应用噬菌体随机七肽库筛选人角质细胞生长因子模拟肽

目的 应用噬菌体随机七肽库生物淘选人角质细胞生长因子(keratinocyte growth factor,KGF)噬菌体模拟肽. 方法 以人KGF单克隆抗体为靶,对噬菌体随机七肽库进行4轮生物淘选,测定噬菌体滴度.随机挑取单克隆噬菌体行ELISA检测其结合力,对结合活性较好的单克隆噬菌体提取噬菌体DNA,测序,并应用Basic BLAST系统行序列相似性和同源性分析. 结果 经过4轮生物淘选获得的噬菌体滴度逐渐升高,特异性噬菌体模拟肽获得了富集,行ELISA检测的单克隆噬菌体滴度均可达到2.0×1014 pfu/mL.取吸光度值显示与特异性抗体具有较好结合性的单克隆噬菌体进行测序,共获得26个与促进分裂、生长有关的碱基序列,其中有2个序列与人KGF相似;同源序列分析显示26个碱基序列的共同序列与人KGF的部分序列相似. 结论 从噬菌体随机七肽库中可淘选到与人KGF DNA序列相似或相关的噬菌体模拟肽,有望改善人KGF性能,促进创面愈合和改善组织工程皮肤替代物的性能.     

利用噬菌体肽库筛选高转移潜能人肝癌细胞结合肽

目的筛选高转移潜能人肝癌细胞特异性结合肽。方法以高转移潜能人肝癌细胞系HCCLM3作为靶细胞，低转移潜能人肝癌细胞系MHCC97L为吸附细胞对噬菌体随机7肽库进行差减筛选，采用噬菌体结合实验和抗噬菌体免疫细胞化学染色对阳性克隆的特异性进行鉴定。结果经3轮筛选，随机挑选48个噬菌体克隆进行DNA序列分析，展示AWYPLPP肽的噬菌体被高度富集77％（37／48）；噬菌体结合实验显示，与低转移潜能人肝癌细胞系MHCC97L、PLC／PRE／5和无转移潜能入肝癌细胞系Hep3B以及正常入肝细胞系CCLl3相比，AWYPLPP噬菌体特异性与高转移潜能人肝癌细胞系HCCLM3、HCCLM6、MHCC97H和MHCC97结合（P〈0．01）；抗噬菌体免疫细胞化学染色证实AWYPLPP噬菌体特异性靶向高转移潜能人肝癌细胞系HCCLM3、HCCLM6、MHCC97H和MHCC97。结论从噬菌体肽库中获得与高转移潜能人肝癌细胞特异性结合肽，可作为肝癌转移复发靶向治疗研究的载体。     

人结肠癌血管特异结合短肽的噬菌体肽库筛选及鉴定

目的 建立小鼠结肠癌模型,筛选并鉴定能够与人结肠癌血管内皮细胞特异结合的噬菌体呈现短肽.方法 建立小鼠荷瘤模型.鉴定阳性噬菌体的体内归巢能力及内皮细胞结合能力.人工合成噬菌体呈现短肽,通过竞争结合实验观察短肽对其呈现噬菌体的竞争结合抑制作用.并检测短肽与共培养内皮细胞及结肠癌血管的特异性结合能力.结果 鉴定出两种外源肽噬菌体,称为pCV1及pCV2.体内实验显示,pCV1、pCV2均能特异性归巢于人结肠癌移植瘤组织,滴度比值pCV1/pCont、pCV2/pCont分别为15.9、20.1倍,明显高于对照组织.体外细胞酶联免疫吸附试验(ELISA)结果显示pCV2在Co-HUVECs上的结合显著高于在HUVECs上的结合,其比值达2.61.人工合成短肽CV2能特异性竞争抑制pCV2向结肠癌移植瘤内的归巢及与Co-HUVECs的特异性结合.免疫荧光染色结果显示,FITC-CV2特异性结合于Co-HUVECs的胞膜与核周胞质,以及结肠癌移植瘤的血管组织.结论 得到两个能特异性结合于结肠癌移植瘤的噬菌体单克隆pCV1及pCV2.pCV2能够特异性结合于Co-HUVECs,pCV2所呈现的环状九肽CV2介导了它与Co-HUVECs的特异结合.短肽CV2具有与Co-HUVECs及结肠癌移植瘤血管特异性结合的能力,有可能用于结肠癌的血管靶向治疗.

