Abstract
The development of methods for effective diagnosis and monitoring of colorectal cancer (CRC) treatment is one of the basic scientific problem. The circulating plasma contains extracellular nanovesicles (EVs) secreted mainly by blood and endothelial cells. The minor fraction of plasma EVs is produced by cells of various tissues, including cells of the intestinal epithelium. The biochemical composition of such vesicles should have tissue-specific features. Presented study was aimed to identify surface markers of EVs secreted by intestinal epithelium cells and to assess the possibility of isolating and quantification of such vesicles for the diagnosis of CRC. The cell cultures (HCT-116, HT-29, COLO-320, HuTu-80, SW837), plasma of CRC patients and healthy donors were used in the study. The methods of nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), dot-blotting and flow cytometry were applied for EVs characterization. With the original technology of immunosorption we have demonstrated an increased amount of CLRN3, GAL4 and Meprin A, i.e. positive EVs in plasma of CRC patients comparing to healthy donors. Based on the quantitative analysis of such EVs, new methods of diagnostics and monitoring of CRC therapy can be developed.
Highlights
The development of methods for effective diagnosis and monitoring of colorectal cancer (CRC) treatment is one of the basic scientific problem
Presented study was aimed to identify surface markers of extracellular nanovesicles (EVs) secreted by intestinal epithelium cells and to assess the possibility of isolating and quantification of such vesicles for the diagnosis of CRC
The cell cultures (HCT-116, HT-29, COLO-320, HuTu-80, SW837), plasma of CRC patients and healthy donors were used in the study
Summary
Биологический материал Биологической материал был получен от доноров и пациентов, проходивших обследование или лечение в ФГБУ «НМИЦ онкологии им. Секретируемых клетками in vitro, культуральную среду также очищали от клеточного детрита и крупных мембранных везикул путем дифференциального центрифугирования, затем ультра-центрифугировали при 110 000g в течение ночи при +4°С. Содержащий экзосомы, разводили в 100 мкл ФСБ (Thermo Fisher, США). Дот-блоттинг Для детекции общего содержания «маркерных» протеинов в биологических образцах (соскобах кишечного эпителия, клеточных культурах или ВНВ) материал лизировали с помощью буферного раствора RIPA (Thermo Scientific, США). Образцы наносили на нитроцеллюлозную мембрану (каждый «дот» - 0,5 мкл) с размером пор 0,45 мкм (BioRad Laboratories, США), блокировали (Трис-буферный солевой раствор (ТБС), 0,05% Tween-20, 5% альбумин бычьей сыворотки (все от Sigma, США)) в течение 1 часа. После трехкратной «отмывки» мембраны детекция пероксидазной активности проводилась путем хемилюминесцентной реакции с использованием Pierce ECL Western Blotting Substrate (Thermo Scientific, США) и системы визуализации Invitrogeni Bright FL1500 Imaging System. The list of potential markers of EVs secreted by the cells of CRC
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