非小细胞肺癌T7噬菌体展示文库的构建

背景与目的SEREX(Serological analysis of recombinant expression libraries)是近年来发展起来的一种寻找肿瘤相关抗原的方法,通过与噬菌体展示技术结合,可以有效和快速的筛选肿瘤相关抗原。本研究拟构建人肺鳞癌和肺腺癌T7噬菌体展示cDNA文库,为进一步筛选和鉴定非小细胞肺癌相关抗原打下基础。方法诊断明确的肺鳞癌和腺癌患者手术标本各5例,Trizol试剂提取总RNA,Straight A's系统分离鳞癌和腺癌mRNA,逆转录合成双链cDNA后,与T7Select 10-3载体连接,体外包装并扩增得到人肺鳞癌和腺癌T7噬菌体展示cDNA文库。结果构建肺鳞癌和肺腺癌T7噬菌体展示cDNA文库各一个,原始文库重组克隆数分别为鳞癌1.8×106pfu,腺癌5×106pfu。扩增后滴度为3.2×1010pfu/mL和2.5×1010pfu/mL。随机从2个文库中各抽取24个克隆PCR扩增插入片断,电泳结果显示肺腺癌文库所有克隆均有插入片段,肺鳞癌文库插入率超过95%(23/24)。两个文库插入片断大小均在(300-1500)bp之间。结论成功构建了人肺鳞癌和腺癌T7噬菌体展示cDNA文库,为下一步筛选和鉴定非小细胞肺癌相关基因奠定基础。     

人源性食管鳞癌cDNA文库的构建及鉴定

目的:构建人源性食管鳞癌cDNA噬菌体表达文库。方法:从人食管鳞癌组织中提取总RNA并分离纯化mRNA,用RT-PCR合成cDNA单链,LD-PCR扩增双链cDNA,除去<500bp的小片段,与载体λTriplEx2噬菌体左右臂连接,经体外包装即构成全长cD-NA噬菌体表达文库。转化E.coliXL1-Blue感受态细胞,测定文库的滴度,确定文库的容量大小,用蓝白筛选测定文库的重组率,PCR鉴定cDNA插入片段的大小。结果:构建的人源性食管鳞癌cDNA噬菌体表达文库滴度为1.64×106pfu/mL,重组率为98.6%,cDNA插入片段的大小为0.7～2.5kb,平均长约1.5kb。结论:成功构建1个人源性食管鳞癌cDNA噬菌体表达文库,适合于大批量筛选cDNA克隆的食管鳞癌肿瘤抗原基因。     

T7噬菌体展示文库筛选肺癌早期检测分子标志群

目的从肺癌T7噬菌体展示文库筛选出能用于肺癌早期检测的分子标志群。方法亲和筛选肺癌T7噬菌体展示文库,对纯化后的富集肺癌相关肽的文库进一步行两轮ELISA检测,PCR扩增阳性噬菌体插入片段,测序后经过NCBI上的BLAST序列比对。结果筛选出13个有意义噬菌体,测序结果显示1个为未知功能的新基因,其余的均已确定与癌症相关;且13个基因中包含有三组相同的基因。结论从T7噬菌体构建的肺癌cDNA文库中筛选出的一组阳性噬菌体表达的抗原,可能是潜在的肺癌诊断分子标志群。     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的:利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法:从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞(PBMC),提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链(VH)和轻链(VL)基因,经重叠延伸PCR(SOE-PCR),在体外将两者连接成单链抗体(scFv)基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果:得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论:本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的：利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法：从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞（PBMC）,提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链（VH）和轻链（VL）基因,经重叠延伸PCR（SOE-PCR）,在体外将两者连接成单链抗体（scFv）基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果：得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论：本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。     

人胃癌转移型裸鼠移植瘤cDNA文库构建及分析

从人转移型胃癌裸鼠移植瘤中抽提出总RNA,分离出多聚腺苷酸RNA,合成双链cDNA,接上ECoRI接头,以pT7T3 18U质粒为载体,以大肠杆菌NM522为受体菌,构建了人转移型胃癌裸鼠移植瘤cDNA文库。该文库含1.5×10~6转化子,插入片段cDNA长度为0.4～6.0Kb,重组效率为80％,并用四种癌基因探针筛选了阳性克隆。     