Abstract：

Objective To select, identify and analyze a peptide binding specifically to blood vessels of human colon cancer by phage displayed peptide library in vivo. Methods Animal models were established using sub-renal capsular assay (SRCA) in immunosuppressive mice implanted with human colon cancer xenografts. The phage displayed peptide library was injected intravenously into mice. After 4 rounds of selection, 20 clones were picked up randomly and sequenced individually. The homing ability to human colon cancer xenografts and co-cultured human umbilical vein endothelial cells ( Co-HUVECs with human colon cancer Lovo cells) of the positive phage clones were determined by in vivo binding assay and in vitro cell enzyme linked immunosorbent assay ( ELISA ). The binding ability to Co-HUVECs of peptide-displayed phage clone was identified by immunocytochemical stain. Peptide displayed on the phage was synthesized and competitive binding assays were performed to observe the competitive inhibition effect of the peptide with their phage counterpart. Immunofluorescence microscopy was used to study the binding of synthesized peptides to Co-HUVECs and vascular vessels in colon cancer. Results Two peptide sequences were obtained finally and named CV1 and CV2. In vivo binding assay showed that the homing ability to human colon cancer xenografts of peptides of CV1 or CV2 was higher than that of control organs. In vitro cell ELISA suggested that CV2 phage preferably binded to Co-HUVECs rather than control HUVECs. Then CV2 phage clone was identified further. Immunocytochemical staining revealed that CV2 phage preferably binded to Co-HUVECs rather than the control. Competition binding assays demonstrated a significant competition between the synthesized peptide CV2 and the phage displaying CV2 while binding to Co-HUVECs or human colon cancer xenografts. Under the immunofluorescence microscopy, fluorescence-labeled CV2 peptide was seen on the membrane and in the perinuclear cytoplasm of Co-HUVECs, and bound to colon cancer xenografts rather than control organs. Conclusion Two phage clones displaying CV1 and CV2 peptide could target to human colon cancer xenografts. The peptide CV2 and its displayed phage were identified binding preferably with Co-HUVECs. And the cyclic nonapeptide (CV2) was binding site of the CV2 phage with Co-HUVECs. Synthesized nonapeptide CV2 had specificity to Co-HUVECs and colon cancer vascular endothelial cells. The peptide CV2 could be used in target therapy of tumor angiogenesis.     

噬菌体展示的肽库中N-甲基-D-天门冬氨酸受体2B亚基模拟抗原表位的筛选

目的 从噬菌体展示的随机十二肽库中筛选N-甲基-D-天门冬氨酸受体（NMDAR）2B亚基模拟抗原表位。方法以NMDAR2B单克隆抗体为配基，免疫亲和筛选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机十二肽。经过3轮筛选，从第3轮洗脱物中随机挑选12个单克隆噬菌体扩增后进行ELISA鉴定，用酶标仪测定450nm处的吸光值（A450）。对这12个单克隆噬菌体分别进行扩增、纯化，并对DNA测序，以确定插入十二肽的氨基酸序列。通过细胞竞争ELISA法分析阳性单克隆噬菌体对NMDAR2B天然抗原表位与其特异性抗体结合的竞争性抑制。结果经过3轮筛选后能与NMDAR2B单克隆抗体特异性结合的噬菌体得到了有效富积，12个单克隆噬菌体中有9个单克隆噬菌体的A450高于其它3个。DNA测序结果表明这9个单克隆噬菌体表达了一个共同氨基酸序列：SHPPVMPWPTST，将其命名为阳性克隆噬菌体，该阳性克隆噬菌体可以竞争性抑制细胞表面天然抗原与特异性抗体的结合，抑制率（45土3）％，具有抗原模拟性。结论从噬菌体展示的随机十二肽库中成功筛选到了能与NMDAR2B特异性抗体结合的短肽，该肽模拟了天然抗原的某个表位。     

