EST在克隆新基因中的应用

随着基因定位 (连锁图谱、物理图谱、转录图谱 )和DNA测序及生物信息技术的迅猛发展 ,EST已成为人类寻找新的未知基因以及克隆不同时空差异表达基因和疾病相关基因的重要标志物。随着EST数据库的进一步完善 ,网上克隆和定位候选克隆策略将成为克隆新基因的主要方法 ,并在虚拟的网上空间将模型生物基因组的研究成果成功地应用于人类基因组的研究中     

肝癌组织差异表达基因cDNA序列的筛选与鉴定

目的：筛选并鉴定肝癌组织特异表达基因。方法：通过菌落原位杂交技术筛选用抑制消减杂交法构建肝癌与癌旁肝组织差异表达基因消减cDNA文库，用PCR方法进一步筛选出有插入片段的阳性克隆，将阳性克隆进行DNA测序和同源性比较分析，用Northern印迹方法对新的cDNA序列进行初步鉴定。结果：从消减文库中随机挑取的100个白色克隆中筛选出13个阳性克隆，DNA测序获得11个不同的cDNA序列；同源性比较分析表明，6个cDNA片段与在基因高度同源，5个cDNA片段为新的序列。其长度大于300bp的3个新序列，Norther印迹证实它都来源于肝癌组织。结论：用抑制消减杂交方法构建的肝癌差异表达基因消减cDNA文库富含肝癌特异表达基因，经验证的3个新的cDNA序列可能为肝癌特异的基因序列。     

癌症高表达蛋白在肝细胞肝癌中的表达及意义

目的:研究癌症高表达蛋白(Hec1)在原发性肝细胞肝癌(HCC)中的表达和意义。方法:采用免疫组化SP法检测27例正常肝组织、30例癌旁组织及62例HCC组织中Hec1的表达;用Westernblot检测上述组织中Hec1蛋白的表达。结果:在正常肝、癌旁组织及HCC组织中,Hec1蛋白的阳性表达率分别为0%、20.8%、62.9%,各组织中Hec1蛋白的表达差异均有统计学意义(P<0.05)。Hec1蛋白的表达与HCC的Edmondson分级、侵袭转移显著相关。Westernblot结果表明,在正常肝、癌旁组织及HCC组织中,Hec1蛋白的平均表达量的分别为0、0.12±0.03、0.89±0.16,差异有统计学意义(P<0.05)。结论:Hec1蛋白的过度表达可能在HCC的发生和发展中起重要作用。     

应用EST和电子克隆策略研究血吸虫表达基因谱

目的开展血吸虫表达基因谱的研究,寻找新的疫苗候选分子和药物靶标。方法应用表达序列标签（EST）和电子克隆策略。结果获得了552个EST序列和487个电子延伸序列,其中104个EST序列在延伸前未表现同源性,而延伸后表现出有意义的同源性;获得了日本血吸虫基因表达谱的信息以及发现了有潜在药物和疫苗价值的新基因序列。结论本研究为日本血吸虫基因表达谱的研究提供更有效的研究思路。     

人肝癌和癌旁组织c－myc基因扩增、转录和表达的研究

应用１４例配对的肝切除标本，系统性地探讨了肝癌和癌旁组织ｃ－ｍｙｃ基因扩增、ｍＲＮＡ转录和ｃ－ｍｙｃ基因产物表达水平的变化，肝癌和癌旁组织未见ｃ－ｍｙｃ基因扩增。原位杂交检测可见１１例癌组织，１０例癌旁组织ｍＲＮＡ转录水平增加，Ｐ６２ｍｙｃ免疫组化显示，染色阳性率分别为癌组织８５．７％，癌旁组织９２．９％。结果表明，人肝癌和癌旁组织存在着转录和基因产物水平高频率的超表达。统计学分析表明两者之间有高度的一致性，结论是ｃ－ｍｙｃ基因超表达与基因扩增无关，不伴有扩增的超表达是肝癌在分子水平呈现的一个重要征象。     

