织与正常肾组织间差异表达的cDNA组成的消减文库。　方法　分别从肾癌组织和正常肾组织提取polyA RNA ,合成双链cDNA ,经RsaI酶切后将肾癌cDNA分为两组并加上不同的DNA接头 ,再与过量正常肾组织cDNA进行两次消减杂交及两次抑制性PCR ,PCR产物与T/A载体连接并转化大肠杆菌构建成cDNA消减文库 ,文库扩增后随机挑取克隆进行酶切、测序及同源性分析。　结果　文库共包含 4 14个阳性克隆 ,随机挑取 2 6 5个阳性克隆提取质粒并酶切分析 ,其中 2 4 6个克隆有插入片段。将其中 4 0个克隆进行测序 ,表明 1个克隆为新基因片段 ,其余 39个源于 35个已知基因。　结论　该消减杂交文库质量可靠 ,它的成功构建为进一步筛选、克隆肾癌差异表达基因提供了依据     

肾癌组织消减文库的构建与肾癌特异表达基因克隆

目的 构建人肾癌组织与正常肾组织差异表达的cDNA消减文库,从文中克隆鉴定出肾癌特异性表达的基因奠定基础。方法 应用抑制性消减杂交技术,分别从肾癌及正常肾组织中提取poly(A) RNA;依次合成单链及双链cDNA,分别与2种不同的接头衔接,再与正常肾cDNA进行2次消减杂交及2次抑制性PCR;将产物T／A载体连接接构建成功cDNA消减文库。结果 构成功具有高消减效率的人肾癌组织cDNA消减文库,文库扩增后得到350个阳性克隆,其中95％克隆均含50－400bp插入片段。结论 应用抑制性消减杂交技术所构建的人肾癌组织cDNA消减文库为进一步大批量筛选、克隆肾癌特异性表达的基因奠定了基础。     

用SSH方法构建BTCC患者尿脱落细胞差异表达基因的cDNA消减文库

目的:应用抑制性消减杂交(SSH)方法构建膀胱移行细胞癌(BTCC)患者与正常人尿脱落细胞差异表达基因cDNA消减文库。方法:分别从BTCC患者与正常人尿液中提取总mRNA,用SMART技术反转录成cDNA,经过HaeⅢ酶切后将BTCC尿脱落细胞cDNA分为两组并接上接头,再与过量正常膀胱尿脱落细胞cD-NA进行两轮消减杂交及两轮抑制性聚合酶链反应(PCR),使得差异表达的DNA片段得以富集。PCR产物与T/A载体连接并转化大肠杆菌JM109构建成差异表达基因的cDNA消减文库。文库扩增后,随机挑取克隆进行酶切、测序及同源性分析。结果:PCR鉴定有317个克隆载有主要在200～900bp之间呈随机分布的插入片段,片段插入率达82.6%,证实建库成功。对20个质粒测序结果经同源性比对分析,其中20个片段源于17个已知基因,1个克隆在GenBank中未检索到与其有相似性的基因序列,表明它们可能为BTCC差异表达的新基因。结论:该消减杂交文库质量可靠,其成功构建为进一步筛选、克隆BTCC差异表达基因提供了依据。     

膀胱癌患者尿脱落细胞抑制消减文库的构建及差异基因的初步筛选

目的 应用抑制性消减杂交方法 筛选膀胱移行细胞癌患者与正常人尿脱落细胞差异表达基因.方法 分离膀胱移行细胞癌患者与正常人尿液中总mRNA,用SMART技术反转录成cDNA,经过酶切、接头连接、两轮消减杂交及两轮抑制性PCR,使得差异表达的DNA片段得以富集.PCR产物与T/A载体连接并转化大肠杆菌XL-blue构建差异表达基因的cDNA消减文库.文库扩增后随机挑取克隆进行酶切、测序及同源性分析.结果 PCR鉴定有317个克隆载有主要在200～900bp之间呈随机分布的插入片段,片段插入率达93.2%,证实建库成功.对20个质粒测序结果 经同源性比对分析,其中20个片段源于17个已知基因,1个克隆在GenBank中未检索到与其有相似性的基因序列,表明它们可能为BTCC差异表达的新基因.结论 该消减杂交文库质量町靠,它的成功构建为进一步筛选、克隆膀胱肿瘤差异表达基因提供了依据.也为膀胱肿瘤诊断基因芯片的研究与开发奠定了基础.     