应用噬菌体随机12肽库筛选TβR Ⅱ特异性序列

目的从噬菌体随机12肽库中筛选TGF-β1Ⅱ型受体(TGF-beta receptorⅡ,TβRⅡ)的特异性序列,评估其对瘢痕疙瘩成纤维细胞的作用。方法以人TGF-β1单克隆抗体为靶,在噬菌体随机12肽库中进行4轮生物筛选;应用ELISA法挑选结合活性较好的单克隆噬菌体,进行DNA序列分析;MTT法评估TGF-β1单克隆噬菌体对瘢痕疙瘩成纤维细胞的生物学效应。结果测序共获得5种类似于TβRⅡ的特异性序列。MTT结果显示其中有3种噬菌体模拟肽能够抑制瘢痕疙瘩成纤维细胞增殖。结论具有TβRⅡ特异性序列的噬菌体模拟肽能够抑制瘢痕疙瘩成纤维细胞增殖。     

噬菌体随机十二肽库淘选及鉴定胃癌特异性结合肽

目的利用噬菌体展示随机十二肽库淘选出与胃癌BGC823细胞特异性结合的多肽,为寻找胃癌早期诊断和治疗的标志物打下基础。方法以胃癌细胞BGC823为靶细胞,胃黏膜永生化上皮细胞GES为吸附细胞,在常温下对噬菌体随机十二肽库进行三轮消减淘选,挑取单克隆、扩增纯化噬菌体,并经ELISA和免疫组化法初步鉴定噬菌体克隆亲和力;测序阳性克隆,合成荧光素FITC标记多肽,鉴定其与胃癌细胞和胃癌组织的亲和力及特异性。结果三轮淘选后,经ELISA法初步鉴定,随机挑选的20个单克隆中11号(GC-11)多肽对BGC823细胞亲和力最高。细胞、组织免疫荧光实验进一步证明,荧光素标记的多肽FITC-GC-11对BGC823细胞和胃癌组织具有高亲和力。结论利用噬菌体随机十二肽库成功淘选出与胃癌细胞BGC823和胃癌组织具有较高亲和力的多肽GC-11。     

利用噬菌体展示技术制备人血管生成素2单链抗体

目的 用纯化的人血管生成素2(Ang2)蛋白,通过噬菌体展示技术从非免疫小鼠噬菌体展示抗体库中淘选、鉴定Ang2单链抗体.方法 以纯化的人Ang2蛋白为抗原,对非免疫小鼠噬菌体展示抗体库进行富集和筛选,获得Ang2单链抗体,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot法检测目的蛋白的表达并测序.结果 经过3轮富集和筛选,获得了1个能特异性识别Ang2蛋白的阳性噬菌体克隆,DNA测序表明其含有完整的单链抗体基因片段,大小约750 bp,表达蛋白相对分子质量约33.7×103;通过SDS-PAGE进一步实现Ang2单链抗体(phage-Ang2-scFv)的鉴定.结论 成功分离并鉴定人Ang2-scFv,为进一步的功能学研究奠定了基础.     

肝癌特异性噬菌体多肽的筛选和初步鉴定

目的 利用噬菌体展示肽库筛选与肝癌HepG2细胞特异性结合的多肽,为筛选及明确新的肝癌早期诊断和治疗标志物打下基础.方法 以肝癌细胞HepG2为靶细胞,LO-2为吸附细胞,在37℃条件下对噬菌体随机12肽库进行多轮减性筛选,挑取单克隆扩增并鉴定.利用ELISA初步鉴定克隆亲和力,测定阳性克隆DNA测序并进行同源性及氨基酸分析.结果 经过3轮减性筛选发现,随机挑选的30个单克隆中,其中ZS-9对HepG2具有较高亲和力,氨基酸测序结果表明,该序列与美国国立生物技术信息中心(NCBI)GenBankDNA序列数据库和Swiss-Prot蛋白数据库中的已知基因和蛋白无同源性,而且,国内外文献均未见报道,表明笔者筛选到一新的肝癌相关抗原的配体.结论 利用噬菌体随机12肽库成功筛选到与肝癌细胞HepG2具有较高亲和力的多肽,为筛选鉴定新的肝癌特异的标志物奠定工作基础,也为肝癌的早期诊断和靶向治疗进一步研发确定了靶标.     

