一种快速判定细胞培养中支原体污染的方法——瓜氨酸测定法

目的：开发一种简便、快速、能及时发现细胞培养中支原体污染的方法。方法：用HPLC检测细胞培养中瓜氨酸是否存在及其量的大小。结果：当细胞培养被支原体污染时,培养基中精氨酸量明显下降,同时有瓜氨酸出现;当支原体被消除后,瓜氨酸即消失。结论：在细胞培养中瓜氨酸的出现与支原体污染的关系是特异的,用HPLC在2h内即可检出,表明该方法可靠、简便、快速,可作为细胞培养过程中支原体污染的常规监测手段。     

两种等位基因特异性扩增方法在结肠癌K-ras癌基因点突变检测中的应用比较

针对传统电泳检测方法存在操作复杂、费时等缺点,提出一种用于检测K-ras癌基因点突变的实时荧光等位基因特异性扩增（Allele specific amplification,ASA）方法。该法采用突变型引物对结肠癌基因组中的K-ras基因进行等位基因特异性扩增,只有突变型样品能被顺利扩增出双链DNA产物,该产物能与双链DNA染料SYBR GreenⅠ结合,产生荧光信号从而被检测到。通过对荧光域值和溶解曲线分析来区分不同的基因突变类型。该法可以检测到野生型DNA中含量为1/1 000的突变型DNA,整个检测时间小于1 h。我们用该法检测31例结肠癌样品中K-ras基因密码子12发生的点突变,其中有15例检出为阳性。此外,还采用等位基因特异性扩增结合电泳分析对样品进行了检测,并对两种方法进行了比较。结果显示：实时荧光等位基因特异性扩增方法具有操作简便、快速、检测成本低等优点,为临床诊断基因突变引起的疾病提供了一种可行的手段。     

基于微流控芯片的温度梯度毛细管电泳检测大肠癌石蜡标本中K-ras基因突变

K-ras基因突变检测可用于大肠癌的早期筛查与诊断,并有利于筛选出抗表皮生长因子受体靶向药物治疗有效的大肠癌患者,以实现肿瘤的个体化治疗．采用以倾斜式热辐射原理建立的微流控温度梯度毛细管电泳(temperature gradient capillary electrophoresis,TGCE)基因突变检测系统,实现了对98例石蜡包埋大肠癌组织中K-ras基因突变的高灵敏度筛查,突变阳性检出率为47.96%,显著高于PCR产物直接测序的23.47%．克隆测序显示该方法至少能检测到2.08%的K-ras基因突变体．K-ras基因突变与临床病理学参数的关系分析显示,直肠癌中K-ras基因突变率明显高于结肠癌(P < 0.05),而与年龄、性别、组织学类型和肿瘤分期等无显著相关性．该检测方法为肿瘤早期诊断和指导临床用药提供了一种灵敏度高、检测速度快、便于大规模筛查的有效手段.     

电化学发光PCR方法检测结肠癌中K-ras癌基因点突变

发展了一种可用于快速检测K-ras癌基因点突变的电化学发光PCR(ECL-PCR)分析方法,该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对目的片段进行PCR扩增;随后,采用限制性内切酶MvaI对扩增产物进行酶切,由于突变导致酶切位点的丢失,所以只有野生型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到反应池中进行电化学发光检测。采用该法对20例结肠癌组织中的K-ras癌基因第12位密码子进行点突变分析,得出其中有9例存在点突变,点突变率为45%。该方法操作简便、安全、快速、灵敏,可用于快速检测K-ras癌基因点突变。     

单克隆抗体间接免疫荧光法检测巨细胞病毒分离培养中的抗原

应用抗巨细胞病毒(HCMV)蛋白抗原(分子量为20千道尔顿)的单克隆抗体(McAb-20k)和HCMV IgG特异性阳性血清以间接免疫荧光试验检测28例器官移植病人尿标本接种人胚肺细胞后的HCMV感染情况,结果前法于接种后48小时检测到HCMV阳性病人9例,后者于接种6天检测到阳性病人11例。与病毒分离结果相比较,两法的敏感性分别为81.8％和90.9％,特异性相应为100％和94.12％,符合率均为92.9％,比病毒分离提前数天至数周作出诊断,重复性良好。因此,抗HCMV蛋白抗原的单克隆抗体间接免疫荧光法是一种有效的、早期快速而又敏感特异的诊断方法,操作简便,既使没有单抗,可用HCMV IgG阳性血清代替,也可取得较好效果,值得在一般实验室推广应用。     

