mdr-1基因表达的荧光定量RT-PCR法检测

目的：建立用实时荧光定量RT—PCR法在mRNA水平上检测肿瘤细胞中多药耐药基因（mdr-1基因）表达的方法。方法：利用RT—PCR方法从耐药肿瘤细胞MCF7／ADR中扩增出目的基因片段，用TA克隆的方法将基因片段插入到PGEM-T质粒载体中，在Line-gene荧光定量检测系统上建立检测mdr-1基因表达的标准曲线。结果：成功构建了mdr-1基因的质粒重组子，建立了在mRNA水平定量检测mdr-1基因的标准曲线，相关系数为0．997，可稳定检测出40μL体系中10^2拷贝数的模板。重复5次实验组内和组问变异系数均〈1．0％。结论：用基因克隆法可快速准确地构建mdr-1基因的标准曲线，定量检测mdr-1基因的表达，简单易行，重现性好。     

裸鼠转基因结肠癌肝转移模型的建立

目的 探讨裸鼠转基因结肠癌肝转移模型的建立方法。方法 应用体外转染法和体内转染法建立裸鼠转基因结肠癌肝转移的动物模型。观察肝脏转移率和自然生存期;瘤组织作病理切片;实时荧光定量（Real-Time RT-PCR）检测CD基因表达,Western blot检测目的基因蛋白表达。结果 体外转染法与体内转染法裸鼠肝脏转移率、自然生存期比较差异无统计学意义[100％(15/15),100％(15/15); (40.60±2.2297)d, (41.73±2.4919)d; P>0.05]。实时荧光定量PCR进行相对定量比较,体内转染法CD基因的表达活性低于体外转染法（P＜0.05,t=2.758）。体内转染法瘤组织CD基因蛋白表达低于体外转染法。结论 体外转染法建立裸鼠转基因结肠癌肝转移模型,目的基因在瘤组织中能更稳定、持续表达。     

mdr-1、c-erbB-2 和bcl-2 基因表达与乳腺癌耐药性的研究

 目的　探讨 mdr- 1、c- erb B- 2和 bcl- 2作为乳腺癌耐药相关基因常规检测的可行性 ,并观察三种基因与肿瘤发展预后的关系 ,以期指导治疗。方法　应用 S- P法免疫组化技术对 43例疗前乳腺癌组织标本的常规切片进行检测。结果　 (1 ) c- erb B- 2和 bcl- 2以中高度表达为主 ,mdr- 1则以低表达或无表达为主。 (2 )复发组与未复发组相比 ,mdr- 1表达有显著性差异 (P<0 .0 5 ) ,c-erb B- 2和 bcl- 2则无显著性差异 (P>0 .0 5 )。 (3)复发病例中 c- erb B- 2以高表达为主 ,bcl- 2均为低表达或无表达。结论　三种基因可作为乳腺癌耐药相关基因而用于临床检测并可为术后治疗及判断预后提供依据。     

食管癌、癌旁和淋巴结组织中mdr—1,mrp表达及其预后关系

目的探讨食管癌、癌旁及淋巴结组织中多药耐药基因(mdr-1)和多药耐药相关蛋白基因(mrp)表达的相互关系及其对患者预后的影响。方法采用逆转录-聚合酶链反应(RT-PCR)技术，检测了51例食管癌、癌旁及49枚手术切除淋巴结组织的mdr-1和mrp基因表达。结果食管癌组织中mdr-1、mrp和两基因共表达的阳性率与癌旁组织比较具有显著性差异(P＜0.01)；转移淋巴结组织(N1-2)高于非转移淋巴结(N0)(P＜0.01)；而癌组织mdr-1、mrp阴性的转移和非转移淋巴结组织未发现mdr-1和mp基因表达。随访发现，食管癌组织mdr-1和(或)mrp阳性患者的一、二、三年生存率(72.4％、42.1％和14.3％)低于mdr-1、mrp阴性者(88.9％，63.6％和60％)。结论检测癌组织及其淋巴结中的mdr-1、mrp基因表达可能对指导食管癌的术后化疗、判定预后具有重要意义。     