肾癌特异性配体多肽ZT-2筛选及新标志物的研究

目的 寻找肾癌早期诊断和靶向治疗的标志物. 方法 以肾癌细胞株A498为靶细胞,正常肾细胞株HK-2为吸附细胞,37℃下对噬菌体十二肽库进行4轮减性筛选.挑取单克隆采用ELISA方法 初步鉴定噬菌体克隆亲和力;阳性克隆提取质粒,测序后合成多肽,鉴定多肽的亲和力及特异性.结果 经ELISA初步鉴定,随机挑选20个单克隆中,2号多肽对A498细胞亲和力最高(A_(ZT-2)/A_(对照)比值为3.15),命名为Phage-ZT-2.细胞免疫荧光试验证明,合成的相应多肽FITC-ZT-2对A498细胞具有特异性结合.利用荧光标记的ZT-2检测肾癌组织芯片,肾癌组织ZT-2的荧光吸光度(A)值为0.453±0.123,正常组织及非肾癌炎性组织为0.148±0.075,2组间比较差异有统计学意义(t=2.776,P<0.01).结论 成功筛选出与肾癌细胞特异性结合的多肽ZT-2,为肾癌的早期诊断和靶向治疗提供了实验依据.     

结肠癌中hTERT的表达及其与Survivin和bcl-2表达的关系

目的：研究端粒酶逆转录酶（hTERT）在结肠癌中的表达及其与Survivin和bcl-2表达的关系。方法：应用免疫组织化学SABC法检测52例结肠癌及其相应癌旁正常组织中hTERT、Survivin和bcl-2的表达。结果：52例结肠癌组织中hTERT表达阳性率为80.8%,而相应癌旁正常结肠组织中无一例阳性表达。hTERT表达的上调与患者性别、年龄、肿瘤浸润深度无明显相关（P〉0.05）,而与肿瘤分化程度、淋巴结转移、Dukes分期明显相关（P〈0.05）。在52例结肠癌组织中hTERT与Survivin以及hTERT与bcl-2的表达之间呈正相关（r=0.589,P〈0.05;r=0.559,P〈0.05）。结论：hTERT表达的上调与结肠癌的发生、发展、恶性程度有着密切的关系;结肠癌中hTERT的表达与Survivin和bcl-2的表达密切相关。     

Survivin基因在胆管癌组织中的表达及其与bcl-2和bax相关性

目的 探讨Survivin基因在胆管癌组织中的表达以及与bel-2和bax基因表达的相关性.方法 应用逆转录-聚合酶链反应(RT-PCR)法检测Survivin mRNA在64例胆管癌,22例癌旁组织及10例正常胆管组织中的表达,免疫组织化学方法测定胆管癌组织中bcl-2和bax蛋白的表达.结果 胆管癌组织中Survivin基因阳性表达率65.6%(42/64),显著高于其在癌旁组织(9.1%,P<0.01)和正常胆管(0%,P<0.01)中的表达;Survivin基因表达与胆管癌的性别、年龄、部位、分化程度、临床分期及淋巴结转移无明显相关(P>0.05).bcl-2和bax在胆管癌组织中的表达率分别为56.3%(36/64)N45.3%(29/64),Survivin基因表达与bcl-2表达正相关(r=0.404,P<0.01),与bax表达负相关(r=-0.373,P<0.01).结论 Survivin基因在胆管癌的发生、发展过程中可能起重要作用;Survivin有可能成为胆管癌基因治疗的新靶点;在胆管癌的发展中,Survivin与bel-2和bax分别起协同和拮抗作用.     