人卵巢癌cDNA表达文库的构建

以polyA~ mRNA提取试剂盒从卵巢癌组织中获得高质量的polyA~ mRNA,并以此合成第1、2链cDNA.cDNA两端补平后加EcoR Ⅰ接头,Xbo酶切,通过分级分离去除<400bP的片段.取100ng cDNA与噬菌体表达载体Uni-ZAP XR连接,体外进行噬菌体DNA包装,并立即进行文库的滴定和扩增,获得卵巢癌cDNA表达文库.该文库原始重组子为10~6独立克隆;重组率>99%;并以引物PUCⅠ、PUCⅡ扩增插入片段,平均大小约为1.1kb.证实此cDNA表达文库合格.以PCR从此cDNA表达文库中筛选HLA-DPB和β-actin基因,获得成功.     

杜氏盐藻基因组文库的构建

目的　构建杜氏盐藻基因组文库,旨在进一步分离和研究杜氏盐藻基因的结构和功能。方法　采用改进的SDS裂解法提取杜氏盐藻基因组DNA ,通过限制性内切酶Sau3AI进行部分酶切,用T4DNA连接酶将目的片段与噬菌体EMBL3载体臂进行连接后转染大肠杆菌LE3 92。结果　体外包装构建了杜氏盐藻基因组文库,得到重组子数为2 .2×10 4,经测定文库滴度为2 .2×10 5pfu/mL。结论　构建了杜氏盐藻基因组文库,为进一步筛选盐藻重要基因及其序列分析奠定基础。     

人胃癌细胞定向克隆表达cDNA文库的构建及鉴定

目的：克隆胃癌的相关基因。方法：提取人胃癌细胞总RNA，分离其mRN。以NatI／oligo（dT）12-18为引物合成双链。cDNA，除去小于400bp的cDNA片段后，连接EcoRⅠ人工接头，经NotⅠ酶酶切，插入定向克隆表达载体λExcellNotI／EcoRI／CIP，体外包装后转染大肠杆菌NM522，构建了人胃癌细胞MGC－803定向克隆表达。cDNA文库。结果：文库含1．2×106个重组子，重组率为96．7％。用PCR技术鉴定，文库中插入cDNA的平均大小为1kbc结论：该文库适于筛选各种丰度mRNA的cDNA克隆。     

抗前列腺癌细胞特异抗体库的构建及特异结合抗体的筛选

目的: 获得前列腺癌的噬菌体呈现型单链抗体库,筛选与前列腺癌特异结合的抗体,为前列腺癌的诊断和治疗奠定基础. 方法: 用两种恶性程度较高的前列腺癌细胞PC3,DU145膜蛋白混合物免疫Balb/c小鼠.取脾脏提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区基因(VH和VL), 经Linker连接形成ScFv基因片段,将ScFv基因片段与噬菌粒载体pCANTAB 5E的连接产物转化大肠杆菌TG1.用辅助噬菌体M13KO7进行超感染,获得重组噬菌体抗体.以恶性程度低的前列腺癌细胞LNCap为对照,用PC3细胞对重组噬菌体抗体库进行五轮筛选后,随机挑取克隆,经phage-ELISA筛选特异性结合PC3细胞的ScFv.结果: 用所构建的库容量为3.5×106的单链抗体库筛选特异结合抗体,得到一个与PC3细胞特异结合的噬菌体-单链抗体.结论: 本研究所构建的抗体库中可筛选到与前列腺癌细胞结合特异性较好的抗体,可用于前列腺癌的诊断和治疗中.     

Cell‐free RNA content in urine as a possible molecular diagnostic tool for clear cell renal cell carcinoma