结肠癌转移相关基因1对结肠癌细胞肝转移的影响

目的:研究结肠癌转移相关基因1(metastasis-associated in colon cancer1,MACC1)的表达对结肠癌肝转移的影响。方法:将融合绿色荧光蛋白(green fluorescent proteins,GFP)的慢病毒表达载体分别感染具有不同转移潜能的人结肠癌细胞株SW1116和HCT116。给予BALB/c-nu/nu裸鼠脾脏注射感染后的结肠癌细胞,构建结肠癌肝转移模型。注射后35d,在荧光解剖显微镜下观察肝脏内结肠癌细胞的转移情况,计算肝转移率。应用蛋白质印迹法检测SW1116和HCT116细胞中MACC1蛋白的表达水平,评估MACC1的表达水平与肝转移率的关系。结果:SW1116细胞的肝转移率明显高于HCT116细胞。MACC1蛋白在HCT116细胞中呈低表达,而在SW1116细胞中呈高表达,差异有统计学意义(P<0.01)。结论:MACC1蛋白的表达水平可能与结肠癌的肝转移能力呈正相关。     

组织特异性CD/5-FC系统热化疗治疗裸鼠结肠癌肝转移瘤的安全性

目的: 探讨组织特异性胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)系统热化疗对裸鼠结肠癌肝转移模型治疗的安全性.方法:30只裸鼠经门静脉注射转染CD基因的人结肠癌LOVO细胞,建立结肠癌肝转移模型,随机分为对照组、热化疗组和化疗组,分别经腹腔注射生理盐水、43 ℃前药5-FC和室温前药5-FC[均为500 mg/(kg·d)]进行治疗.治疗21 d后处死裸鼠,取各组裸鼠肝脏转移瘤组织、正常肝组织及胃、肺、胰腺、小肠及大肠组织作病理检测;RT-PCR检测各组织的CD基因表达.结果:常规病理检测显示对照组肝转移瘤组织细胞生长活跃,热化疗组较化疗组肝转移瘤细胞生长受抑制更明显;3组裸鼠正常肝组织及胃、肺、胰腺、小肠和大肠组织均呈正常形态,无明显病理改变.RT-PCR检测显示,3组肝脏转移瘤组织CD基因表达稳定,均见154 bp条带;显示3组裸鼠正常肝组织及胃、肺、胰腺、小肠和大肠组织均无CD基因表达.结论:组织特异性CD/5-FC系统热化疗明显提高了CD基因表达的靶向性,减少了热化疗引起的正常组织损伤,该治疗系统有较好的安全性.     

mdr-1基因在淋巴瘤组织中的表达与临床意义

背景与目的:多药耐药是一种常见的化疗耐药现象。mdr-1是产生多药耐药的编码基因之一,其表达与复发淋巴瘤多药耐药性的相关性目前尚未明确。在对mdr-1表达水平与淋巴瘤疗效的相关性研究方面,未取得一致结论。本文旨在检测淋巴瘤组织P-gp及mdr-1mRNA表达水平,并初步观察淋巴瘤P-gp表达水平与近期化疗疗效的关系。方法:使用流式细胞术检测31例初治与复发淋巴瘤P-gp表达水平,观察其中17例淋巴瘤P-gp表达水平与近期化疗疗效的关系。同时使用荧光定量RT-PCR技术检测淋巴瘤mdr-1mRNA表达水平。结果:19%(4/21)初治淋巴瘤P-gp高表达;60%(6/10)复发淋巴瘤P-gp高表达。复发淋巴瘤P-gp高表达率高于初治淋巴瘤(P=0.012)。46%(6/13)P-gp低表达或不表达病例化疗后完全缓解;仅25%(1/4)P-gp高表达病例化疗后完全缓解。非P-gp高表达组化疗完全缓解率较P-gp高表达组增高21%(P=0.6)。荧光定量RT-PCR检测18例初治淋巴瘤mdr-1mRNA含量为4.00×102～1.32×104copies/μgRNA;10例复发淋巴瘤mdr-1mRNA含量为4×102～4×104copies/μgRNA。复发淋巴瘤的mdr-1mRNA含量高于初治淋巴瘤(P<0.05)。结论:复发淋巴瘤组织过度表达P-gp及mdr-1mRNA;本实验中淋巴瘤P-gp表达水平与近期化疗疗效的相关性尚不明显。     

