Expression Profiles Of GCRG213Protein In Human Gastric Mucosa And Dual-color Immunolfuorescent Labeling Of Associated Proteins With Quantum Dots

Background:GCRG213(gastric cancer related gene213) is a variant of functional LINE-1endonuclease. It has been reported that GCRG213played an important role in thegastric carcinogenesis. Previous study using traditional immunohistochemistry indicatedthat GCRG213protein expressed in the cytoplasm of gastric cancer cells, but not in thenoncancerous tissues of resection margin, or covering epithelium of mucosa. However,the expression profiles of GCRG213protein in various glands of normal tissue isunknown exactly.QD (Quantum Dot) is a kind of novel nanomaterial. Comparing with traditionalorganic fluorescence dyes, QD has advantages at many aspects, such as board andcontinuous excitation spectrum, narrow and symmetrical emission spectrum, bleachingresistance, different QDs can be excited with one certain light source and emitteddifferent fluorescences, and so on. Given those excellent qualities, QDs basedimmunofluorescence can be used in direct and indirect labeling. In specific, two or morebiological target components can be detected simultaneously through different size ofQDs particles. To date, QDs immunofluorescence technology has became one of themost important applications in molecular biology and cytohistology.Objective:First, to detect the expression of GCRG213protein in human gastric mucosa, andto investigate the natural distribution of positive cells in mucosa. The second, to detectthe expression of GCRG213and Ki67proteins in the gastric mucosa tissues individualyand simultaneously, by the QDs immunofluorescence probes. Methods:1. Expression profiles of GCRG213in human gastric mucosaDetect GCRG213protein expression in serial sections of normal human mucosa byimmunohistochemistry staining using the mouse anti human antibody, and definepositive cells by HE staining.2．Dual-color immunofluorescent labeling of associated proteins with quantum dots(1) Conjugating QDs with anti-Ki67antibody by EDC(1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride). QDs-anti-Ki67conjugates were applied into gastric cancer tissues.(2) Using QDs-streptavidin system to detect GCRG213protein and Ki67protein inhuman gastric mucosa respectively.(4) GCRG213protein expression in cell cytoplasm and Ki67protein expression innucleus were detected with QDs-SA and QDs-IgG co-staining, simultaneously.Results:GCRG213protein was positively expressed in the cell cytoplasm of gastric cancer.It was in consistent with previous study. And most importantly and interestingly, wefound that GCRG213was substantially expressed in some inherent glands which secreteacid in normal human mucosa. In detail, based on HE staining, the positively expressedcells in the glands are chief cells, while parietal cells, mucous neck cells, surfacemucous cells and pyloric glands are negatively expressed.The agarose electrophoresis showed that QDs was conjugated to anti-Ki67antibody successfully. The QDs-anti-Ki67probes were applied to the gastric cancertissue, the positive signal located in the nucleus.Expression of GCRG213and Ki67proteins in normal human mucosa and gastriccancer tissues were detected by QDs-SA based immunofluorescence. They showed apattern similar to the brown signal from the traditional IHC.QDs based multiple-color immunofluorescent labeling was successfully applied inGCRG213and Ki67proteins detection. GCRG213expression in the cytoplasm with redQDs fluorescence and Ki67expression in the nucleolus with green QDs fluorescence were exhibited simultaneously. The fluorescence light of quantum dots was stable andthe samples could be kept for long time.Conclusions:It is the first report that GCRG213, a variant of functional LINE-1endonuclease,expressed in some inherent glands which secret acid of the normal human mucosa,mainly in chief cells. The gastric cancer associated protein Ki67was successfullydetected with QDs-anti-Ki67conjugate in gastric cancer tissue. Last but the mostimportant is that we successfully detected the co-expression of GCRG213and Ki67proteins in gastric cancer tissue simultaneously by QDs based dual-colorimmunofluorescent labeling. Our study has important significance to promotenanotechnology in clinical medicine.