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1.
Intensive studies of molecular mechanisms responsible for tumor transformation results in identification of new proteins and their genes involved into tumor development. These proteins may be used as markers of tumor transformation of cells and the level of their expression may be evaluated by means of modern highly sensitive and technological methods of analysis. This review summarized literature data on currently used immunohistochemical and molecular genetic markers of gastric cancer. It highlights genetic and epigenetic changes detected in nucleic acids of tumor tissue cells in malignant and benign gastric diseases as well as in the level of DNA circulating in blood of patients with gastric cancer.  相似文献   

2.
迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。  相似文献   

3.
Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.  相似文献   

4.
Breast cancer is the leading cause of cancer death among women worldwide. Multiple extrinsic and intrinsic factors are associated with this disease’s development. Various research groups worldwide have reported the presence of human papillomavirus (HPV) DNA in samples of malignant breast tumors. Although its role in mammary carcinogenesis is not fully understood, it is known that the HPV genome, once inserted into host cells, has oncogenic capabilities. The present study aimed to detect the presence of HPV DNA in 116 breast tissue biopsies and classify them according to their histology. It was found that 50.9% of the breast biopsies analyzed were malignant neoplasms, of which 74.6% were histologically classified as infiltrating ductal carcinoma. In biopsies with non-malignant breast disease, fibroadenoma was the most common benign neoplasm (39.1%). Detection of HPV DNA was performed through nested PCR using the external primer MY09/11 and the internal primer GP5+/6+. A hybridization assay genotyped HPV. HPV DNA was identified in 20.3% (12/59) of malignant neoplasms and 35% non-malignant breast disease (16/46). It was also detected in 27.3% (3/11) of breast tissue biopsies without alteration. However, there are no statistically significant differences between these groups and the existence of HPV DNA (p = 0.2521). Its presence was more frequent in non-malignant alterations than in malignant neoplasias. The most frequent genotypes in the HPV-positive samples were low-risk (LR) HPV-42 followed by high-risk (HR) HPV-31.  相似文献   

5.
Exosomes are tiny vesicles (30–150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.  相似文献   

6.
Circulating tumor cells (CTCs) are seeds for cancer metastasis and are predictive of poor prognosis in breast cancer patients. Whether CTCs and primary tumor cells (PTCs) respond to chemotherapy differently is not known. Here, we show that CTCs of breast cancer are more resistant to chemotherapy than PTCs because of potentiated DNA repair. Surprisingly, the chemoresistance of CTCs was recapitulated in PTCs when they were detached from the extracellular matrix. Detachment of PTCs increased the levels of reactive oxygen species and partially activated the DNA damage checkpoint, converting PTCs to a CTC-like state. Inhibition of checkpoint kinases Chk1 and Chk2 in CTCs reduces the basal checkpoint response and sensitizes CTCs to DNA damage in vitro and in mouse xenografts. Our results suggest that DNA damage checkpoint inhibitors may benefit the chemotherapy of breast cancer patients by suppressing the chemoresistance of CTCs and reducing the risk of cancer metastasis.  相似文献   

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Cancer is one of the most important causes of death worldwide. The onset of cancer may be initiated due to a variety of factors such as environment, genetics or even due to personal lifestyle choices. To counteract this tremendous increase, the demand for a new technology has risen. By this means, the use of digital polymerase chain reaction (dPCR) has been shown to be a promising methodology in the early detection of many types of cancers. Furthermore, several researchers confirmed that the use of tumor cell‐free DNA (cfDNA) and circulating tumor cells (CTC) in peripheral blood is essential in revealing an early prognosis of such diseases. Besides this, it was established that dPCR might be used in a much more efficient, accurate, and reliable manner to amplify a variety of genetic material up to the identification of mutations in hematological diseases. Therefore, this article demonstrates the differences between conventional PCR and dPCR as a molecular technique to detect the early onset of cancer. Furthermore, CTC and cfDNA were officially approved by the Food and Drug Administration as new biological biomarkers in cancer development and monitoring.  相似文献   

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The mitochondrial free radical theory of aging suggests that accumulating oxidative damage to mitochondria and mitochondrial DNA (mtDNA) plays a central role in aging. Circulating cell‐free mtDNA (ccf‐mtDNA) isolated from blood may be a biomarker of disease. Extracellular vesicles (EVs) are small (30–400 nm), lipid‐bound vesicles capable of shuttling proteins, nucleic acids, and lipids as part of intercellular communication systems. Here, we report that a portion of ccf‐mtDNA in plasma is encapsulated in EVs. To address whether EV mtDNA levels change with human age, we analyzed mtDNA in EVs from individuals aged 30–64 years cross‐sectionally and longitudinally. EV mtDNA levels decreased with age. Furthermore, the maximal mitochondrial respiration of cultured cells was differentially affected by EVs from old and young donors. Our results suggest that plasma mtDNA is present in EVs, that the level of EV‐derived mtDNA is associated with age, and that EVs affect mitochondrial energetics in an EV age‐dependent manner.  相似文献   

11.
环状RNA是由前体RNA通过反向剪接形成的一类共价闭合环状分子.在过去,环状RNA被认为是DNA转录的"噪音",不参与生物代谢过程.然而,最近研究表明,环状RNA的异常表达可影响包括三阴性乳腺癌在内的多种恶性肿瘤的发生发展.该文综述了环状RNA在肿瘤中的分子机制及其在三阴性乳腺癌细胞增殖、凋亡、迁移、侵袭和药物抗性中的...  相似文献   