arresten基因转染对裸鼠实验性结肠癌肝转移的抑制作用

背景与目的:肝转移是晚期结肠癌患者最常见的致死原因,也是最常见的内脏转移,高达50%以上的结肠癌患者都会出现肝转移.本研究探讨arresten基因转染对人结肠癌LoVo细胞形成的裸鼠实验性结肠癌肝转移的影响.方法:通过脂质体转染法将arresten基因导入LoVo细胞,RT-PCR、Western blot分别检测arresten在mRNA、蛋白水平的表达,四甲基噻唑蓝(MTT)比色法检测arresten对LoVo细胞增殖的影响;通过建立裸鼠实验性结肠癌肝转移模型了解arresten对肿瘤转移的抑制作用;FⅧRag多克隆抗体染色的免疫组化方法检测肿瘤组织的微血管密度(microvessel density,MVD).结果:RT-PCR、Western blot结果显示arresten基因成功导入LoVo细胞并有arresten蛋白的表达.MTT比色法显示不同浓度的arresten对LoVo细胞增殖的影响差异无统计学意义(P＞0.05).导入arresten基因的LoVo细胞转移率为(25.1±2.1)%,低于未导入arresten基因的LoVo细胞的(87.1±1.2)%和对照组的(87.1±1.5)%,其差异均有统计学意义(P值均＜0.05).pSecTag2-arresten组裸鼠形成的肿瘤结节数为4.5 0.5,低于另外两组的19.6±2.5和20.4±2.5,其差异均有统计学意义(P值均＜0.05).pSeeTag2-arresten组形成的肿瘤MVD为15.3±3.5,低于另外两组(分别为42.2±2.6、45.6 5.1),其差异均有统计学意义(P值均＜0.05).结论:arresten能抑制结肠癌肝转移,其作用机制可能与arresten抑制肿瘤血管生成有关.     

组织特异性CD/5-FC系统热化疗治疗裸鼠结肠癌肝转移瘤的安全性

目的:探讨组织特异性胞嘧啶脱氨酶/5氟胞嘧啶(CD/5FC)系统热化疗对裸鼠结肠癌肝转移模型治疗的安全性。方法： 30只裸鼠经门静脉注射转染CD基因的人结肠癌LOVO细胞,建立结肠癌肝转移模型,随机分为对照组、热化疗组和化疗组,分别经腹腔注射生理盐水、43 ℃前药5FC和室温前药5FC\[均为500 mg/（kg·d）\]进行治疗。治疗21 d后处死裸鼠,取各组裸鼠肝脏转移瘤组织、正常肝组织及胃、肺、胰腺、小肠及大肠组织作病理检测; RTPCR检测各组织的CD基因表达。结果：常规病理检测显示对照组肝转移瘤组织细胞生长活跃,热化疗组较化疗组肝转移瘤细胞生长受抑制更明显;3组裸鼠正常肝组织及胃、肺、胰腺、小肠和大肠组织均呈正常形态,无明显病理改变。RTPCR检测显示,3组肝脏转移瘤组织CD基因表达稳定,均见154 bp条带;显示3组裸鼠正常肝组织及胃、肺、胰腺、小肠和大肠组织均无CD基因表达。结论：组织特异性CD/5FC系统热化疗明显提高了CD基因表达的靶向性,减少了热化疗引起的正常组织损伤,该治疗系统有较好的安全性。     

组织特异性CD/5-FC系统热化疗对裸鼠结肠癌肝转移的疗效

目的:探讨组织特异性胞嘧啶脱氨酶/5-氟胞嘧啶(cytisine deaminase/5-fluorocytosine,CD/5-FC)系统热化疗对结肠癌肝转移裸鼠模型的治疗作用.方法:将含CEA启动子调控CD基因表达的逆转录病毒载体进行扩增、纯化、包装,并收集病毒上清.45只裸鼠经门静脉注射人结肠癌LoVo细胞,成瘤后2 d腹腔注射病毒上清(0.2 ml/次,每天1次,共5 d).随机分为对照组、常温化疗组和热化疗组,分别经腹腔注射生理盐水、室温前药5-FC和43℃前药5-FC[均为500 mg/(kg·d)]进行治疗.治疗21 d后处死裸鼠,观察肝脏转移率和转移结节数,RT-PCR检测CD基因在肿瘤组织的表达,光镜及电镜下观察肿瘤病理学的变化.结果:病毒滴度为5.6×106CFU/L.CD基因在移植瘤组织中有效表达.热化疗组的肝转移率与转移结节数均低于常温化疗组[13.3%vs 40.0%,(0.20±0.56)个vs(0.80±1.01)个;均P＜0.05].光镜下见对照组肝转移瘤组织细胞生长活跃,热化疗组较化疗组肝转移瘤细胞生长受抑制更明显.电镜下见化疗组、热化疗组肝转移瘤细胞有不同程度的凋亡改变.结论:组织特异性CD/5-FC系统热化疗对裸鼠结肠癌肝转移瘤有明显的抑制作用.     