Changes in the Cell Volume of Didinium nasutum During Population Increase*

SYNOPSIS. In 3 species of carnivorous protozoa, the rate of individual food intake per generation declines with an increase in the density of the population. In all, the rate of division remains constant. Three hypotheses may be proposed to explain these phenomena: the individual size decreases, thus bringing about a decline in food need; the individuals in the earlier stages of population growth consume excess food which is passed on to later generations to supplement their food intake; an increase in density within the carnivore population decreases the activity rate, thus reducing the food requirement. Experiments using Didinium nasutum as the carnivore and Paramecium aurelia, syngen 4, as the food source were conducted to test these 3 hypotheses. The results contradicted the 1st hypothesis and part of the 2nd. A new working hypothesis based on the remaining hypotheses is proposed.     

Changes in Cell Size and Shape Associated with Changes in the Replication Time of the Chromosome of Escherichia coli

Average cell mass is shown to be inversely related to the concentration of thymine in the growth medium of a thy⁻ strain of Escherichia coli. The kinetics of the transition from one steady-state average cell mass to another was followed in an attempt to determine the relationship between the chromosome replication time and the time between completion of a round of chromosome replication and the subsequent cell division. Differences in average cell mass are shown to be associated with similar differences in average cell volume. Changes in volume associated with changes in thymine concentration are shown to be due primarily to differences in the width of cells. It is proposed that extension in length of the cell envelope occurs at a linear rate which is proportional to the growth rate and which doubles at the time of termination of rounds of replication. Changes in volume not associated with a change in growth rate are therefore accommodated by a change in cell width. Conditions are described under which average cell mass can continue to increase in successive generations and no steady-state average cell mass is achieved.     

Signal-Dependent Control of Autophagy and Cell Death in Colorectal Cancer Cell: The Role of the p38 Pathway

Autophagy is a vacuolar process leading to the degradation of long-lived proteins and cytoplasmic organelles in eukaryotes. This process has an important role in normal and cancer cells during adaptation to changing environmental conditions, cellular and tissue remodeling, and cell death.

To date, several signaling cascades have been described to regulate autophagy in a cell type-specific and signal-dependent manner.

We found that pharmacological blockade of the p38 pathway in colorectal cancer cells, either by the inhibitor SB202190 or by genetic ablation of p38α kinase, causes cell cycle arrest and autophagic cell death. In these cells, a complex network of intracellular kinase cascades controls autophagy and survival since the effect of p38α blockade is differentially affected by the pharmacological inhibition of MEK1, PI3K class I and III, and mTOR or by the differentiation status.

Collectively, our results suggest an opportunity for exploiting the pharmacological manipulation of the p38α pathway in the treatment of colorectal cancer. Given the number of drugs, currently available or under development, that target the p38 pathway, it stands to reason that elucidating the molecular mechanisms that link p38 and autophagy might have an impact on the clinical translation of these drugs.

Addendum to:

A Novel Cell Type-Specific Role of p38α in the Control of Autophagy and Cell Death in Colorectal Cancer Cells

F. Comes, A. Matrone, P. Lastella, B. Nico, F.C. Susca, R. Bagnulo, G. Ingravallo, S. Modica, G. Lo Sasso, A. Moschetta, G. Guanti and C. Simone