抗人肝癌单克隆抗体HAb18 Fab基因的克隆和表达

目的：克隆抗人肝癌单抗(mAb)HAb18的Fab基因，并在大肠杆菌中表达。方法：采用RT-PCR法，利用设计的引物扩增mAb Fd和κ链全长基因，并分别克隆到T载体中进行序列分析。然后，亚克隆到原核表达载体pComb3中，转化感受态大肠杆菌后以IPTG诱导表达。采用ELISA和免疫荧光法检测表达产物的特异性。结果：克隆了mAb HAb18的Fab基因，并在大肠杆菌中获得表达。竞争性ELISA和免疫荧光检测证实，表达产物具有与相应抗原特异结合的活性。结论：成功地构建了抗人肝癌小分子Fab抗体，为其进一步在肝癌诊断与治疗中的应用奠定了基础。     

肝癌细胞凋亡相关基因APG的克隆、表达及其与肝癌相关性研究

目的 研究和克隆新的肝癌凋亡相关基因,探索肝癌发生机制。方法 采用同源筛选、RT-PCR等克隆肝癌凋亡相关基因APG,分析其在肝癌及癌旁组织中表达,测序比对,研究其与肝癌之间相关性。结果 克隆了一个肝癌凋亡相关基因,cDNA全长为563bp。其在肝癌及癌旁组织中序列存在一定差异。50例肝癌中,16%(8/50)为上调表达,84%(42/50)为下调表达(P<0.01)。AGP表达与性别、AFP大小无关(P>0.05),但与肿瘤大小(P<0.05)、HBsAg(P<0.01)、分化程度(P<0.01)、包膜侵犯(P<0.01)、临床分期(P<0.01)、血管癌栓(P<0.01)、癌旁卫星灶(P<0.01)、Ki-67蛋白表达(P<0.01)、细胞凋亡(P<0.05)、P53蛋白表达(P<0.01)密切相关。结论 APG是肝癌凋亡相关的下调基因,其表达与肝癌的某些临床病理特征有关。     

肝细胞肝癌中CENPF的表达及临床意义

目的 探讨CENPF在肝细胞肝癌(hepatocellular carci-noma,HCC)中的表达及临床意义.方法 基于GEO数据库、TCGA、Oncomine平台及临床HCC样本探索CENPF基因在HCC中的表达情况,运用GEPIA平台绘制患者生存曲线;基于TCGA数据库,采用GSEA软件分析CENPF表达相关的...     

利用外显子捕获技术从基因组片段中分离表达序列

本实验旨在以外显子捕获（ｅｘｏｎ－ｔｒａｐｐｉｎｇ）技术从人类５号染色体短臂（５ｐ）＂猫叫综合征＂区域分离表达序列。将来自５ｐ的基因组片段克隆进ｐＳＰＬ３剪接载体，用以转染ＣＯＳ－７细胞。经ＲＴ－ＰＣＲ扩增后，以ＵＤＧ法克隆剪接产物，并进行基因组来源、组织表达特异性及序列分析等鉴定。结果表明，基因组片段在实验系统中能被有效剪接，并得到了确为５ｐ来源的表达序列。证明外显子捕获技术能有效用于表达序列的筛选，但现有的剪接载体仍有较为严重的隐含剪接问题。     

肝细胞性肝癌中MAZ基因的表达及意义

目的探讨MAZ基因在肝细胞性肝癌(hepatocellular carcinoma,HCC)及其癌旁正常组织中的表达,分析其与HCC各临床病理特征及预后的关系。方法采用组织芯片技术和免疫组化法检测75例HCC及其对应癌旁正常组织中MAZ基因的表达,分析其与HCC临床病理特征及与患者预后的关系。结果 MAZ在HCC中的表达(48%,36/75)明显高于其癌旁正常组织(22.67%,17/75),组间比较差异有统计学意义(P0.05)。MAZ表达与HCC患者性别、年龄、病理分级、临床分期、TNM分期、是否合并肝硬化、是否合并乙肝病毒感染、是否有肝癌家族史以及血清AFP、CEA、γ-GT、ALT、AST和ALB水平、是否有淋巴结转移等均无明显相关性,而与肿瘤直径、吸烟与否、饮酒与否显著相关(P0.05)。HCC中MAZ阳性组和阴性组的累计生存率差异有显著性(P0.05),MAZ阳性组患者的术后无瘤生存时间明显低于阴性组,提示MAZ表达上调可能导致患者的预后更差。结论 MAZ基因表达上调可能与HCC的发生、发展密切相关。     