Oncogénétique et psycho-oncologie, une collaboration recommandée...

Résumé: La possibilité depuis 1994, de connaître la probabilité individuelle de développer certains cancers a permis de proposer de nouvelles modalités de prévention, de traitements et contribué au développement actuel de loncogénétique. Une meilleure connaissance des répercussions psychologiques tant pour les patients que pour les apparentés est désormais possible et limplication des psycho-oncologues dans ce cadre de la réalisation des tests prédictifs, recommandée. La mission de «messager» qui incombe au «cas-index» doit faire lobjet dune attention particulière. La complexité de linformation et la dimension paradoxale que peut avoir parfois la communication à propos des choix, rend difficile lévaluation de la qualité du consentement. La situation particulièrement délicate dune aide à la décision à légard de la chirurgie prophylactique, exige une collaboration étroite des généticiens et des psycho-oncologues.Les soins de support en oncologie     

The role of papillomaviruses in human cancers

This review comprehensively evaluates the influence of gene-gene, gene-environment and multiple interactions on the risk of colorectal cancer (CRC). Methods of studying these interactions and their limitations have been discussed herein. There is a need to develop biomarkers of exposure and of risk that are sensitive, specific, present in the pathway of the disease, and that have been clinically tested for routine use. The influence of inherited variation (polymorphism) in several genes has been discussed in this review; however, due to study limitations and confounders, it is difficult to conclude which ones are associated with the highest risk (either individually or in combination with environmental factors) to CRC. The majority of the sporadic cancer is believed to be due to modification of mutation risk by other genetic and/or environmental factors. Micronutrient deficiency may explain the association between low consumption of fruit/vegetables and CRC in human studies. Mitochondrial modulation by dietary factors influences the balance between cell renewal and death critical in colon mucosal homeostasis. Both genetic and epigenetic interactions are intricately dependent on each other, and collectively influence the process of colorectal tumorigenesis. The genetic and environmental interactions present a good prospect and a challenge for prevention strategies for CRC because they support the view that this highly prevalent cancer is preventable.     

Antifungal combination therapy in localized candidosis

A mouse model of localized candidosis in air-filled subcutaneous cysts imitating thrush has been developed. We have now tested various antifungal combinations in this animal model. Flucytosine (5-FC) + amphotericin B (Amph B) showed the highest efficacy, a clear additive or even synergistic effect was seen. The combination of 5-FC + imidazole or triazole derivative was less efficacious, an additive effect was rare. The combination of 5-FC + Amph B was also tested against Candida albicans strains showing various degrees of 5-FC-resistance. A significant reduction in 5-FC-resistant mutants was seen after the treatment with the combination.     

Les génériques en cancérologie: à molécule identique, médicaments identiques? Les biosimilaires, des super-génériques?

Résumé: Les biosimilaires vont bientôt voir leur apparition en Europe. Comment un laboratoire peut-il aborder le développement de son dossier dAMM? Quelles sont les bases légales et les recommandations officielles? Comment la similarité et/ou le caractère générique peuvent-ils être démontrés? Les règles sont-elles identiques à celles des produits chimiques conventionnels pour lesquels, notamment en cancérologie, il existe des médicaments génériques? Comment faire pour que la sécurité et lefficacité des médicaments biosimilaires soient assurées pour les patients?     