Cell Death Differ 2007; 14: 693-702     

抑癌基因DLC-1在结肠癌细胞sw480中表达的研究

目的:初步探讨DLC-1基因在结肠癌细胞sw480中的表达改变及其可能机制;方法:采用RT-PCR和Western blot分析结肠癌细胞CaCo2、sw480的DLC-1表达情况;应用去甲基化药物5—氮杂胞苷处理DLC-1基因阴性表达的细胞株,观察用药前后该细胞DLC-1基因的表达水平、细胞周期、细胞增殖力和侵袭力的影响。结果:CaCo2细胞高表达DLC-1 mRNA,而sw480则呈现表达缺失。经去甲基化药物5—氮杂胞苷处理sw480后,DLC-1 mRNA恢复表达,比较用药前后sw480细胞增殖力下降、细胞周期被阻滞在G2期、细胞侵袭力减弱。结论:DLC-1基因可影响结肠癌细胞sw480的增殖能力和侵袭力,并使结肠癌细胞sw480细胞周期阻滞于G2期,DNA甲基化可能是导致DLC-1在sw480细胞中的失活的一个重要原因。     

A Comparison of Positron Emission Tomography and Colonoscopy for the Detection of Advanced Colorectal Neoplasms in Subjects Undergoing a Health Check-Up

Background & Aims

There is no agreement as to whether F-18 fluorodeoxyglucose positron emission tomography and computed tomography (FDG PET/CT) screening for advanced colorectal neoplasms is meaningful. This retrospective study was undertaken to determine whether FDG PET/CT may be a valuable screening tool for the detection of advanced colorectal neoplasms.

Methods

A retrospective review of the records of 1,109 FDG PET/CT scans acquired from January 2007 to December 2011 was performed. Colonoscopy and FDG PET/CT imaging were performed within two days of each other. The results of colonoscopy were taken as the gold standard, either with or without the results of the histopathological examination. An advanced neoplasm was defined as the presence of a malignant tumor, an adenoma ≥1 cm, or histological evidence of high-grade dysplasia or significant villous components.

Results

A total of 36 subjects had advanced colorectal neoplasms detected by colonoscopy (totaling 38 neoplasms). Six of the 38 neoplasms were also detected by FDG PET/CT. The sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy of FDG PET/CT in the detection of advanced colorectal neoplasms were 15.8% (6/38), 99.1% (1063/1073), 37.5% (6/16), 97.1% (1063/1095), and 96.2% (1069/1111) respectively. The presence of lesions with an endoscopic size ≤1.5 cm (P<0.001) and low-grade dysplasia (P<0.001) were the main predictors of false-negative FDG PET/CT findings.

Conclusions

We conclude that FDG PET/CT screening of advanced colorectal neoplasms is unwarranted, especially in the presence of lesions with an endoscopic size ≤1.5 cm or low-grade dysplasia.     

Discovery of a 29-Gene Panel in Peripheral Blood Mononuclear Cells for the Detection of Colorectal Cancer and Adenomas Using High Throughput Real-Time PCR

Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform.     

脾加骨髓细胞移植治疗小鼠大肠癌的实验研究

目的:探讨半相合脾加骨髓细胞移植治疗小鼠大肠癌的效果及其对嵌合体水平和移植物抗宿主病(GVHD)的影响。方法:以接种CT26大肠癌细胞的BALB/c×C57BL/6杂交Fl代雌性小鼠为受鼠,以健康雌性Fl、雄性C57BL/6、雄性C3H小鼠为MHC全相合、半相合、不相合供鼠,观察移植后的抑瘤情况;另设5只化疗联合半相合脾加骨髓细胞移植的小鼠为监测组,用来作嵌合体的分析,观察各组GVHD的情况。结果:经化疗预处理的脾加骨髓细胞移植组小鼠肿瘤明显缩小,与单纯化疗未进行移植组比较,差异具有统计学意义(P0.05);化疗联合半相合脾加骨髓细胞移植的小鼠于移植3周后达完全供者植入,于移植后第10天左右出现纳差、倦怠、步态不稳、脱毛、腹泻、体重明显下降等GVHD的症状。结论:化疗预处理联合脾加骨髓细胞移植能对CT26大肠癌细胞产生GVT效应,并伴随着GVHD及嵌合率的变化。     

Polymorphism of the Thymidylate Synthase Gene and Thymidylate Synthase Levels in Colon Cancer Cell Lines and Different Tissues of Colorectal Cancer Patients

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.     