转化生长因子β_1刺激肝星状细胞差异表达下调基因筛选

目的筛选并克隆转化生长因子β1(TGF-β1)刺激肝星状细胞差异表达下调基因,阐明TGF-β1导致肝纤维化的分子生物学机制。方法以TGF-β1及磷酸盐缓冲液分别刺激大鼠肝星状细胞(即实验组和对照组),提取mRNA并逆转录为cDNA,经RsaⅠ酶切后,将对照组细胞cDNA分成两组,分别与两种不同的接头衔接,再与实验组细胞cDNA进行2次消减杂交及2次抑制性PCR扩增,将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转染大肠埃希菌进行文库扩增,随机挑选克隆经PCR扩增后进行测序及同源性分析。结果成功构建了TGF-β1刺激肝星状细胞差异表达基因的cDNA消减文库。文库扩增后得到98个阳性克隆,进行菌落PCR分析,均得到200～1000bp插入片段。选取含有插入片段的35个克隆进行测序,并通过生物信息学分析获得了19种已知基因序列和2个未知功能基因。结论应用抑制性消减杂交技术成功构建了TGF-β1刺激的肝星状细胞差异表达基因的cDNA消减文库,为进一步阐明TGF-β1参与肝纤维化的分子生物学机制提供了理论依据。     

抑制性消减杂交技术克隆肾癌差异表达基因

目的 应用抑制性消减杂交技术构建人吕组织与正常肾组织差异表达的ｃＤＮＡ消减文库，并从中克隆鉴定出蛑癌特异性表达的新基因。方法 分别从肾癌及正常肾组织中提取ｍＲＮＡ并合成ｃＤＮＡ，经酶切后将肾癌ｃＤＮＡ分为两组，分别与两种不贩接头衔接，再与正常肾组织（ｃＤＡＮ）进行两次的消减杂交及两次抑制性ＰＣＲ产物与Ｔ／Ａ载体连接构建成功ｃＤＮＡ消减文库，并转染大肠杆菌进行文库扩增，随机挑取克隆进行酶切、测序分析     

应用抑制性消减杂交方法构建斑秃毛乳头差异表达基因文库

目的：构建斑秃区与正常毛囊毛乳头细胞（DPC）差异表达的cDNA正向和反向消减杂交文库，为从中克隆鉴定出斑秃特异性表达和生长期DPC特异性表达的基因奠定基础。方法：应用抑制性消减杂交技术，分别从斑秃区DPC及正常头皮DPC提取总mRNA；依次合成单链及双链cDNA，分别与2种不同的接头连接，再进行正向和反向的2次消减杂交及2次抑制性PCR，将产物与T／A载体连接构建cDNA消减文库。结论：构建成功具有高消减效率的斑秃区及正常头皮DPC cDNA消减文库，文库扩增后得到120个阳性克隆，其中90个克隆含有100－500bp插入片段，结论：应用抑制性消减杂交技术所构建的斑秃区及正常头皮DPC cDNA消减文库，为进一步批量筛选，克隆斑秃区及正常头皮DPC特异性表达的基因奠定了基础。     

肾癌抑制性消减文库构建及差异表达基因鉴定

目的：应用抑制性消减杂交技术筛选人肾癌差异表达基因。方法：以786-0和HK-2为消减杂交对象构建人肾癌抑制性消减文库,挑选阳性克隆进行测序及Genbank BLAST分析。结果：文库包含362个有插入片段的阳性克隆,随机分析50个克隆,其中2个为新基因,另48个源于36个已知基因,这些差异表达基因与肿瘤细胞的转录、翻译、增殖、凋亡、代谢、信号转导、血管新生、膜受体表达异常等有关。结论：该文库质量可靠,筛选出包括低峰度和新基因在内的肾癌差异表达基因,为进一步研究肾癌发生、发展机制奠定了基础。     

肾癌差异表达基因GYLZ-RCC18的全长克隆及意义

目的 克隆并鉴定肾癌与下沉肾组织之间差异表达的基因。为研究肾癌发生发展机制提供新的突破口。方法 应用抑制性消减杂交技术,构建人肾癌组织与正常肾组织差异表达的ｃＤＮＡ消减文库,并从中克隆鉴定出肾癌特异表达的基因。结果：构建成功高消减效率的人肾癌组织ｃＤＮＡ消减文库,对其中１０个克隆的插入ｃＤＮＡ片段进行测序后,经基因库检索表明１０个片段均为未知新序列,其中ＧＹＬＺ－ＲＣＣ１８基因为５个拷贝,这提示以上１０个ｃＤＮＡ片段可能来自６个新基因。差异分析显示ＧＹＬＺ－ＲＣＣ１８的肾癌组织中有明显表达,而在正常肾组织中无表达,应用ＳＭＡＲＴ ＲＡＣＥ技术获得ＧＹＬＺ－ＲＣＣ１８基因的全长,并证明ＧＹＬＺ－ＲＣＣ１８是一个５′端有Ｄ３种不同剪切方式亚型的基因家族。结论 ＧＹＬＺ－ＲＣＣ１８基因是肾癌特异表达的新基因。人肾癌ｃ     