12.
Immunization of animals with DNA leads to the production of anti-DNA antibodies (Abs) demonstrating both DNase and RNase activities. It is currently not known whether anti-RNA Abs can possess nuclease activities. In an attempt to address this question, we have shown that immunization of three rabbits with complex of RNA with methylated BSA (mBSA) stimulates production of IgGs with RNase and DNase activities belonging to IgGs, while polyclonal Abs from three non-immunized rabbits and three animals immunized with mBSA are catalytically inactive. Affinity chromatography of IgGs from the sera of autoimmune (AI) patients on DNA-cellulose usually demonstrates a number of fractions, all of which effectively hydrolyze both DNA and RNA, while rabbit catalytic IgGs were separated into Ab subfractions, some of which demonstrated only DNase activity, while others hydrolyzed RNA faster than DNA. The enzymic properties of the RNase and DNase IgGs from rabbits immunized with RNA distinguish them from all known canonical RNases and DNases and DNA- and RNA-hydrolyzing abzymes (Abzs) from patients with different AI diseases. In contrast to RNases and AI RNA-hydrolyzing Abs, rabbit RNase IgGs catalyze only the first step of the hydrolysis reaction but cannot hydrolyze the formed terminal 2',3'-cyclophosphate. The data indicate that Abzs of AI patients hydrolyzing nucleic acids in part may be Abs against RNA and its complexes with proteins. Copyright (c) 2008 John Wiley & Sons, Ltd.  相似文献   

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mRNA profiles of circulating tumour cells (CTCs) were analysed in patients with triple‐negative breast cancer (TNBC) (pts) before (BT) and after therapy (AT) to identify additional treatment options. 2 × 5 mL blood of 51 TNBC pts and 24 non‐TNBC pts (HR+/HER2?; HR?/HER2+) was analysed for CTCs using the AdnaTest EMT‐2/Stem Cell Select?, followed by mRNA isolation and cDNA analysis for 17 genes by qPCR PIK3CA, AKT2, MTOR and the resistance marker AURKA and ERCC1 were predominantly expressed in all breast cancer subtypes, the latter ones especially AT. In TNBC pts, ERBB3, EGFR, SRC, NOTCH, ALK and AR were uniquely present and ERBB2+/ERBB3 + CTCs were found BT and AT in about 20% of cases. EGFR+/ERBB2+/ERBB3 + CTCs BT and ERBB2+/ERBB3 + CTCs AT significantly correlated with a shorter progression‐free survival (PFS; P = 0.01 and P = 0.02). Platinum‐based therapy resulted in a reduced PFS (P = 0.02) and an induction of PIK3CA expression in CTCs AT. In non‐TNBC pts, BT, the expression pattern in CTCs was similar. AURKA+/ERCC1 + CTCs were found in 40% of HR?/HER2 + pts BT and AT. In the latter group, NOTCH, PARP1 and SRC1 were only present AT and ERBB2 + CTCs completely disappeared AT. These findings might help to predict personalized therapy for TNBC pts in the future.  相似文献   

15.
血行播散是乳腺癌转移的重要途径,检测腋窝淋巴结已经不能完全准确判断乳腺癌患者是否存在转移。肿瘤细胞进入外周血是肿瘤远处转移的前提,对乳腺癌患者循环肿瘤细胞的检测将有助于判断预后,指导治疗,监测治疗效果。简要综述了乳腺癌循环肿瘤细胞及其检测方法的研究进展。  相似文献   

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Abstract In order to better understand bacterial growth in the natural environment, a tracer experiment was carried out by adding [14C]bicarbonate to samples from a eutrophic lake and enclosures which were constructed inside the lake to provide different substrate conditions. It was shown that extracellular dissolved organic carbon (EDOC) released from photosynthetic phytoplankton was one of the most important carbon sources for the growth of planktonic bacteria in the aquatic environment studied here. About 5% of photosynthetically fixed 14C was transferred to the bacterial fraction between 4 and 8 h after the addtion of the [14C]bicarbonate. This increased to 10% during the following 16 h in the dark. Between 50% and 60% of bacterial 14C from EDOC was transformed to bacterial macromolecules. Analysis of cellular components showed that 36–39% of macromolecules produced were DNA during the light incubation, although the proportion of DNA relative to total macromolecules decreased in the dark. RNA synthesis increased with time, from 25% to 38% during the dark incubation. There was a correlation between bacterial growth rates and the ratio of RNA/DNA synthesized from EDOC (r = 0.81). However, the RNA/DNA ratio was very small, 1.8 for all samples examined, compared with previous data obtained for the laboratory bacteria. This is probably due to the very slow growth rate of the natural planktonic bacteria.  相似文献   

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以吖啶橙为细胞DNA荧光探针,用阿达玛(Hadamard)变换显微图象分析仪和显微荧光光度计分别测定了4例乳腺肿瘤的细胞DNA含量(倍性),并对分析结果进行了比较,二者的分析结果均与病理学诊断结论相吻合.阿达玛变换显微图象分析仪作为一种新的细胞定量分析仪器,其分析结果的准确度可与显微荧光光度计相媲美,而且阿达玛变换显微图象分析仪还具有其独特的优点,如信噪比高、具有同时分析多个细胞和同步扣除背景信号的能力等.  相似文献   

20.
We previously reported on the purification and characterization of a functional multi‐protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry‐out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30‐fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7–10 ribonucleotides long and DNA primers that are 20–40 deoxyribonucleotides long using a poly(dT) template of exogenous single‐stranded DNA. The synthesome‐catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double‐stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10–20 nt in length and the primers are covalently link to DNA to form RNA‐primed nascent DNA of 100–200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins. J. Cell. Biochem. 106: 798–811, 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

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