两株新的抗肝细胞肝癌单克隆抗体的制备及特异性鉴定

以外科手术切除的肝癌标本制备的细胞悬液和新建株的肝癌细胞为免疫原。籍多重免疫途经致敢ＢＡＬＢ／ｃ小鼠，经２０次融合，从近１０，０００个融合孔中，筛选出两株（ＨＡｂ２５，ＨＡｂ２７）肝癌单克隆抗体，在９４例肝癌、１４１例其他肝病组织，２６种正常组织，１２９例各组织系统的肝外恶性肿瘤组织上做了交叉反应。除ＨＡｂ２７与毛细胆管有弱的反应外，两株抗体与肝癌组织结合的阳性率高（８３．１５％，８５．１１％），仅与少数消化道肿瘤有交叉．１３１Ⅰ标记抗体进行放射免疫显像，最佳显像时间为７２ｈ，瘤／肝、瘤／血比值分别为５．０７～６．８４、１．２６～２．１３，可望为肝癌导向药物研究，提供新的载体。     

基质金属蛋白酶及其基因多态性与肝癌的发生发展

肝癌是威胁人类健康的主要疾病。基质金属蛋白酶(MMPs)是一类Zn2 依赖的蛋白水解酶,能参与细胞外基质的代谢。已有的研究表明,MMPs在肝癌的生长、血管形成、侵袭和转移等多个阶段中发挥重要作用,由此,MMPs成为极佳的肝癌候选易感基因,其基因多态性可能调节肝癌的易感性。然而,目前针对肝癌的遗传易感研究还不多见。随着人类基因组计划和单倍型图谱计划的完成,进行肝癌易感基因的鉴定对于指导肝癌的早期预防和治疗具有重要意义。     

原发性肝癌合并糖尿病的介入治疗

目的研究原发性肝癌合并糖尿病的介入治疗方法。方法临床明确诊断的42例肝癌合并糖尿病患者在介入治疗前后通过口服降糖药或注射胰岛素将血糖、尿糖控制在安全范围内,并对隐性感染或感染源进行治疗,然后再行肝动脉化疗灌注栓塞术。结果42例患者介入治疗前血糖均降到9.0mmol/L以下,尿糖控制在-～ ,术后均未发生严重并发症,仅有2例出现慢性胆囊炎急性发作,经抗炎、利胆治疗后安全出院。结论原发性肝癌合并糖尿病患者在血糖、尿糖降到一定的范围内,消除潜在的感染,行肝动脉化疗灌注栓塞治疗是安全可行的。     

Challenge and Hope in Radiotherapy of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most critical global health issues. With frequent association of viral liver disease, HCC is highly complex, harboring both cancer and chronic liver disease. The tumor stage and underlying liver function are both major determinants of the treatment selection as well as prognosis in HCC patients, thus allowing no more than a 20% chance for potentially curative therapies. Radiotherapy technology has been evolved remarkably during the past decade, and radiation can be precisely delivered, thereby permitting higher doses to the tumour and reduced doses to surrounding normal tissues. There has been increasing interest in the merits of radiotherapy in HCC over the past few years, as indicated by a Pub Med search. Radiotherapy has been used as the definitive therapy with curative intent in early stage tumours. It has been used also in combination with TACE for intermediate stage tumours. In locally advanced tumours, radiotherapy has been combined with systemic agents. Despite its efficacy, radiotherapy has not yet been incorporated into the standard management guidelines of HCC. The lack of high evidence level data, especially randomized controlled trials, has posed an obstacle in including radiotherapy into the routine treatment schema of HCC. Therefore, well-designed prospective studies are strongly recommended using developing technology for radiotherapy alone or combination therapies. Also, many issues such as the optimal dose-fractionation, intra- or extrahepatic metastasis after radiotherapy, and radiation-induced hepatic dysfunction remain to be solved. In this review, current status of radiotherapy for HCC will be discussed with regard to technical consideration and combination strategy. The limitation and future perspectives will also be discussed.     