ox-LDL特异性人血管平滑肌细胞cDNA消减文库的构建

目的:构建ox-LDL特异性人血管平滑肌细胞cDNA消减文库。方法:35％ug/ml ox-LDL刺激培养的人血管平滑肌细胞,利用消减杂交技术,构建cDNA消减文库,并运用蓝白斑筛选和影印杂交进一步确定文库克隆的特异性。结果:成功构建ox-LDL特异性血管平滑肌细胞cDNA消减文库,约2000个白色克隆。影印杂交后共获82个差异表达克隆。结论:消减文库的构建为进一步克隆ox-LDL特异性基因cDNA片段和全长奠定了基础。     

Sprouty2蛋白表达对肾癌细胞功能影响的实验研究

目的研究Sprouty2蛋白的表达水平对肾癌细胞增殖、侵袭等生物学功能的影响。方法采用RNAi技术,构建质粒转染肾癌细胞系786-O,建立Sprouty2低表达细胞株,通过MTT、Transwell等技术研究Sprouty2蛋白的表达水平对肾癌细胞增殖、侵袭等生物学行为的影响。结果成功构建Sprouty2低表达肾癌细胞系。当Sprouty2表达水平下调后,肾癌细胞的增殖、侵袭能力增强,与对照组及空质粒转染组比较差异有显著性（P〈0.05）。结论 Sprouty2的表达对肾癌细胞的功能有一定影响,Sprouty2表达越低,肾癌细胞的增殖及侵袭力越强。     

白藜芦醇对肾癌细胞体外增殖与侵袭能力的影响

目的探讨白藜芦醇对肾癌786-O与ACHN细胞体外迁增殖与侵袭能力的影响及可能的机制。方法 MTT检测白藜芦醇对肾癌细胞增殖能力的影响,划痕实验检测白藜芦醇对786-O与ACHN细胞体外迁移能力的影响,Transwell实验检测白藜芦醇对786-O与ACHN细胞体外侵袭能力的影响,RT-PCR与Western bolt检测白藜芦醇对MMP-2与MMP-9蛋白表达水平的影响。结果白藜芦醇可抑制膀胱癌细胞体外生长能力,且呈剂量依赖性。30μmol/L白藜芦醇处理786-O与ACHN细胞24h后,划痕实验发现白藜芦醇可抑制786-O与ACHN细胞体外迁移能力,Transwell实验证实白藜芦醇可抑制肾癌细胞786-O与ACHN的体外侵袭能力。RT-PCR与Western bolt结果表明,白藜芦醇可从mRNA与蛋白水平抑制MMP-2与MMP-9的表达。结论白藜芦醇能抑制肾癌细胞体外增殖能力,可能通过下调MMP-2与MMP-9的表达而抑制肾癌细胞786-O与ACHN体外迁移与侵袭能力,有望成为治疗肾癌的新策略。     

Biological role of HGF/MET pathway in renal cell carcinoma

PURPOSE: Several lines of evidence show that hepatocyte growth factor (HGF) and its receptor MET play a significant role in the progression of various cancers including renal cell carcinoma (RCC). Our objectives were to evaluate the gene expression of HGF and MET in RCC, and to examine the effect of HGF on the biological activities of cultured RCC cells. MATERIALS AND METHODS: We examined the gene expression of HGF and MET in 27 primary RCC tumors by quantitative competitive RT-PCR. The effects of HGF on in vitro chemoinvasion assay and the expression of matrix metalloproteinase-9 (MMP-9), and the induction of Fas-induced apoptosis were studied by transfection of HGF cDNA to cultured RCC cells, Caki-1. RESULTS: HGF mRNA and MET mRNA were detected in all surgical specimens. The level of expressed HGF mRNA was proportional with the volume of tumor (r = 0.50, p = 0.015). Caki-1 cells overexpressing HGF cells showed enhanced in vitro invasiveness in the chemoinvasion assay and increased activity of 92 kDa type IV collagenase (MMP-9). The sensitivity to Fas-induced cell death was reduced in HGF transfectants, which was reversed by the presence of anti-HGF antibody. CONCLUSIONS: HGF enhanced the invasive properties of cultured RCC cells and inhibited Fas-induced apoptosis in vitro. Both HGF and MET mRNA were expressed in RCC tissues tested. Our results indicate that HGF/MET pathway may have a significant role in the progression of RCC.     

