Study Expression Of Artocarpus Lingnanensis Lectin Receptor In Gastric Tissue

Background and objective:Currently malignant tumor is the primary causes of deaths. Glycoprotein and glycolipide on the cell membrane often show changes in the process of cell canceration, infiltration and metastasis. On the basis of lectin’s highly specific recognition of chitosan and its conjugates, lectin probe was used to study the changes of membrane structure during malignant process at the molecular level. In this research, Artocarpus lingnanensis lectin (ALL) was labeled with horseradish peroxidase and fluorescein isothiocyanate succssfully, and the expression of ALL receptors were observed in gastric cancer tissue and normal gastric tissue respectively by lectin histochemistry. The aim of the study is to lay a good theoretical and experimental foundation for the exploration of the tumor development and application of ALL in the tumor diagnosis.Method:1. ALL was purified from the seeds of artocarpus lingnanensis as described [1,2]. Briefly, ALL was purified from the seeds of Artocarpus lingnanensis by ammonium sulfate fractional precipitation and affinity chromatography on Gal-Sepharose 6B, which was eluted with 0.2mol/L Gal, then dislysis and ultrafiltration. The activity of Artocarpus lingnanensis lectin was examined by red cell agglutination test.2. The preparation of ALL-FITC and ALL-HRP2.1 The preparation of ALL-FITC:2.1.1 Dialysis method:first, the concentration Artocarpus Lengnanensis Lectin solution was determined, then, FITC was added to the solution, and mixed thoroughly. The well-mixed solution was dialysed, and the F/P value of conjugate was measured.2.1.2 Solidification method:first, the solid phase absorption between ALL and Sepharose4B was conducted, then, FITC solution was dropped into the gel, and ALL-FITC was eluted with Gal-PBS.2.1.3 Compare F/P of the two methods.2.2 The preparation of ALL-HRP:HRP was dialysed in sodium periodate solution and acetic acid buffer solution, then, ALL was added to the solution. The mixed solution was dialysed and stored in a dark place.3. Expression of ALL receptors:3.1 Collection of cases:Gastric adenocarcinoma cancerous tissue sections were collected from 32 patients, and normal gastric tissue sections were collected from 25 health people.3.2 ALL-FITC Staining:Paraffin section was dewaxed and hydrated. The tissue section was first stained with ALL-FITC solution and then stained with DAPI. The expression of Artocarpus Lengnanensis Lectin receptors was observed using inverted fluorescence microscope.3.3 ALL-HRP Staining:Paraffin section was dewaxed and hydrated. The tissue section was first stained with ALL-HRP solution and then stained with haematine. The expression of Artocarpus Lengnanensis Lectin receptors was observed using microscope.4.The Preparation of ALL-Sepharose 6B:The Sepharose 6B was first activated, then affinity adsorbed with ALL, and the other active sites of Sepharose 6B was sealed. The haemagglutination activity of conjugate was examined.Results:1. The agglutinating activity of Artocarpus lingnanensis lectin is 29-211.2. The F/P valueis of conjugates which is made by dialysis method is 0.758, while conjugates which is made by solid phase method is 1.02. We acquirded good effects when stained paraffin sections with the conjugates prepared by the two methods.3. The expression of ALL receptors3.1 ALL-FITC Staining:In the cytoplasm, the weak green fluorescence can be seen in normal gastric tissue while bright green fluorescence in the gastric adenocarcinoma tissue under fluorescence microscope. These results indicated that ALL receptors are expressed both in normal gastric tissue and the gastric adenocarcinoma tissue, and mainly located in cytoplasm. The positive rate of gastric adenocarcinoma tissue is 68.8% while the positive rate of normal stomach tissue is 24%. The difference between two groups has statistically significances (P=0.001), indicating that the expression of ALL receptors in gastric adenocarcinoma tissue is significantly higher than that in normal gastric tissue.3.2 ALL-HRP Staining:In the cytoplasm, the light yellow can be seen in normal gastric tissue while brown in the gastric adenocarcinoma tissue under microscope. These results indicated that ALL receptors are expressed both in normal gastric tissue and the gastric adenocarcinoma tissue, and mainly located in cytoplasm. The positive rate of gastric adenocarcinoma tissue is 75% while the positive rate of normal stomach tissue is 16%. The difference between two groups has statistically significances (P=0.001), indicating that the expression of Artocarpus Lengnanensis Lectin receptors in gastric adenocarcinoma tissue is significantly higher than that in normal gastric tissue.4. The agglutination test showed that ALL-Sepharose 6B has the agglutination activity.Conclusion:1. Solidification method is better than dialysis method in labeling ALL with FTIC.2. Both ALL-FITC and ALL-HRP prepared in this experiment can be used to analyze the expression of ALL receptors.3.The expression of ALL receptors in gastric adenocarcinoma tissue is up-regulated, which are mainly distributed in the cytoplasm of cancer cells.4. The ALL-Sepharose 6B can be used for the further isolation and purification of glycoprotein.