Cancer immunotherapy

Unprecedented progress has seen made in the last decade in the field of cancer immunotherapy. The recent approval of nivolumab （Opdivo）, the first anti-programmed cell death-1 （PD-1） antibody, for metastatic melanoma in Japan, marked a milestone in the rapidly advancing field of cancer immunotherapy. Nivolumab together with ipilimumab （Yervoy）, the anti-cytotoxic T-lymphocyte-associated antigen-4 （CTLA-4） antibody, are the first 2 drugs in the class of ＂immune checkpoint inhibitors＂ that have delivered impressive responses in patients with metastatic melanoma and renal cell cancer （RCC） as well as a variety of solid tumors.     

Effect of tumor therapeutic irradiation on the mechanical properties of teeth tissue

Tumor irradiation of the head-neck area is accompanied by the development of a so-called radiation caries in the treated patients. In spite of conservative therapeutic measures, the process results in tooth destruction. The present study investigated the effects of irradiation on the demineralization and remineralization of the dental tissue. For this purpose, retained third molars were prepared and assigned either to a test group, which was exposed to fractional irradiation up to 60 Gy, or to a non-irradiated control group. Irradiated and non-irradiated teeth were then demineralized using acidic hydroxyl-cellulose gel; afterwards the teeth were remineralized using either Bifluorid12 or elmex gelee. The nanoindentation technique was used to measure the mechanical properties, hardness and elasticity, of the teeth in each of the conditions. The values were compared to the non-irradiated control group. Irradiation decreased dramatically the mechanical parameters of enamel and dentine. In nonirradiated teeth, demineralization had nearly the same effects of irradiation on the mechanical properties. In irradiated teeth, the effects of demineralization were negligible in comparison to non-irradiated teeth. Remineralization with Bifluorid12 or elmex gelee led to a partial improvement of the mechanical properties of the teeth. The enamel was more positively affected by remineralization than the dentine.     

Consultation multidisciplinaire pour les patients atteints d’une pathologie tumorale (carcinome infiltrant ou lésion précancéreuse) liée à HPV : la consultation multidisciplinaire papillomavirus

Given the recent increase in the number of human papillomavirus (HPV)-induced cancers in other locations than gynaecological, the number of patients with two cancers at distinct sites, and because of the lack of exhaustive data, we decided to create a multidisciplinary network around an HPV consultation at the Georges-Pompidou European Hospital (HEGP). This network aims to set up the best tools for detecting HPV-associated “multisite” precancerous lesions in order to determine the possible impact of dedicated care for this at-risk population. This monthly consultation was created at the HEGP in June 2014. It is currently organized around five consultations: gynaecological, ENT, urological, digestive and immunological. Every patient who has been diagnosed with HPV-related cancer and whose care is provided at the HEGP is offered this particular follow-up: systematically, once the initial lesion has been treated, the patient is convened annually for a day during which it benefits from the consultations mentioned above. A consultation with a psychologist is systematically proposed. Local samples are taken at each site: a cytological examination, the analysis of known predictive and prognostic virological markers are carried out. This study fits more broadly in a theme of clinical and fundamental research around cancers related to HPV.     

Differentiation state and invasiveness of human breast cancer cell lines

Summary Eighteen breast cancer cell lines were examined for expression of markers of epithelial and fibroblastic differentiation: E-cadherin, desmoplakins, ZO-1, vimentin, keratin and ₁ and ₄ integrins. The cell lines were distributed along a spectrum of differentiation from epithelial to fibroblastic phenotypes. The most well-differentiated, epithelioid cell lines contained proteins characteristic of desmosomal, adherens and tight junctions, were adherent to one another on plastic and in the basement membrane matrix Matrigel and were keratin-positive and vimentin-negative. These cell lines were all weakly invasive in anin vitro chemoinvasion assay. The most poorly-differentiated, fibroblastic cell lines were E-cadherin-, desmoplakin- and ZO-1-negative and formed branching structures in Matrigel. They were vimentin-positive, contained only low levels of keratins and were highly invasive in thein vitro chemoinvasion assay. Of all of the markers analyzed, vimentin expression correlated best within vitro invasive ability and fibroblastic differentiation. In a cell line with unstable expression of vimentin, T47D_CO, the cells that were invasive were of the fibroblastic type. The differentiation markers described here may be useful for analysis of clinical specimens and could potentially provide a more precise measure of differentiation grade yielding more power for predicting prognosis.