TGFβ_1及PCNA在肾细胞癌中的表达及其临床意义

目的 :探讨肾细胞癌中转化生长因子 β1 (TGFβ1 )及增殖细胞核抗原 (PCNA)的表达及其价值。方法 :采用免疫组织化学技术 (SP法 )对 46例肾细胞癌和 11例正常肾组织标本中 TGFβ1 、PCNA进行检测 ,并结合临床资料进行分析。结果 :肾癌组 TGFβ1 表达量较正常组高 (P <0 .0 5 ) ;PCNA在肾癌组高表达 (P <0 .0 1) ,且在高分期、高分级肾细胞癌中表达量较低分期、低分级肾细胞癌高 (P <0 .0 5 ) ;TGFβ1 表达量与 PCNA表达量呈负相关关系 (P <0 .0 1) ;TGFβ1 表达量低及 PCNA表达量高的肾癌患者预后差 (P <0 .0 5 )。结论 :TGFβ1 对处于增殖状态的肾细胞癌是一种重要负性调节因子 ,可抑制肾癌发展 ;TGFβ1 及 PCNA表达可作为判断肾细胞癌预后的指标     

Increasing expression of extracellular matrix metalloprotease inducer in renal cell carcinoma: Tissue microarray analysis of immunostaining score with clinicopathological parameters

AIM: Renal tumor cell invasion is responsible for both local tissue destruction and distant metastasis. Invasion is largely mediated by matrix metalloproteases that are thought to be induced by tumor cell-derived extracellular matrix metalloprotease inducer (EMMPRIN) in surrounding fibroblasts. We hypothesized that EMMPRIN and matrix metalloproteinase-9 (MMP-9) are over-expressed in renal cell carcinoma. METHODS: Immunohistochemical analysis of EMMPRIN and MMP-9 was performed in tissue microarrays of 79 renal cell carcinomas including 12 cases of chromophobe renal cell carcinoma (ChRCC), 53 cases of clear cell renal cell carcinoma (CRCC), 8 cases of papillary renal cell carcinoma (PRCC), and 6 cases of carcinoma of the collecting ducts of Bellini (CoRCC). RESULTS: All renal cell carcinomas showed significant immunohistochemical expression of EMMPRIN. The EMMPRIN score in ChRCC (321+/-21) was significantly higher than in other histological subtypes of RCC (166+/-19 for CRCC; 276+/-24 for PRCC; 98+/-17 for CoRCC). MMP-9 was mainly expressed in tumor stromal cells and not in non-cancerous fibrovascular regions. The percent positive staining of MMP-9 at the invasive front of tumor cells was significantly higher in CRCC than in ChRCC, PRCC, or CoRCC. Higher EMMPRIN scores in CRCC were associated with shorter survival time, and correlated with higher T staging and nuclear grading. CONCLUSIONS: Our findings demonstrate for the first time that EMMPRIN is over-expressed in renal cell carcinomas. Increased expression of EMMPRIN in tumor cells is associated with poor prognosis of patients with CRCC.     

Cyst-associated renal cell carcinoma: Clinicopathologic characteristics and evaluation of prognosis in 27 cases

BACKGROUND: No consistent clinicopathologic characteristics of cyst-associated renal cell carcinoma (CRCC) have previously been determined. METHODS: In total, 768 patients with renal cell carcinoma (RCC) underwent radical or partial nephrectomy. Renal cell carcinoma was classified as CRCC in 27 of these patients (3.5%, subdivided into RCC originating in a cyst and cystic RCC), clear-cell RCC in 662 patients (86.2%), chromophobe cell renal carcinoma in 36 patients (4.7%) and papillary RCC in 43 patients (5.6%) according to the criteria of the World Health Organization. RESULTS: The pathologic stage and nuclear grade were usually lower in those with CRCC (low stage/low grade; 89%/96%) or chromophobe cell renal carcinoma (low stage/low grade; 89%/80%) than in those with clear-cell RCC (low stage/low grade; 59%/65%) or papillary RCC (low stage/low grade; 53%/69%). Of the 27 CRCC patients, only 19 (70%) could be diagnosed through preoperative imaging studies. Patients with CRCC showed a favorable prognosis (survival rate: 95% at 1 year, 89.7% at 3 years and 84.4% thereafter) and, especially among the patients with RCC originating in a cyst, no cancer-related death was observed. Comparing the survival among four types of RCC, a favorable outcome was observed in cases of CRCC or chromophobe cell renal carcinoma compared with clear-cell RCC or papillary RCC (clear vs chromophobe: P = 0.002; chromophobe vs papillary: P = 0.019; clear vs cyst-associated: P = 0.001; papillary vs cyst-associated: P = 0.00079). CONCLUSIONS: In cases of CRCC, the disease was usually detected at lower stages and grades and therefore the prognosis was better than in cases of other types of RCC. Preoperative diagnosis of this disease was very difficult, especially in cases of RCC originating in a cyst.     