There is limited research on cell‐free RNA (cf‐RNA) in the urine of cancer patients. The present study was performed to detect the cf‐RNA in the urine of patients with clear cell renal cell carcinoma (ccRCC). Ninety‐five urine samples from ccRCC patients and 50 urine samples from control subjects were analyzed. The cf‐RNA integrity index was calculated by using quantitative real‐time RT‐PCR assays of the small‐sized fragment (106 bp) and the big‐sized fragment (416 bp) in GAPDH mRNA. The initial analysis showed that cf‐RNA was stable and detectable in the urine. The mean cf‐RNA integrity index was significantly lower in the urine of ccRCC patients (mean: 0.07, 95%CI: 0.05–0.10) when compared with the urine from control subjects (mean: 0.25, 95%CI: 0.16–0.33) (p < 0.001). The value of the area under the receiver‐operating characteristic curve by using the cf‐RNA integrity index for the diagnosis of ccRCC was 0.858 with a sensitivity of 68.0% and a specificity of 92.6%. Moreover, the small‐sized VEGF mRNA fragment (98 bp) was detected in 31 of 50 urine samples of patients with ccRCC and in only 2 of 50 urine samples of control subjects (p < 0.001) while the detection of the big‐sized (420 bp) VEGF mRNA fragment was an infrequent event. Our findings suggest that the small‐sized cf‐RNA in urine was more abundant in cancer patients. The tumor‐related gene VEGF mRNA fragment was detectable in the urine of cancer patients. Our finding may provide a new molecular assay for the diagnosis of renal cell carcinoma.     

Distribution of Vascular Patterns in Different Subtypes of Renal Cell Carcinoma. A Morphometric Study in Two Distinct Types of Blood Vessels

To analyze the presence of mature and immature vessels as a prognostic factor in patients with renal cell carcinoma and propose a classification of renal cancer tumor blood vessels according to morphometric parameters. Tissue samples were obtained from 121 renal cell carcinoma patients who underwent radical nephrectomy. Staining with CD31 and CD34 was used to differentiate between immature (CD31+) and mature (CD34+) blood vessels. We quantified the microvascular density, microvascular area and different morphometric parameters: maximum diameter, minimum diameter, major axis, minor axis, perimeter, radius ratio and roundness. We found that the microvascular density was higher in CD31+ than CD34+ vessels, but CD34+ vessels were larger than CD31+ vessels, as well as being strongly correlated with the ISUP tumor grade. We also identified four vascular patterns: pseudoacinar, fascicular, reticular and diffuse. Pseudoacinar and fascicular patterns were more frequent in clear cell renal cell carcinoma (37.62 and 35.64% respectively), followed by reticular pattern (21.78%), while in chromophobe tumors the reticular pattern predominated (90%). The isolated pattern was present in all papillary tumors (100%). In healthy renal tissue, the pseudoacinar and isolated patterns were differentially found in the renal cortex and medulla respectively. We defined four distinct vascular patterns significantly related with the ISUP tumor grade in renal cell carcinomas. Further studies in larger series are needed in order to validate these results. Analysis of both mature and immature vessels (CD34+ and CD31+) provides additional information when evaluating microvascular density.     

Differential expression of the enzyme that esterifies retinol, lecithin:retinol acyltransferase, in subtypes of human renal cancer and normal kidney.

PURPOSE: Retinoids, a group of compounds, including vitamin A (retinol), and related metabolites, have been shown to regulate the growth and differentiation of many types of cells. IFN-alpha and either 13-cis-retinoic acid or liposomal all-trans retinoic acid have been used in the treatment of patients with metastatic renal cell carcinoma. We knew that samples from renal cell carcinomas contained greatly reduced levels of retinol and retinyl esters relative to samples from normal human kidney. This prompted us to examine the levels of LRAT (lecithin:retinol acyltransferase) protein in various subtypes of human kidney cancers relative to normal human kidney by immunohistochemistry. EXPERIMENTAL DESIGN: We examined 31 partial or radical nephrectomy specimens diagnosed with kidney tumors between 1997 and 1998. Representative paraffin-embedded tissue blocks from each tumor, with each containing adjacent nonneoplastic renal parenchyma, were used for immunohistochemical analysis with affinity purified antibodies to human LRAT protein. RESULTS: LRAT protein was detected at high levels in the epithelial cells in the tubules and the lining of Bowman's capsule in the glomeruli of normal, nonneoplastic kidney sections. Among the 31 tumors, there were 13 cases of conventional (clear cell) renal cell carcinoma (RCC; including 2 multilocular cystic RCCs), 7 papillary RCC, 6 chromophobe RCC, 1 RCC, unclassified, and 4 renal oncocytoma. All tumors showed diffuse immunoreactivity for LRAT. In each case, the staining was uniform throughout the tumor, with only minimal variation in the staining intensity between different areas. All 4 renal oncocytomas, 2 of 6 chromophobe RCCs, 1 conventional (clear cell) carcinoma, 1 RCC, unclassified, and 2 conventional RCCs, which were of the multilocular cystic-type stained strongly (3+) for LRAT. In contrast, the remaining conventional RCCs and the papillary RCC samples stained much less intensely for LRAT. Of the 10 tumors that stained 3+ for LRAT in the study, 9 were either benign tumors or tumors with low malignant potential. CONCLUSIONS: These data show that LRAT expression is higher in renal tumors with an indolent biological behavior. Additional studies will ascertain if LRAT possesses any prognostic or therapeutic role in renal cancer.     