AFP增强子驱动的HSV-TK/GCV自杀基因系统对肝癌细胞杀伤的实验研究

目的探讨人AFP增强子驱动的单纯疱疹病毒胸苷激酶/丙氧鸟苷（HSV-TK/GCV）自杀基因系统体外靶向杀伤肝癌细胞效应。方法构建人AFP增强子驱动的pAFP-CDNA3.1-TK自杀基因真核表达质粒,脂质体转染肝癌细胞,检测TK mRNA和蛋白表达,MTT法检测GCV对肝癌细胞的杀伤作用。结果成功构建pAFP-CDNA3.1-TK自杀基因真核表达质粒,在AFP阳性HepG2细胞中检测到TK mRNA和蛋白表达,添加GCV可特异性地杀伤HepG2细胞,而AFP阴性的SMMC7721细胞生长不受影响。结论 AFP增强子驱动的TK/GCV自杀基因系统可以靶向杀伤AFP阳性肝癌细胞。     

Vascular Changes in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most vascular solid tumors, in which angiogenesis plays an important role. The status of angiogenesis in HCC correlates with the disease progression and prognosis, and thus provides a potential therapeutic target. This review summarizes the vascular changes and molecular and cellular basis of angiogenesis in HCC. Development of HCC is characterized by arterialization of its blood supply and sinusoidal capillarization. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays a critical role in mediating angiogenesis in HCC. The VEGF can function on various types of cells, such as endothelial cells, hepatic stellate cells, endothelial progenitor cells and hemangiocytes, to induce vascular changes in HCC. Therefore, blockade of VEGF‐mediated pathways, either by anti‐VEGF neutralizing antibody or tyrosine kinase inhibitors that target VEGF receptors, suppresses carcinogenesis and angiogenesis in HCC. In addition to VEGF, several other angiogenic factors in HCC have recently been identified. These factors can also regulate angiogenic processes through interaction with VEGF or VEGF‐independent pathways. Despite the fact that treatment of HCC remains a tough task due to lack of effective systemic therapy, antiangiogenic therapy has already entered clinical trials in HCC patients and sheds light on a promising novel treatment for this disease. Anat Rec, 291:721–734, 2008. © 2008 Wiley‐Liss, Inc.     

IL12 Gene Polymorphism in Association with Hepatocellular Carcinoma in HCV-infected Egyptian Patients

Background: Hepatocellular carcinoma has been recorded the commonest cancer in Egypt. This increasing incidence may be attributed to the high prevalence of hepatitis C virus with its complications, so this study aimed to investigate the association between the potentially functional polymorphisms of IL12A and IL12B genes as a risk factor of development of hepatocellular carcinoma (HCC) on top of hepatitis C virus (HCV) infection in an Egyptian population.

Materials and methods: We genotyped two loci of IL12 which were rs568408 (3’UTR G>A) for IL12A and rs3212227 (3’UTR A>C) for IL12B in 78 patients with HCC on top of chronic HCV infection. In addition, 64 cancer-free chronic HCV patients were studied, besides 92 healthy subjects who were included as control.

Results: Study of rs568408 (G>A) gene polymorphism showed that the A allele is higher while the G allele is lower in HCC cases than cancer-free chronic HCV patients (p = 0.006*). The A-containing genotypes AG and (AG+AA) were higher while the GG was lower (p = 0.009* and p = 0.005*), respectively. The study of the rs3212227 (A>C) polymorphism showed neither statistically significant differences between the C and A allele (p = 0.2) nor between CC, AC, or AC+CC in HCC cases and cancer-free chronic HCV patients (p = 0.7, p = 0.2, and p = 0.29), respectively.