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.Colorectal cancer (CRC)¹ is the second most prevalent cancer in the western world. The development of this disease takes decades and involves multiple genetic events. CRC remains a major cause of mortality in developed countries because most of the patients are diagnosed at advanced stages because of the reluctance to use highly invasive diagnostic tools like colonoscopy. Actually only a few proteins have been described as biomarkers in CRC (carcinoembryonic antigen (CEA), CA19.9, and CA125 (1–3)), although none of them is recommended for clinical screening (4). Proteomics analysis is actively used for the identification of new biomarkers. In previous studies, the use of two-dimensional DIGE and antibody microarrays allowed the identification of differentially expressed proteins in CRC tissue, including isoforms and post-translational modifications responsible for modifications in signaling pathways (5–8). Both approaches resulted in the identification of a collection of potential tumoral tissue biomarkers that is currently being investigated.However, the implementation of simpler, non-invasive methods for the early detection of CRC should be based on the identification of proteins or antibodies in serum or plasma (9–13). There is ample evidence of the existence of an immune response to cancer in humans as demonstrated by the presence of autoantibodies in cancer sera. Self-proteins (autoantigens) altered before or during tumor formation can elicit an immune response (13–19). These tumor-specific autoantibodies can be detected at early cancer stages and prior to cancer diagnosis revealing a great potential as biomarkers (14, 15, 20). Tumor proteins can be affected by specific point mutations, misfolding, overexpression, aberrant glycosylation, truncation, or aberrant degradation (e.g. p53, HER2, NY-ESO1, or MUC1 (16, 21–25)). In fact, a number of tumor-associated autoantigens (TAAs) were identified previously in different studies involving autoantibody screening in CRC (26–28).Several approaches have been used to identify TAAs in cancer, including natural protein arrays prepared with fractions obtained from two-dimensional LC separations of tumoral samples (29, 30) or protein extracts from cancer cells or tissue (9, 31) probed by Western blot with patient sera, cancer tissue peptide libraries expressed as cDNA expression libraries for serological screening (serological analysis of recombinant cDNA expression libraries (SEREX)) (22, 32), or peptides expressed on the surface of phages in combination with microarrays (17, 18, 33, 34). However, these approaches suffer from several drawbacks. In some cases TAAs have to be isolated and identified from the reactive protein lysate by LC-MS techniques, or in the phage display approach, the reactive TAA could be a mimotope without a corresponding linear amino acid sequence. Moreover, cDNA libraries might not be representative of the protein expression levels in tumors as there is a poor correspondence between mRNA and protein levels.Protein arrays provide a novel platform for the identification of both autoantibodies and their respective TAAs for diagnostic purposes in cancer serum patients. They present some advantages. Proteins printed on the microarray are known “a priori,” avoiding the need for later identifications and the discovery of mimotopes. There is no bias in protein selection as the proteins are printed at a similar concentration. This should result in a higher sensitivity for biomarker identification (13, 35, 36).In this study, we used commercially available high density protein microarrays for the identification of autoantibody signatures and tumor-associated antigens in colorectal cancer. We screened 20 CRC patient and control sera with protein microarrays containing 8000 human proteins to identify the CRC-associated autoantibody repertoire and the corresponding TAAs. Autoantibody profiles that discriminated the different types of CRC metastasis were identified. Moreover a set of TAAs showing increased or decreased expression in cancer cell lines and paired tumoral tissues was found. Finally an ELISA was set up to test the ability of the most immunoreactive proteins to detect colorectal adenocarcinoma. On the basis of the antibody response, combinations of three antigens, PIM1, MAPKAPK3, and ACVR2B, showed a great potential for diagnosis.     