Her2 amplification is significantly more frequent in lymph node metastases from urothelial bladder cancer than in the primary tumours

Background

Her2, an alias for the protein of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian), might be an attractive therapeutic target in metastasising bladder cancer. Genotype and phenotype of primary tumours and their metastases may differ.

Objectives

Determine Her2 status in both tumour components to better assess the potential of anti-Her2 therapies.

Design, setting, and participants

Histologic examination revealed lymph node metastases in 150 patients with urothelial bladder cancer clinically staged as N0M0. A tissue microarray was constructed with four tumour samples per patient: two from the primary tumour and two from nodal metastases. Her2 status was determined at the gene level by fluorescence in situ hybridisation (FISH) and at the protein level by immunohistochemistry (IHC).

Interventions

All patients underwent cystectomy and standardised extended lymphadenectomy.

Measurements

Overall survival was assessed according to HER2 gene status and protein expression in primary bladder cancers and lymph node metastases.

Results and limitations

Her2 amplification was significantly more frequent in lymph node metastases (15.3%) than in matched primary bladder cancers (8.7%; p = 0.003). Her2 amplification in primary tumours was highly preserved in the corresponding metastases as indicated by only one amplified primary tumour without amplification of the metastasis. There was a high concordance in HER2 FISH results between both samples from the primary tumour (κ = 0.853) and from the metastases (κ = 0.930). IHC results were less concordant (κ = 0.539 and 0.830). FISH and IHC results were poorly correlated in primary tumours (κ = 0.566) and metastases (κ = 0.673). While Her2 amplification in the primary tumour significantly predicted poor outcome (p = 0.044), IHC-based survival prediction was unsuccessful.

Conclusions

Her2 amplification in metastasising bladder cancer is relatively frequent, is homogeneous in each tumour component, and predicts early death. This suggests a high potential for anti-Her2 therapies. For patient selection, FISH might be more accurate than IHC.     

Survivin、Bcl-2在壶腹癌组织中的表达及临床意义

目的 探讨Survivin和Bcl-2在壶腹癌组织中的表达及其临床意义.方法 用免疫组化EnVision法对40例壶腹癌组织进行Survivin和Bcl-2的检测,并对8例正常壶腹组织进行对照研究.结果 Survivin和Bcl-2在壶腹癌组织中的阳性表达率均显著高于正常壶腹组织(82.5％,72.5％ vs 0,12.5％),差异有统计学意义(P＜0.05).壶腹癌组织中Survivin和Bcl-2表达与患者性别、年龄、肿瘤大小、组织学类型、临床分期差异无统计学意义(P＞0.05);Survivin和Bcl-2表达均与十二指肠浸润、胰腺浸润、淋巴结转移差异有统计学意义(P＜ 0.05);经Spearman等级相关性分析表明,Survivin与Bcl-2表达呈正相关(r=0.647,P＜0.05).结论 Survivin和Bcl-2的高表达可能共同参与壶腹癌的发生、发展,联合检测Survivin和Bcl-2可能有助于判断壶腹癌的恶性程度及进展情况.     

Survivin and COX-2 expression in male breast carcinoma

Male breast carcinoma (Male BC) is a distinct biological subtype of breast cancer. We previously reported an association between 5-year overall survival and strong intratumoral aromatase (ITA) expression in Male BC. In this study, we investigate survivin and COX-2 expression and their relationship to ITA. Clinical data were abstracted for all Male BC patients diagnosed between 1985 and 2005 in Nova Scotia, Canada. Archival tissues were retrieved for tissue microarray (TMA) and immunohistochemistry (IHC). Of 54 Male BCs identified, 39 cases had tissue available for IHC. Survivin expression was noted in 27 cases (69%); 12 nuclear and 26 cytoplasmic while strong COX-2 expression was observed in 14 cases (36%). Cytoplasmic survivin and COX-2 expressions were positively associated with ITA expression. However, survivin and COX-2 expression were not predictive of survival. Additional studies are needed to further elucidate the correlation between cytoplasmic survivin, COX-2 and ITA expression in Male BC considering their potential therapeutic implications.