Vimentin在肾透明细胞癌中的表达及临床意义

目的:探讨波形蛋白(Vimentin)在人肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)和癌旁组织样本中的表达情况,并分析其与 ccRCC 临床分期、病理分级和侵袭转移等恶性生物学特性的关系及临床意义。方法:Western blot方法检测29例新鲜肾透明细胞癌及相应癌旁组织中Vimentin的表达;免疫组织化学方法检测120例肾透明细胞癌及相应癌旁组织中Vimentin的表达情况,分析Vimentin在肾透明细胞癌组织中及癌旁正常肾脏组织中的表达水平差异及Vimentin的表达与肿瘤大小、临床分期、组织病理分级、远处转移的关系。结果:Vimentin在肾透明细胞癌及相应癌旁组织中均有表达,前者的表达水平较后者显著升高（P＜0.01）;免疫组织化学显示肾透明细胞癌组织中,Vimentin的阳性表达率高达90.0%,与癌旁组织存在明显差异（P＜0.01）;Vimentin在肾透明细胞癌组织中以胞膜表达为主,其表达随肾癌的临床分期、肾包膜受侵、癌栓形成或远处转移有升高趋势,但组间比较经统计学分析均无显著性差异。结论:Vimentin在肾透明细胞癌组织中的表达明显高于癌旁正常肾组织,提示Vimentin在肾透明细胞癌的发生发展过程中发挥重要的作用。Vimentin在肾透明细胞癌组织中过表达,与肾癌的发展密切相关,可以作为肾细胞癌早期诊断、预后和复发评估的参考指标之一。     

线粒体蛋白Bcl-2和Bax在肿瘤发生中作用的初步探讨

目的:研究线粒体蛋白(MAB1273)、Bcl-2、Bax在肾细胞癌(RCC)组织内的表达及相关性分析.方法:采用免疫组织化学SP方法检测9例嗜酸细胞瘤、6例嫌色细胞癌、23例透明细胞癌以及12例正常肾组织中MABl273、Bcl-2和Bax蛋白的表达.结果:MABl273和Bel-2在嗜酸细胞瘤、嫌色细胞癌、透明细胞癌中表达明显高于正常肾组织(P=0.006,P=0.008).Bax在各组间表达无明显差异(P=0.057).通过秩相关分析,MAB1273的表达与Bcl-2的表达呈正相关(r=0.341,P=0.015),而Bcl-2表达与Bax表达呈负相关(r=-0.287,P=0.043).结论:线粒体蛋白及Bcl-2的高表达、Bax低表达可能共同参与了肾嗜酸细胞瘤、嫌色细胞癌及透明细胞癌的发生,提示线粒体蛋白表达异常参与RCC细胞凋亡调控过程.     

Nucleostemin mRNA is expressed in both normal and malignant renal tissues

Nucleostemin (NS), a p53-binding protein, has been shown essential for stem and cancer cell proliferation and implicated in oncogenesis. To explore potential contributions of NS to the development of clear cell renal cell carcinomas (ccRCCs), we determined NS expression in ccRCC cell lines, and in paired normal and malignant renal tissues from 31 patients with ccRCC. Nucleostemin mRNA and/or protein expression was observed in all four cell lines and 27 of 31 (87%) tumour specimens. Surprisingly, 16 of 31 (52%) adjacent normal renal samples also expressed NS mRNA and its levels in four of them were comparable with those in paired tumour tissues. Three of the patients had detectable NS mRNA in their normal renal tissues whereas lacked its expression in the matched tumours. Compared to the oncogene c-MYC expression in these same samples, NS expression showed a much less specificity for ccRCC. We further demonstrated that NS mRNA expression was closely associated with cellular proliferation in normal fibroblasts or T lymphocytes and renal cell carcinoma cell lines. Collectively, NS expression widely occurs in normal and malignant renal tissues, and is likely a proliferation marker rather than a unique regulator of cell proliferation and survival in stem and cancer cells.     