Evaluation of the Right Ventricular Function in Pneumoconiosis Patients Using Volume–Time Curves Obtained by Real-Time Three-Dimensional Echocardiography

The study was aimed to evaluate the right ventricular function in pneumoconiosis patients by real-time three-dimensional echocardiography. A total of 80 individuals including 44 consecutive pneumoconiosis patients and 36 age- and gender-matched healthy volunteers as controls were prospectively recruited for the study. All the patients underwent two- and three-dimensional echocardiography. Measurements of the right ventricle included tricuspid regurgitation pressure (TRPG), anterior and posterior wall thickness and range of motion (TH1, TH2, M1, M2), right end-diastolic volume and end-systolic volume. The right ventricular ejection fraction (RVEF) was also calculated. The RVEF of healthy volunteers ranged from 50 to 78 %, whereas that of the pneumoconiosis patients varied from 29 to 73 %. An increase in TRPG caused a significant (p = 0.006) decrease in RVEF (by 77.3 %), suggesting the two variables were negatively correlated (r = ?0.643, p < 0.01). In comparison with normal, the volume–time curves of the pneumoconiosis patients showed a lower trough. Use of real-time three-dimensional echocardiography provides with added clinical information needed to evaluate right ventricular function in pneumoconiosis patients.     

Changes in the Composition of Cell Wall Polysaccharides of Suspension-cultured Vinca rosea Cells during Culture

In a suspension culture of Vinca rosea L. three distinct growth phases were distinguished: a cell division phase, a cell expansion phase, and a stationary phase. At various stages of the growth cycle, extra-cellular polysaccharides (ECP) and cell wall were isolated and their compositions were investigated. ECP are mainly composed of xyloglucan, 3,6-linked arabinogalactan, and polyuronide. From the results of gas chromatographic analyses of sugars and partially methylated sugars in each fraction of cell wall polysaccharides, the following changes were observed in the composition of cell wall polysaccharides during culture: (1) a decrease in polyuronide content, (2) a decrease in non-cellulosic glucan content, (3) an increase in xyloglucan content, and (4) an increase in a-cellulose content. The significance of the changes is discussed in relation to the growth of cells.     

Three-Dimensional Spatiotemporal Modeling of Colon Cancer Organoids Reveals that Multimodal Control of Stem Cell Self-Renewal is a Critical Determinant of Size and Shape in Early Stages of Tumor Growth

We develop a three-dimensional multispecies mathematical model to simulate the growth of colon cancer organoids containing stem, progenitor and terminally differentiated cells, as a model of early (prevascular) tumor growth. Stem cells (SCs) secrete short-range self-renewal promoters (e.g., Wnt) and their long-range inhibitors (e.g., Dkk) and proliferate slowly. Committed progenitor (CP) cells proliferate more rapidly and differentiate to produce post-mitotic terminally differentiated cells that release differentiation promoters, forming negative feedback loops on SC and CP self-renewal. We demonstrate that SCs play a central role in normal and cancer colon organoids. Spatial patterning of the SC self-renewal promoter gives rise to SC clusters, which mimic stem cell niches, around the organoid surface, and drive the development of invasive fingers. We also study the effects of externally applied signaling factors. Applying bone morphogenic proteins, which inhibit SC and CP self-renewal, reduces invasiveness and organoid size. Applying hepatocyte growth factor, which enhances SC self-renewal, produces larger sizes and enhances finger development at low concentrations but suppresses fingers at high concentrations. These results are consistent with recent experiments on colon organoids. Because many cancers are hierarchically organized and are subject to feedback regulation similar to that in normal tissues, our results suggest that in cancer, control of cancer stem cell self-renewal should influence the size and shape in similar ways, thereby opening the door to novel therapies.     

Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

Recently we found that ATP5J was over-expressed in tissue samples from patients with colorectal cancer. However, the clinical significance and function of the over-expression of ATP5J in these patients remains unclear. We investigated these issues in the current study. Our results indicated that expression of ATP5J was significantly higher in colorectal cancer tissue than in adjacent tissue, and it was also significantly higher in metastatic lymph nodes than in primary cancer tissue (P<0.05). A correlation between ATP5J expression and tumor differentiation was detected, but no correlation with gender, age, T stage, lymph node metastasis, or survival status was observed. Down-regulation of ATP5J expression attenuated the ability of cell migration and increased the sensitivity to 5-fluorouracil (5-Fu) in cells of the DLD1 cell line. Inversely, up-regulation of ATP5J expression enhanced cell migration and decreased 5-Fu sensitivity, suggesting that the function of ATP5J in colorectal cancer might involve cell migration and 5-Fu sensitivity.