长链非编码RNA MALAT-1通过EZH2-β-catenin信号通路调控肾癌细胞的增殖、凋亡及侵袭

目的探究长链非编码RNA肺腺癌转移相关转录因子1(LncRNA MALAT-1)在肾癌组织及肾细胞癌(RCC)细胞株中的表达情况及其在调控RCC细胞增殖、凋亡及侵袭过程中的作用机制。方法利用实时荧光定量聚合酶链反应(qRT-PCR)实验检测正常和RCC组织以及HK-2、786-O、ACHN及Caki-1细胞中MALAT-1的表达情况;将786-O细胞随机分为3组,分别转染MALAT-1-siRNA沉默载体(si-MALAT-1组)及MALAT-1-siRNA阴性表达载体(si-NC组),空白对照组加入PBS(Blank组)。采用CCK-8法、流式细胞术法、Transwell法检测细胞增殖、凋亡及侵袭能力;利用Western blot法检测Zeste基因同源物增强子2(EZH2)及β-catenin的蛋白表达水平。结果与正常组织及细胞株HK-2相比,肾癌组织及细胞株786-O、ACHN及Caki-1中MALAT-1的表达水平明显增加(P<0.05);与Blank组和si-NC组相比,si-MALAT-1组RCC细胞活性和侵袭能力明显降低、细胞凋亡率明显增加、EZH2及β-catenin的蛋白表达水平明显升高(P<0.05)。结论LncRNA MALAT-1在肾癌组织及RCC细胞中表达上调;抑制MALAT-1的表达能够抑制RCC细胞的增殖和侵袭,促进凋亡,其机制可能与下调EZH2-β-catenin信号通路的表达有关。     

肾癌并静脉癌栓的影像学诊断与手术方法选择

目的：探讨肾癌并静脉癌栓的影像学诊断与治疗及方法的选择。方法：回顾性分析我科收治的肾癌伴静脉癌栓患者21例的临床资料。结果：MRI精确地诊断出癌栓的范围；20例肾癌根治性切除加癌栓取出术的患者取得了满意的效果。结论：MRI可替代创伤性大、不良反应多的下腔静脉造影，用于确诊肾癌并静脉癌栓；应依据癌栓的类型选择手术方法。     

Impact of tumor size on the predictive ability of the pT3a primary tumor classification for renal cell carcinoma

PURPOSE: The accuracy of the pT3a primary tumor classification for renal cell carcinoma has been questioned recently. We investigated the association of perinephric and renal sinus fat invasion with death from renal cell carcinoma independent of tumor size. MATERIALS AND METHODS: We identified 2,165 patients treated with open radical nephrectomy or nephron sparing surgery for clinically localized, sporadic pT1a, pT1b, pT2 or pT3a renal cell carcinoma between 1970 and 2002. Patients with pT3a disease were then subdivided into 3 groups according to tumor size to match the size definitions for the pT1a, pT1b and pT2 tumor classifications. RESULTS: There were 834 patients with pT1a RCC, 674 with pT1b, 494 with pT2 and 163 with pT3a RCC. At last followup 317 patients died of RCC at a median of 3.8 years following surgery. The median followup among the 1,087 patients still alive at last followup was 7.8 years (range 0 to 34). The risk ratios (95% CI) for the association between fat invasion and death from RCC among patients with tumors 4 cm or smaller, 4 to 7 cm and more than 7 cm were 6.15 (1.84-20.50, p = 0.003), 4.12 (2.50-6.78, p <0.001) and 2.13 (1.53-2.97, p <0.001), respectively. These associations remained statistically significant in a multivariate analysis that included nuclear grade and histological coagulative tumor necrosis. CONCLUSIONS: Peripheral perinephric and renal sinus fat invasion was associated with death from RCC independent of tumor size. Our data contradict reports suggesting that pT3a tumors should be reclassified according to tumor size only.