Detection of Multidrug Resistance (MDR1) Gene RNA Expression in Human Tumors by a Sensitive Ribonuclease Protection Assay

The human MDR 1 gene encoding P-glycoprotein, an energy-dependent drug-efflux pump, was initially isolated from a multidrug-resistant KB carcinoma cell. When a 3 kb genomlc sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR 1 gene was compared to the equivalent fragment from KB cells, the MDR 1 gene from KB carcinoma cells was found to have a point mutation in the first exon. Although this mutation does not affect the downstream promoter sequence or the coding sequence of the MDR 1 gene, it creates a single base mismatch between the 5' KB genomic fragment previously used for RNase protection analysis of MDR 1 RNA expression in normal tissues and thereby reduces the sensitivity of this assay. Using the DNA fragment from normal tissues rather than KB cells, we have reanalyzed MDR 1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR 1 RNA levels are as high in these tumors as in the multidrug-resistant cell line, KB-8–5. The ribonuclease protection assay indicated that the major downstream promoter was mainly used in these clinical samples including two samples of RNA from metastatic renal cancer. This assay appears to be a very sensitive and specific assay for detecting MDR 1 mRNA levels and mRNA initiation sites in clinical samples.     

Detection of multidrug resistance (MDR1) gene RNA expression in human tumors by a sensitive ribonuclease protection assay

The human MDR1 gene encoding P-glycoprotein, an energy-dependent drug-efflux pump, was initially isolated from a multidrug-resistant KB carcinoma cell. When a 3 kb genomic sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR1 gene was compared to the equivalent fragment from KB cells, the MDR1 gene from KB carcinoma cells was found to have a point mutation in the first exon. Although this mutation does not affect the downstream promoter sequence or the coding sequence of the MDR1 gene, it creates a single base mismatch between the 5' KB genomic fragment previously used for RNase protection analysis of MDR1 RNA expression in normal tissues and thereby reduces the sensitivity of this assay. Using the DNA fragment from normal tissues rather than KB cells, we have reanalyzed MDR1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR1 RNA levels are as high in these tumors as in the multidrug-resistant cell line, KB-8-5. The ribonuclease protection assay indicated that the major downstream promoter was mainly used in these clinical samples including two samples of RNA from metastatic renal cancer. This assay appears to be a very sensitive and specific assay for detecting MDR1 mRNA levels and mRNA initiation sites in clinical samples.     

血清lncRNA HLA-DRB6对宫颈鳞癌预后的预测价值

目的:探讨血清HLA-DRB6在宫颈鳞癌无创筛查和预后预测中的价值。方法:回顾性选择2013年2月就诊于我科的3例宫颈鳞癌患者,选取同期就诊的3例妇科疾病患者（子宫内膜异位症）作为对照组,对两组间的血清样本进行长链非表达RNA（long non-coding RNA,lncRNA）表达谱分析,筛选差异表达lncRNA。然后挑选出HLA-DRB6,进一步搜集2013年2月至2013年10月就诊于我科的92例宫颈鳞癌患者的血清标本进行验证,并与另54例妇科疾病患者（子宫内膜异位症）对照组进行比较,探讨其诊断效能。根据血清HLA-DRB6表达水平进行分组,比较HLA-DRB6高表达组与HLA-DRB6低表达组的预后。结果:对3例宫颈鳞癌患者和3例对照者的血清lncRNA表达谱进行比较分析,共筛选出764个差异lncRNA,在宫颈鳞癌组上调lncRNA共431个,下调lncRNA共333个。其中HLA-DRB6差异倍数为2.58,在宫颈鳞癌患者血清中表达升高（P=0.001 737）。进一步扩大临床样本量验证,结果提示宫颈鳞癌患者的血清HLA-DRB6高于对照组（5.5±1.1 vs 4.2±1.1,P=0.001）。ROC曲线分析提示其曲线下面积为0.801,灵敏度为89.6%,特异度为62.3%。以6.1为截点分成HLA-DRB6高表达组和HLA-DRB6低表达组,发现HLA-DRB6高表达组患者的5年OS低于HLA-DRB6低表达组（8.9% vs 76.4%,P=0.001）。结论:本研究成功筛选了血清HLA-DRB6作为宫颈鳞癌预后预测的无创血清标记物,并与宫颈鳞癌的较高分期有关。