Conclusion: Our findings showed that IL12A rs568408 (G>A) polymorphism may contribute to the risk of HCC on top of chronic HCV infection, whereas that of IL12B rs3212227 (A>C) do not.     

Induced Differentiation of Hepatocellular Carcinoma by Natural Products

Hepatocellular carcinoma is one of the most common malignant tumors worldwide. For the difficulty of the giving sufficient dose because of the poor liver function and the low sensitivity of hepatoma cells for the chemotherapeutic agents, chemotherapy adds little to overall survival of hepatocellular carcinoma patients. The induction of terminal differentiation in tumor cells represents a possible therapeutic strategy with less toxicity. Gekko sulfated polysaccharides, isoverbascoside, Ginsenoside-Rh2, Camptothecin, 9-nitro-camptothecin, tachyplesin, Matrine, tylophorine, 7-OH-4-CH (3)- coumarin and arsenic trioxide are known to have a differentiation-inducing capability on hepatocellular carcinoma in vitro and/or in vivo. Although the therapeutic effect of the differentiation-inducing agents may not be potent when compared with that of conventional chemotherapeutic agents, they have multiple therapeutic targets, low toxicity and less probability of drug resistance. More data are required on the molecular mechanisms of therapeutic effects, dose response and potential toxicities.     

实时荧光定量RT-PCR检测肝细胞癌中删基因的表达

目的探讨PTCH基因在肝细胞癌组织中的表达及其与肝癌病理分级的关系。方法提取手术切除18例不同病理分级的人肝癌、癌旁组织和6例胆管结石手术切除的正常肝组织中的总RNA并逆转录为cDNA。应用实时荧光定量RT-PCR法观察人肝细胞癌组织与癌旁组织中PTCH基因的相对表达水平,以及观察人正常肝组织中PTCH基因的表达水平。结果PTCH在所测18例肝细胞癌组织与癌旁组织中均有表达,在6例正常肝组织中不表达，且在病理分级为Ⅰ和Ⅱ级的癌组织中PTCH的mRNA表达水平显著高于癌旁组织（P〈0．01），而在病例分级为Ⅲ级的癌组织中PTCH的mRNA表达水平显著低于癌旁组织（P=0．024）。结论随着肝癌恶性程度的增加PTCH的mRNA表达量呈递减趋势，提示PTCH可能参与了肝细胞的早期癌变过程。     

Tissue Levels of Stefin A and Stefin B in Hepatocellular Carcinoma

Stefins have been reported to be associated with the progression and metastasis of various malignant tumors. However, the expressions of stefins in hepatocellular carcinoma (HCC) have not been well‐defined. In this study, the protein levels of stefin A and stefin B were assessed by immunohistochemical staining, and the mRNA levels were quantified by real‐time polymerase chain reaction in 85 primary HCC tissues, 85 surrounding non‐cancerous tissues, and 9 normal hepatic tissues. The immunohistochemical staining of cathepsin B and cathepsin D, and the ratio of cathepsins to stefins were assessed. The mRNA expressions of stefin A and stefin B in HCC tissues were significantly higher than surrounding noncancerous tissues and normal hepatic tissues, respectively. A significant positive relationship of stefin A and stefin B was found with node metastasis, tumor size, and Edmondson grade for HCC. Univariate and multivariate analyses revealed that Edmondson grade and stefin B expression were independent factors associated with the risk of lymph node metastasis in HCC. The ratios of cathepsin B to stefin A, cathepsin D to stefin A, cathepsin B to stefin B and cathepsin D to stefin B of the HCC group were significantly higher than that of the surrounding noncancerous group. A significant positive correlation between the ratio of cathepsins to stefins (cathepsin B/stefin A, cathepsin B/stefin B and cathepsin D/stefin B) and node metastasis was demonstrated. We concluded that high expressions of stefin A and stefin B may be an important factor contributing to the development and metastasis of HCC. Anat Rec, 299:428–438, 2016. © 2016 Wiley Periodicals, Inc.